1 00:00:06,222 --> 00:00:07,523 >> HELLO, EVERYBODY. 2 00:00:07,523 --> 00:00:10,059 WELCOME TO THE STETTEN LECTURE. 3 00:00:10,059 --> 00:00:13,062 THIS LECTURE IS BEING RECORDED. 4 00:00:13,062 --> 00:00:20,970 MY NAME IS JOHN LO LORSCH. 5 00:00:20,970 --> 00:00:23,139 THIS IS PART OF THE DIRECTOR'S 6 00:00:23,139 --> 00:00:27,143 WEDNESDAY AFTERNOON LECTURE 7 00:00:27,143 --> 00:00:30,646 SERIES. 8 00:00:30,646 --> 00:00:32,248 HE SPENT MOST OF HIS CAREER AT 9 00:00:32,248 --> 00:00:34,083 NIH, SERVING IN TWO INSTITUTES 10 00:00:34,083 --> 00:00:36,853 AS WELL AS THE OFFICE OF THE NIH 11 00:00:36,853 --> 00:00:37,520 DIRECTOR. 12 00:00:37,520 --> 00:00:38,621 AND HE WAS PARTICULARLY 13 00:00:38,621 --> 00:00:40,957 WELL-KNOWN AS THE CO-AUTHOR OF 14 00:00:40,957 --> 00:00:43,659 THE EARLY EDITIONS OF THE 15 00:00:43,659 --> 00:00:44,627 TEXTBOOK, PRINCIPLES OF 16 00:00:44,627 --> 00:00:45,161 BIOCHEMISTRY. 17 00:00:45,161 --> 00:00:47,663 IN RECOGNITION OF HIS MANY 18 00:00:47,663 --> 00:00:48,865 CONTRIBUTIONS TO NIH, BOTH 19 00:00:48,865 --> 00:00:52,034 WITHIN AND OUTSIDE OF THE NIH, 20 00:00:52,034 --> 00:00:55,471 HE HAS THE RARE DISTINCTION OF 21 00:00:55,471 --> 00:00:56,739 HAVING TWO NIH ENTITIES NAMED 22 00:00:56,739 --> 00:00:59,242 AFTER HIM: THIS LECTURE AND THE 23 00:00:59,242 --> 00:01:00,676 DEWITT STETTEN JR. MUSEUM OF 24 00:01:00,676 --> 00:01:01,711 MEDICAL RESEARCH, WHICH YOU 25 00:01:01,711 --> 00:01:02,845 PROBABLY ACTUALLY SAW WHEN YOU 26 00:01:02,845 --> 00:01:04,180 WALKED IN, BECAUSE IT'S 27 00:01:04,180 --> 00:01:05,314 DISTRIBUTED AROUND THE CAMPUS, 28 00:01:05,314 --> 00:01:07,250 MOST OF IT, I THINK ACTUALLY IN 29 00:01:07,250 --> 00:01:07,817 THIS BUILDING. 30 00:01:07,817 --> 00:01:09,218 SO THE EXHIBIT OUT THERE AS WELL 31 00:01:09,218 --> 00:01:12,522 AS THE ONE AROUND THE CORNER OF 32 00:01:12,522 --> 00:01:14,390 MARSHAL NUREMBERG AND THE 33 00:01:14,390 --> 00:01:21,931 DISCOME RETHEDISCOVERY OF THE G. 34 00:01:21,931 --> 00:01:24,834 THE WAY THE LECTURE WORKS IS THE 35 00:01:24,834 --> 00:01:26,002 SPEAKER WILL GIVE A 50 MINUTE 36 00:01:26,002 --> 00:01:27,503 TALK ON HER RESEARCH FOLLOWED BY 37 00:01:27,503 --> 00:01:28,538 A 10 MINUTE QUESTION AND ANSWER 38 00:01:28,538 --> 00:01:29,438 SESSION. 39 00:01:29,438 --> 00:01:31,140 YOU CAN ASK YOUR QUESTIONS BY 40 00:01:31,140 --> 00:01:32,975 USING THE MICS IN THE ROOM. 41 00:01:32,975 --> 00:01:34,977 IF YOU'RE IN THE ROOM. 42 00:01:34,977 --> 00:01:37,547 OR BY SELECTING THE "SEND LIVE 43 00:01:37,547 --> 00:01:39,348 FEEDBACK" BUTTON ON YOUR 44 00:01:39,348 --> 00:01:40,182 VIDEOCAST SCREEN IF YOU'RE 45 00:01:40,182 --> 00:01:40,716 ONLINE. 46 00:01:40,716 --> 00:01:42,685 IF YOU ARE SEEKING A CONTINUING 47 00:01:42,685 --> 00:01:45,421 MEDICAL EDUCATION CREDIT OR CME, 48 00:01:45,421 --> 00:01:54,397 THE CODE IS 57663. 49 00:01:54,397 --> 00:01:55,531 WE INVITE THOSE OF YOU HERE IN 50 00:01:55,531 --> 00:01:58,000 PERSON TO JOIN US OUTSIDE OF THE 51 00:01:58,000 --> 00:01:59,535 AMPHITHEATER FOR A BRIEF 52 00:01:59,535 --> 00:02:00,703 RECEPTION AFTER THE LECTURE, 53 00:02:00,703 --> 00:02:02,505 JUST RIGHT OUT THERE. 54 00:02:02,505 --> 00:02:06,409 SO TODAY'S SPEAKER, SUDHA 55 00:02:06,409 --> 00:02:08,477 CHAKRAPANI, JOINS A VERY 56 00:02:08,477 --> 00:02:11,247 DISTINGUISHED LIST OF STETTEN 57 00:02:11,247 --> 00:02:12,682 LECTURERS, PROFESSOR AND CHAIR 58 00:02:12,682 --> 00:02:13,416 IN THE DEPARTMENT OF 59 00:02:13,416 --> 00:02:14,584 PHARMACOLOGY AT CASE WESTERN 60 00:02:14,584 --> 00:02:15,618 RESERVE UNIVERSITY AND SERVES AS 61 00:02:15,618 --> 00:02:17,186 DIRECTOR OF THE CLEVELAND CENTER 62 00:02:17,186 --> 00:02:20,423 FOR MEMBRANE AND STRUCTURAL 63 00:02:20,423 --> 00:02:21,090 BIOLOGY. 64 00:02:21,090 --> 00:02:22,458 SUDHA HAS DEDICATED HER CAREER 65 00:02:22,458 --> 00:02:24,226 TO INVESTIGATING ION CHANNELS 66 00:02:24,226 --> 00:02:25,595 THAT MEDIATE FAST SYNAPTIC 67 00:02:25,595 --> 00:02:27,797 TRANSMISSION AT THE NEURONAL AND 68 00:02:27,797 --> 00:02:29,732 NEUROMUSCULAR JUNCTION. 69 00:02:29,732 --> 00:02:31,601 SHE LEADS A LABORATORY THAT 70 00:02:31,601 --> 00:02:33,569 UTILIZES CRYOELECTRON MICROSCOPY 71 00:02:33,569 --> 00:02:35,938 TO INVESTIGATE ION TRANSPORT IN 72 00:02:35,938 --> 00:02:38,441 HEALTHY AND DISEASED CONDITIONS 73 00:02:38,441 --> 00:02:40,676 AND HOW MEDICINES CAN TARGET TO 74 00:02:40,676 --> 00:02:41,777 TREAT SPECIFIC DISEASES. 75 00:02:41,777 --> 00:02:45,615 DURING TODAY'S LECTURE, SUDHA 76 00:02:45,615 --> 00:02:47,216 WILL DISCUSS THE CONFORMATIONAL 77 00:02:47,216 --> 00:02:49,518 CHANGES THEY GO THROUGH BASED ON 78 00:02:49,518 --> 00:02:50,653 DIFFERENT FACTORS, THEIR 79 00:02:50,653 --> 00:02:52,455 REGULATION AND THE POTENTIAL TO 80 00:02:52,455 --> 00:02:54,423 DEVELOP SUBTYPE-SPECIFIC 81 00:02:54,423 --> 00:02:57,126 THERAPIES FOR VARIOUS DISEASE 82 00:02:57,126 --> 00:02:57,760 STATES. 83 00:02:57,760 --> 00:03:00,329 SUDHA RECEIVED HER MASTERS IF 84 00:03:00,329 --> 00:03:02,098 BIOMEDICAL ENGINEERING FROM THE 85 00:03:02,098 --> 00:03:02,698 INDIAN INSTITUTE OF TECHNOLOGY 86 00:03:02,698 --> 00:03:04,467 AND A PH.D. IN BIOPHYSICS FROM 87 00:03:04,467 --> 00:03:05,701 THE UNIVERSITY OF BUFFALO IN 88 00:03:05,701 --> 00:03:07,370 NEW YORK. 89 00:03:07,370 --> 00:03:09,672 I'M PLEASED TO NOTE THAT NIGMS 90 00:03:09,672 --> 00:03:11,907 SUPPORTS SUDHA'S WORK THROUGH 91 00:03:11,907 --> 00:03:13,709 OUR MAXIMIZING INVESTIGATORS 92 00:03:13,709 --> 00:03:16,412 RESEARCH AWARD, AND NOW PLEASE 93 00:03:16,412 --> 00:03:23,219 HELP ME WELCOME HER FOR HER TALK 94 00:03:23,219 --> 00:03:26,856 ON MOLECULAR MECHANISMS 95 00:03:26,856 --> 00:03:28,357 UNDERLYING GLYCINERGIC 96 00:03:28,357 --> 00:03:30,459 NEUROTRANSMISSION. 97 00:03:30,459 --> 00:03:31,193 >> ALL RIGHT. 98 00:03:31,193 --> 00:03:32,695 GOOD AFTERNOON, EVERYONE. 99 00:03:32,695 --> 00:03:34,530 AND THANK YOU, JON, FOR THE VERY 100 00:03:34,530 --> 00:03:35,464 KIND INTRODUCTION. 101 00:03:35,464 --> 00:03:38,067 AND FOR THE INVITATION TO 102 00:03:38,067 --> 00:03:39,268 PRESENT THE STETTEN LECTURE HERE 103 00:03:39,268 --> 00:03:39,935 TODAY. 104 00:03:39,935 --> 00:03:41,504 IT'S A TREMENDOUS HONOR FOR ME 105 00:03:41,504 --> 00:03:45,074 TO BE HERE AND TO ALSO PRESENT 106 00:03:45,074 --> 00:03:46,375 SOME WORK THAT WE'VE BEEN DOING 107 00:03:46,375 --> 00:03:50,346 IN THE LAST 10 YEARS OR SO THAT 108 00:03:50,346 --> 00:03:52,848 HAS BEEN CONTINUOUSLY FUNDED BY 109 00:03:52,848 --> 00:03:54,617 NIGMS THROUGH R01 AND R35 110 00:03:54,617 --> 00:03:55,051 AWARDS. 111 00:03:55,051 --> 00:03:57,253 I MUST ALSO MENTION THAT OUR 112 00:03:57,253 --> 00:03:59,789 WORK WHICH HEAVILY RELIES ON 113 00:03:59,789 --> 00:04:03,292 CRYO-EM AS A TECHNIQUE HAS BEEN 114 00:04:03,292 --> 00:04:04,093 SUPPORTED BY THE INFRASTRUCTURE 115 00:04:04,093 --> 00:04:06,929 AT THE NATIONAL CENTERS THAT 116 00:04:06,929 --> 00:04:08,397 FUNDED BY COMMON FUND PROGRAMS 117 00:04:08,397 --> 00:04:13,502 AND NOW THROUGH N IF NIGMS. 118 00:04:13,502 --> 00:04:15,037 SO DELIGHTED TO SHARE THIS WORK 119 00:04:15,037 --> 00:04:17,039 WITH YOU AND THANK YOU ALL FOR 120 00:04:17,039 --> 00:04:17,873 JOINING ME HERE TODAY. 121 00:04:17,873 --> 00:04:22,311 SO WHAT I WANT TO TELL YOU ABOUT 122 00:04:22,311 --> 00:04:23,913 IS SOMEWHAT OF A JOURNEY THAT 123 00:04:23,913 --> 00:04:26,949 I'VE HAD THROUGH STUDYING HOW 124 00:04:26,949 --> 00:04:28,317 CHEMICAL SYNAPSES BEHAVE, AND 125 00:04:28,317 --> 00:04:31,420 WHAT ARE SOME OF THE INTRICATE 126 00:04:31,420 --> 00:04:33,856 UNDERPINNINGS OF RECEPTORS THAT 127 00:04:33,856 --> 00:04:35,591 MEDIATE FAST COMMUNICATION. 128 00:04:35,591 --> 00:04:39,462 SO THESE RECEPTORS, THE ONES WE 129 00:04:39,462 --> 00:04:42,198 STUDY BELONG TO A FAMILY OF ION 130 00:04:42,198 --> 00:04:42,531 CHANNELS. 131 00:04:42,531 --> 00:04:45,234 THEY GOVERN A WIDE RANGE OF 132 00:04:45,234 --> 00:04:46,435 PHYSIOLOGIES, FROM MUSCLE 133 00:04:46,435 --> 00:04:49,505 MOVEMENT TO LEARNING MEMORY, 134 00:04:49,505 --> 00:04:50,940 GUT-BRAIN SIGNALING, PAIN 135 00:04:50,940 --> 00:04:53,843 PERCEPTION AND SO ON. 136 00:04:53,843 --> 00:04:56,545 AMONG THIS CLASS OF CHANNELS IS 137 00:04:56,545 --> 00:04:57,313 GLYCINE RECEPTORS WHICH I WILL 138 00:04:57,313 --> 00:04:59,482 BE TALKING ABOUT TODAY. 139 00:04:59,482 --> 00:05:01,450 AND SINCE I STARTED, MY MASTER'S 140 00:05:01,450 --> 00:05:03,385 DEGREE AND GOING THROUGH MY 141 00:05:03,385 --> 00:05:04,920 GRADUATE EDUCATION, THIS JOURNEY 142 00:05:04,920 --> 00:05:06,689 OF STUDYING THESE RECEPTORS HAS 143 00:05:06,689 --> 00:05:08,824 TAKEN ME THROUGH A WIDE RANGE OF 144 00:05:08,824 --> 00:05:10,826 APPROACHES, STARTED AS A SINGLE 145 00:05:10,826 --> 00:05:12,027 CHANNEL ELECTROPHYSIOLOGIST, 146 00:05:12,027 --> 00:05:13,796 THEN MOVING ON TO STRUCTURAL 147 00:05:13,796 --> 00:05:20,569 BIOLOGY, STUDYING EPR ELECTRON 148 00:05:20,569 --> 00:05:21,036 PARAMAGNETIC RESONANCE 149 00:05:21,036 --> 00:05:22,438 SPECTROSCOPY AND NOW CRYO-EM. 150 00:05:22,438 --> 00:05:24,807 SO IT'S BEEN A FUN RIDE SO FAR. 151 00:05:24,807 --> 00:05:26,809 SO ON TO GLYCINE RECEPTORS. 152 00:05:26,809 --> 00:05:28,878 SO THESE RECEPTORS PLAY A VERY 153 00:05:28,878 --> 00:05:30,513 IMPORTANT ROLE IN REGULATING 154 00:05:30,513 --> 00:05:32,982 SENSORY PATHWAYS AND MOTOR 155 00:05:32,982 --> 00:05:33,482 COORDINATION. 156 00:05:33,482 --> 00:05:43,526 THE SIGNAL THAT COMES FROM THE 157 00:05:43,526 --> 00:05:45,494 PERIPHERAL TERMINALS BROUGHT IN 158 00:05:45,494 --> 00:05:48,297 BY NOCICEPTOR NEURONS OR SENSORY 159 00:05:48,297 --> 00:05:50,099 NEURONS EFFICIENTLY GO TO THE 160 00:05:50,099 --> 00:05:51,000 SOMATOSENSORY CORE TECH, WHERE 161 00:05:51,000 --> 00:05:52,835 IT IS PERCEIVED AS PAIN, AND THE 162 00:05:52,835 --> 00:05:56,705 SIGNAL IS REGULATED AN IN THE 163 00:05:56,705 --> 00:05:58,607 DORSAL HORN OF THE SPINAL CORD 164 00:05:58,607 --> 00:05:59,942 BY THE GLYCINERGIC RECEPTORS 165 00:05:59,942 --> 00:06:00,609 HERE. 166 00:06:00,609 --> 00:06:01,844 SIMILARLY, THE SIGNAL THAT COMES 167 00:06:01,844 --> 00:06:03,245 FROM THE BRAIN THAT GOES TO THE 168 00:06:03,245 --> 00:06:05,414 MOTOR NEURON THAT MEDIATES 169 00:06:05,414 --> 00:06:06,949 MUSCLE CONTRACTION AGAIN IS 170 00:06:06,949 --> 00:06:09,251 REGULATED BY GLYCINE RECEPTORS. 171 00:06:09,251 --> 00:06:11,020 SO LET'S ZOOM IN TO THIS REGION 172 00:06:11,020 --> 00:06:15,157 OF THE SPINAL CORD. 173 00:06:15,157 --> 00:06:15,691 RIGHT HERE. 174 00:06:15,691 --> 00:06:26,235 SO THE SIGNAL BROUGHT IN SO THE 175 00:06:27,002 --> 00:06:29,138 SIGNAL BROUGHT IN BY THE 176 00:06:29,138 --> 00:06:30,539 TERMINALS EVENTUALLY GOES TO THE 177 00:06:30,539 --> 00:06:33,375 DORSAL ROOT PROJECTION ROOT 178 00:06:33,375 --> 00:06:35,344 CAVITY TO THE BRAIN AND THIS 179 00:06:35,344 --> 00:06:37,213 SIGNAL IS MODULATED BY 180 00:06:37,213 --> 00:06:38,914 INHIBITORY INTERNEURONS THAT 181 00:06:38,914 --> 00:06:42,985 RELEASE GLYCINE AND GABA AS 182 00:06:42,985 --> 00:06:43,586 NEUROTRANSMITTERS. 183 00:06:43,586 --> 00:06:47,223 THESE DIFFUSE ACROSS THE 184 00:06:47,223 --> 00:06:49,892 SYNAPTIC CLEFT AND BIND THE 185 00:06:49,892 --> 00:06:50,793 RECEPTORS ON THE MEMBRANE RIGHT 186 00:06:50,793 --> 00:06:51,060 HERE. 187 00:06:51,060 --> 00:06:52,561 AS YOU WILL NOTE, THESE 188 00:06:52,561 --> 00:06:55,231 RECEPTORS ACTUALLY COME IN TWO 189 00:06:55,231 --> 00:06:56,198 FLAVORS. 190 00:06:56,198 --> 00:06:57,700 THEY ARE EITHER ON THE POST 191 00:06:57,700 --> 00:06:59,869 SYNAPTIC MEMBRANE, THEY'RE ALSO 192 00:06:59,869 --> 00:07:01,937 PRESENT IN PRESYNAPTIC AND EXTRA 193 00:07:01,937 --> 00:07:04,039 SYNAPTIC SPACES. 194 00:07:04,039 --> 00:07:06,275 THE GLYCINE RECEPTORS HAVE A 195 00:07:06,275 --> 00:07:07,276 SLIGHTLY DIFFERENT ARCHITECTURE 196 00:07:07,276 --> 00:07:10,279 THAT POPULATE THESE SPACES, THEY 197 00:07:10,279 --> 00:07:12,982 MEDIATE TONIC INHIBITION. 198 00:07:12,982 --> 00:07:14,450 RECEPTORS ON THE POST SYNAPTIC 199 00:07:14,450 --> 00:07:15,985 MEMBRANE APPEAR TO BE MORE 200 00:07:15,985 --> 00:07:18,988 CLUSTERED BY A VERY ECK WHICH IS 201 00:07:18,988 --> 00:07:21,023 SIT MECHANISM I'LL TOUCH ON 202 00:07:21,023 --> 00:07:22,324 LATER AND THESE MAINTAIN THE 203 00:07:22,324 --> 00:07:26,595 POST SYNAPTIC INHIBITION. 204 00:07:26,595 --> 00:07:28,130 SO THE GLYCINE RECEPTORS SH 205 00:07:28,130 --> 00:07:29,832 THERE ARE FOUR GENES THAT CODE 206 00:07:29,832 --> 00:07:31,634 FOR THE ALPHA SUBUNIT AND ONE 207 00:07:31,634 --> 00:07:33,002 GENE THAT CODES FOR THE BETA 208 00:07:33,002 --> 00:07:33,535 SUBUNIT. 209 00:07:33,535 --> 00:07:35,304 THE ALPHA SUBUNITS ARE THE ONES 210 00:07:35,304 --> 00:07:37,206 THAT FORM FUNCTIONAL CHANNELS, 211 00:07:37,206 --> 00:07:40,876 THE ALPHA-1, 2 AND 3 SUBTYPES. 212 00:07:40,876 --> 00:07:41,911 THESE SUBUNITS CAN ASSEMBLE ON 213 00:07:41,911 --> 00:07:46,382 THEIR OWN TO FORM A HOMO PEN TOE 214 00:07:46,382 --> 00:07:48,117 MEERK CHANNELS FOUND IN THE 215 00:07:48,117 --> 00:07:49,685 PRESYNAPTIC AND EXTRA SYNAPTIC 216 00:07:49,685 --> 00:07:50,653 MEMBRANES. 217 00:07:50,653 --> 00:07:54,189 WHEREAS THE BETA SUBUNIT CANNOT 218 00:07:54,189 --> 00:07:55,891 FORM A PEN TOE MER ON ITS OWN SO 219 00:07:55,891 --> 00:07:57,693 IT ASSEMBLES WITH ONE OF THE 220 00:07:57,693 --> 00:07:58,761 ALPHA SUBUNITS. 221 00:07:58,761 --> 00:08:01,664 AND THE BETA SUBUNIT IS PART OF 222 00:08:01,664 --> 00:08:03,232 THE ASSEMBLIES OF GLYCINE 223 00:08:03,232 --> 00:08:04,867 RECEPTORS IN THE POST SYNAPTIC 224 00:08:04,867 --> 00:08:05,134 MEMBRANE. 225 00:08:05,134 --> 00:08:08,370 AND THE REASON FOR THAT IS THAT 226 00:08:08,370 --> 00:08:09,838 THE BETA SUBUNIT HAS A SEQUENCE 227 00:08:09,838 --> 00:08:12,207 THAT BINDS TO A PROTEIN CALLED 228 00:08:12,207 --> 00:08:14,910 DP. EFFRIN, A POST SYNAPTIC 229 00:08:14,910 --> 00:08:15,678 SCAFFOLDING PROTEIN. 230 00:08:15,678 --> 00:08:18,547 THIS IS ESSENTIAL FOR THE POST 231 00:08:18,547 --> 00:08:19,448 SYNAPTIC ARCHITECTURE. 232 00:08:19,448 --> 00:08:21,183 SO LOOKING AT IT AT THE 233 00:08:21,183 --> 00:08:23,652 INDIVIDUAL LEVEL, EACH OF THE 234 00:08:23,652 --> 00:08:26,088 ALPHA SUBUNITS SHARE A VERY 235 00:08:26,088 --> 00:08:27,723 STRONG SEQUENCE SIMILARITY, AND 236 00:08:27,723 --> 00:08:29,458 INCLUDING THE BETA SUBUNIT, THIS 237 00:08:29,458 --> 00:08:33,729 IS ABOUT 85 TO 90% SEQUENCE AND 238 00:08:33,729 --> 00:08:35,831 IN THE BETA, YOU HAVE ABOUT 50% 239 00:08:35,831 --> 00:08:37,466 SEQUENCE INVERITY TO THESE. 240 00:08:37,466 --> 00:08:39,501 SO EACH OF THESE SUBUNITS AS YOU 241 00:08:39,501 --> 00:08:42,538 CAN SEE HAS THREE MODULAR 242 00:08:42,538 --> 00:08:43,639 DOMAINS. 243 00:08:43,639 --> 00:08:44,974 THE EXTRACELLULAR DOMAIN WHICH 244 00:08:44,974 --> 00:08:46,442 HOUSES THE NEUROTRANSMITTER 245 00:08:46,442 --> 00:08:49,545 BINDING POCKET, IT IS A SET OF 246 00:08:49,545 --> 00:08:51,146 BETA STRANDS THAT FOLD TO FORM 247 00:08:51,146 --> 00:08:52,247 THE SCAFFOLD. 248 00:08:52,247 --> 00:08:54,116 THE TRANSMEMBRANE REGION HAS 249 00:08:54,116 --> 00:09:02,791 FOUR HE HELICES, IT'S A SITE FOR 250 00:09:02,791 --> 00:09:05,394 SELECTIVITY AND ITS GATING 251 00:09:05,394 --> 00:09:05,661 MECHANISM. 252 00:09:05,661 --> 00:09:07,162 THE REGION WE DON'T SEE HERE IN 253 00:09:07,162 --> 00:09:08,364 THE SCAFFOLD IS THE 254 00:09:08,364 --> 00:09:08,964 INTRACELLULAR DOMAIN. 255 00:09:08,964 --> 00:09:11,166 WE THINK THAT THIS IS A VERY 256 00:09:11,166 --> 00:09:12,901 INTERESTING REGION, BUT 257 00:09:12,901 --> 00:09:17,239 STRUCTURALLY, REALLY HARD TO 258 00:09:17,239 --> 00:09:19,241 WORK WITH BECAUSE THIS IS HIGHLY 259 00:09:19,241 --> 00:09:20,776 UNSTRUCTURED AND WHAT YOU WILL 260 00:09:20,776 --> 00:09:22,478 SEE IN THE SLIDES THAT I'M GOING 261 00:09:22,478 --> 00:09:23,879 TO SHOW ARE WE HAVEN'T REALLY 262 00:09:23,879 --> 00:09:26,582 BEEN ABLE TO GET A HANDLE ON THE 263 00:09:26,582 --> 00:09:27,483 DOMAIN. 264 00:09:27,483 --> 00:09:29,551 BUT WHAT MAKES THEM VERY 265 00:09:29,551 --> 00:09:31,754 INTERESTING IS THAT THIS 266 00:09:31,754 --> 00:09:32,888 INTRACELLULAR DOMAIN IS THE 267 00:09:32,888 --> 00:09:35,224 REGION THAT BINDS TO GEFFRIN 268 00:09:35,224 --> 00:09:37,793 THAT I MENTIONED, THE POST 269 00:09:37,793 --> 00:09:39,528 SYNAPTIC SCAFFOLDING PROTEIN BUT 270 00:09:39,528 --> 00:09:41,797 ALSO INTERACTS WITH SEVERAL 271 00:09:41,797 --> 00:09:43,232 OTHER PROTEINS THAT MODIFY THESE 272 00:09:43,232 --> 00:09:44,033 RECEPTORS AND MODULATE THEIR 273 00:09:44,033 --> 00:09:44,333 FUNCTION. 274 00:09:44,333 --> 00:09:47,036 SO THERE IS A LOT THAT WE NEED 275 00:09:47,036 --> 00:09:49,204 TO STILL DO TO UNDERSTAND THIS 276 00:09:49,204 --> 00:09:52,074 DOMAIN. 277 00:09:52,074 --> 00:09:54,276 SO ALSO IN THIS -- I WANTED TO 278 00:09:54,276 --> 00:09:56,945 MENTION THAT THE ALPHA SUBUNIT, 279 00:09:56,945 --> 00:09:59,515 ALPHA-1 TYPE, IS EXPRESSED 280 00:09:59,515 --> 00:10:01,550 THROUGHOUT THE GREY MATTER OF 281 00:10:01,550 --> 00:10:03,252 THE SPINAL CORD WHEREAS THE 282 00:10:03,252 --> 00:10:05,354 ALPHA THREE SUBTYPE IS ONLY 283 00:10:05,354 --> 00:10:06,922 PRESENT IN THE DORSAL HORN. 284 00:10:06,922 --> 00:10:07,823 THE ALPHA-1 BRINGS ABOUT THE 285 00:10:07,823 --> 00:10:09,625 MOTOR FUNCTIONS, WHEREAS THE 286 00:10:09,625 --> 00:10:11,093 ALPHA 3 IS INVOLVED IN PAIN 287 00:10:11,093 --> 00:10:13,896 PERCEPTION, SO THEY HAVE 288 00:10:13,896 --> 00:10:16,065 DISTINCT EXPRESSION PATTERNS AND 289 00:10:16,065 --> 00:10:17,433 FUNCTIONAL BEHAVIOR. 290 00:10:17,433 --> 00:10:19,234 SO THE ALPHA 2 IS THE FIRST OF 291 00:10:19,234 --> 00:10:21,537 THE GLYCINE ALPHA TYPES TO BE 292 00:10:21,537 --> 00:10:21,837 EXPRESSED. 293 00:10:21,837 --> 00:10:23,906 THEY ARE EXPRESSED BEFORE THE 294 00:10:23,906 --> 00:10:25,974 SYNAPSES ARE FORMED, AND THEY 295 00:10:25,974 --> 00:10:27,810 PLAY A VERY IMPORTANT ROLE IN 296 00:10:27,810 --> 00:10:31,580 THE INTERNEURON MIGRATION, AND 297 00:10:31,580 --> 00:10:33,515 THESE, IF ALPHA 2 IS KNOCKED 298 00:10:33,515 --> 00:10:35,818 OUT, THE NUMBER OF DORSAL ROOT 299 00:10:35,818 --> 00:10:37,619 PROJECTION NEURON NUMBER IS 300 00:10:37,619 --> 00:10:38,120 PRETTY LOW. 301 00:10:38,120 --> 00:10:40,155 AND SO THEY PLAY AN IMPORTANT 302 00:10:40,155 --> 00:10:43,058 ROLE IN HAVING SYNAPSES DEVELOP 303 00:10:43,058 --> 00:10:44,927 AND ANOTHER INTERESTING FEATURE 304 00:10:44,927 --> 00:10:47,729 IS THAT DURING DEVELOPMENT, THE 305 00:10:47,729 --> 00:10:49,865 CHLORIDE GRADIENT IS ACTUALLY 306 00:10:49,865 --> 00:10:51,233 OPPOSITE, SO WHEN THESE CHANNELS 307 00:10:51,233 --> 00:10:52,668 OPEN, THEY DEPOLARIZE THE 308 00:10:52,668 --> 00:10:53,235 MEMBRANE. 309 00:10:53,235 --> 00:10:56,505 SO THEY DEPOLARIZE THE MEMBRANE, 310 00:10:56,505 --> 00:10:57,239 ACTIVATE CALCIUM CHANNEL AND 311 00:10:57,239 --> 00:11:00,109 EVENTUALLY CAUSES THE ACTIN 312 00:11:00,109 --> 00:11:03,345 MYOSIN ENGAGEMENT LEADING TO 313 00:11:03,345 --> 00:11:04,413 NEURON MIGRATION. 314 00:11:04,413 --> 00:11:07,649 BUT AS THE SYNAPSES DEVELOP, YOU 315 00:11:07,649 --> 00:11:09,985 HAVE THIS EXQUISITE ARCHITECTURE 316 00:11:09,985 --> 00:11:13,755 THAT I WAS TALKING ABOUT, WHICH 317 00:11:13,755 --> 00:11:15,891 IS AN INTERACTION, YOU HAVE 318 00:11:15,891 --> 00:11:20,462 GLYCINE RECEPTORS WHICH IN TURN 319 00:11:20,462 --> 00:11:21,964 INTERACTS WITH OTHERS SO THIS IS 320 00:11:21,964 --> 00:11:26,135 A NETWORK OF PROTEIN THAT 321 00:11:26,135 --> 00:11:32,774 MAINTAINS SYNAPTIC ARCHITECTURE. 322 00:11:32,774 --> 00:11:36,411 ALSO LS WHAT YOU SEE IS ACTIVITY 323 00:11:36,411 --> 00:11:37,546 DEPENDENT SO IF YOU BLOCK THE 324 00:11:37,546 --> 00:11:38,647 FUNCTIONALITY OF GLYCINE 325 00:11:38,647 --> 00:11:40,449 RECEPTORS, YOU SEE THAT THESE 326 00:11:40,449 --> 00:11:42,251 RECEPTORS DIFFUSE AWAY IN THE 327 00:11:42,251 --> 00:11:44,286 SYNAPTIC ARCHITECTURE IS 328 00:11:44,286 --> 00:11:46,955 MODIFIED. 329 00:11:46,955 --> 00:11:48,423 SO THERE ARE A NUMBER OF 330 00:11:48,423 --> 00:11:50,993 MUTATIONS THAT ARE KNOWN IN 331 00:11:50,993 --> 00:11:54,096 GLYCINE RECEPTORS THAT IMPAIR 332 00:11:54,096 --> 00:11:55,864 GLYCINERGIC NEUROTRANSMISSION, 333 00:11:55,864 --> 00:11:57,766 AND MANY OF THESE ARE IN THE 334 00:11:57,766 --> 00:12:00,269 ALPHA ONE SUBUNIT. 335 00:12:00,269 --> 00:12:01,036 IT'S DYSFUNCTION IS ASSOCIATED 336 00:12:01,036 --> 00:12:07,309 WITH A DISEASE CALLED 337 00:12:07,309 --> 00:12:07,743 HYPEREKPLEXIA. 338 00:12:07,743 --> 00:12:10,479 IN THIS SITUATION, THE BABIES 339 00:12:10,479 --> 00:12:13,215 EXPERIENCE AN EXAGGERATED 340 00:12:13,215 --> 00:12:16,351 STARTLE REFLEX AND SEIZURE-LIKE 341 00:12:16,351 --> 00:12:16,952 SYMPTOMS. 342 00:12:16,952 --> 00:12:18,420 AND IF YOU LOOK AT WHERE THESE 343 00:12:18,420 --> 00:12:19,888 MUTATIONS ARE, THEY ARE LOCATED 344 00:12:19,888 --> 00:12:22,658 THROUGHOUT THE PROTEIN, AND MOST 345 00:12:22,658 --> 00:12:24,726 OF THE IMPAIRMENT THAT YOU SEE 346 00:12:24,726 --> 00:12:27,296 COMES FROM A DECREASED GLYCINE 347 00:12:27,296 --> 00:12:30,132 RECEPTOR EXPRESSION AND INCREASE 348 00:12:30,132 --> 00:12:30,866 IN DESENSITIZATION RATE. 349 00:12:30,866 --> 00:12:33,068 YOU SEE A REDUCTION IN GLYCINE 350 00:12:33,068 --> 00:12:35,437 SENSITIVITY AND CHLORIDE 351 00:12:35,437 --> 00:12:41,310 CONDUCTANCE AND ALSO A LOSS OF 352 00:12:41,310 --> 00:12:42,344 ZINC POTENTIATION. 353 00:12:42,344 --> 00:12:43,812 ANOTHER KIND OF NEUROLOGICAL 354 00:12:43,812 --> 00:12:45,280 DISORDER IS THE STIFF PERSON 355 00:12:45,280 --> 00:12:47,716 SYNDROME DISORDER AND ANOTHER 356 00:12:47,716 --> 00:12:50,452 ONE WHICH IS THE PROGRESSIVE 357 00:12:50,452 --> 00:12:51,853 ENCEPHALOMYELITIS, WHICH IS 358 00:12:51,853 --> 00:12:53,889 SEVERE FORM OF THE STIFF PERSON 359 00:12:53,889 --> 00:12:54,423 SYNDROME DISORDER. 360 00:12:54,423 --> 00:12:59,428 IN THIS CASE, THE PERSON MAKES 361 00:12:59,428 --> 00:13:00,896 ANTIBODIES TOWARDS GLYCINE 362 00:13:00,896 --> 00:13:01,697 RECEPTORS, AND AS A RESULT, YOU 363 00:13:01,697 --> 00:13:03,799 HAVE A DROP IN GLYCINE RECEPTOR 364 00:13:03,799 --> 00:13:05,667 DENSITY IN THE POST SYNAPTIC 365 00:13:05,667 --> 00:13:09,371 MEMBRANE. 366 00:13:09,371 --> 00:13:11,206 SO IN PATHOPHYSIOLOGY, THEY ARE 367 00:13:11,206 --> 00:13:13,542 ASSOCIATED WITH ALPHA 3 GLYCINE 368 00:13:13,542 --> 00:13:14,743 RECEPTORS, COME FROM THEIR ROLE 369 00:13:14,743 --> 00:13:17,379 IN INFLAMMATORY PAIN STATES. 370 00:13:17,379 --> 00:13:26,021 SO DURING INFLAMMATION, 371 00:13:26,021 --> 00:13:27,155 CYCLOOXYGENASE, EVENTUALLY 372 00:13:27,155 --> 00:13:32,127 RELEASE OF PROSTAGLANDIN 373 00:13:32,127 --> 00:13:33,228 ACTIVATES PROSTAGLANDIN 374 00:13:33,228 --> 00:13:37,366 RECEPTORS, WHICH ACT VAIK 375 00:13:37,366 --> 00:13:38,934 CYCLIC -- ACTIVATES FOR PROTEIN 376 00:13:38,934 --> 00:13:41,670 KINASE A WHICH PHOSPHORYLATES 377 00:13:41,670 --> 00:13:43,205 ALPHA 3 GLYCINE RECEPTORS. 378 00:13:43,205 --> 00:13:44,072 PHOSPHORYLATION OF THESE 379 00:13:44,072 --> 00:13:45,807 RECEPTORS THEN DECREASES OR 380 00:13:45,807 --> 00:13:47,209 DIMINISHES GLYCINE RECEPTOR 381 00:13:47,209 --> 00:13:47,476 FUNCTION. 382 00:13:47,476 --> 00:13:48,644 AS A RESULT, YOU HAVE 383 00:13:48,644 --> 00:13:51,213 HYPERACTIVATION OF THESE NEURONS 384 00:13:51,213 --> 00:13:53,915 AND TRANSITION TO PAIN STATE. 385 00:13:53,915 --> 00:13:59,821 TO SUPPORT THIS MODEL OF 386 00:13:59,821 --> 00:14:00,789 PHOSPHODEFICIENT MUTANT THAT 387 00:14:00,789 --> 00:14:03,158 DOES NOT CARRY THIS 388 00:14:03,158 --> 00:14:04,192 PHOSPHORYLATED BY PK, YOU CAN 389 00:14:04,192 --> 00:14:07,129 SEE THE IMPACT BY THESE MICE 390 00:14:07,129 --> 00:14:09,364 LOSE SENSITIVITY TO 391 00:14:09,364 --> 00:14:11,633 PROSTAGLANDINS. 392 00:14:11,633 --> 00:14:13,869 SO IN TERMS OF PHARMACOLOGY, 393 00:14:13,869 --> 00:14:15,337 GLYCINE RECEPTORS HAVE A VERY 394 00:14:15,337 --> 00:14:17,506 DIVERSE AND RICH PHARMACOLOGY TO 395 00:14:17,506 --> 00:14:19,474 TAKE A LOOK RIGHT HERE. 396 00:14:19,474 --> 00:14:21,476 SO THERE ARE A NUMBER OF KNOWN 397 00:14:21,476 --> 00:14:22,911 BINDING POCKETS, WHICH ARE 398 00:14:22,911 --> 00:14:24,946 MODULATED BY A RANGE OF 399 00:14:24,946 --> 00:14:26,381 ENDOGENOUS AND EXOGENOUS 400 00:14:26,381 --> 00:14:29,785 LIGANDS, AND MANY OF THESE MAY 401 00:14:29,785 --> 00:14:31,753 HAVE THERAPEUTIC VALUE, SO THERE 402 00:14:31,753 --> 00:14:35,457 ARE LIGANDS THAT WERE PURSUED BY 403 00:14:35,457 --> 00:14:39,895 AM GENERGEN AND PFIZER THAT BINO 404 00:14:39,895 --> 00:14:42,631 THE EXTRACELLULAR DOMAIN THAT 405 00:14:42,631 --> 00:14:45,100 ARE CANNABINOIDS, NEUROSTEROIDS, 406 00:14:45,100 --> 00:14:46,835 ALCOHOLS, IVERMECTIN AND 407 00:14:46,835 --> 00:14:47,903 ANESTHETICS BIND TO THESE 408 00:14:47,903 --> 00:14:49,004 VARIOUS POCKETS AND ALSO 409 00:14:49,004 --> 00:14:51,106 MODULATED BY ENDOGENOUS 410 00:14:51,106 --> 00:14:53,108 REGULATORS LIKE PH AND ZINC. 411 00:14:53,108 --> 00:14:54,176 THE ISSUES THAT WE FACE WITH 412 00:14:54,176 --> 00:14:59,081 USING ANY OF THESE RIGHT NOW IS 413 00:14:59,081 --> 00:15:01,583 THAT THESE COMPOUNDS ARE FAIRLY 414 00:15:01,583 --> 00:15:02,818 NON-SELECTIVE FOR GLYCINE 415 00:15:02,818 --> 00:15:05,153 RECEPTORS, THEY TARGET A WIDE 416 00:15:05,153 --> 00:15:07,322 RANGE OF RECEPTORS, SO THIS IS A 417 00:15:07,322 --> 00:15:08,290 GOOD STARTING POINT. 418 00:15:08,290 --> 00:15:10,258 IT'S A GREAT STARTING POINT TO 419 00:15:10,258 --> 00:15:10,892 UNDERSTAND THE FUNDAMENTAL 420 00:15:10,892 --> 00:15:12,561 MECHANISM OF HOW THESE 421 00:15:12,561 --> 00:15:14,629 MODULATORS AFFECT FUNCTION, BUT 422 00:15:14,629 --> 00:15:17,065 WE REALLY ARE ON THE LOOKOUT FOR 423 00:15:17,065 --> 00:15:18,467 BETTER MODULATORS OF THESE 424 00:15:18,467 --> 00:15:20,535 RECEPTORS. 425 00:15:20,535 --> 00:15:22,704 SO WHAT DO WE KNOW ABOUT THE 426 00:15:22,704 --> 00:15:24,373 DIFFERENT SUBTYPES THAT WE CAN 427 00:15:24,373 --> 00:15:26,408 HARNESS TO TARGET THEM? 428 00:15:26,408 --> 00:15:27,976 SO THE ALPHA -- EVEN THOUGH THEY 429 00:15:27,976 --> 00:15:30,612 HAVE A PRETTY STRONG SEQUENCE 430 00:15:30,612 --> 00:15:31,680 SIMILARITIES, YOU CAN SEE THAT 431 00:15:31,680 --> 00:15:33,181 THEY HAVE DIFFERENCES IN 432 00:15:33,181 --> 00:15:34,616 SENSITIVITY TO GLYCINE AS YOU 433 00:15:34,616 --> 00:15:37,853 CAN SEE ALPHA-1 IS MUCH HIGHER 434 00:15:37,853 --> 00:15:39,221 SENSITIVE TO GLYCINE AND ALSO TO 435 00:15:39,221 --> 00:15:41,790 PARTIAL AGONIST COMPARED TO THAT 436 00:15:41,790 --> 00:15:43,558 OF THE ALPHA 3 SUBUNIT SO YOU 437 00:15:43,558 --> 00:15:45,560 CAN SEE THAT IN THE SHIFT IN THE 438 00:15:45,560 --> 00:15:46,128 DOSE-RESPONSE. 439 00:15:46,128 --> 00:15:49,865 YOU ALSO SEE THE ASSEMBLIES ALSO 440 00:15:49,865 --> 00:15:51,900 HAVE DIFFERENCES IN SENSITIVITY 441 00:15:51,900 --> 00:15:53,869 TO GLYCINE AS YOU CAN SEE IN THE 442 00:15:53,869 --> 00:15:55,437 SHIFT IN THE DOSE-RESPONSE, NOT 443 00:15:55,437 --> 00:15:59,207 ONLY THAT, THEY ALSO DISPLAY 444 00:15:59,207 --> 00:16:00,709 VERY DISTINCT SINGLE CHANNEL 445 00:16:00,709 --> 00:16:01,009 BEHAVIOR. 446 00:16:01,009 --> 00:16:02,878 YOU CAN SEE THE ALPHA-1 447 00:16:02,878 --> 00:16:05,781 POPULATES AT LEAST FIVE 448 00:16:05,781 --> 00:16:06,481 DIFFERENT SUBCONDUCT TANT 449 00:16:06,481 --> 00:16:09,684 STATES, WHEREAS THE ALPHA-1 BETA 450 00:16:09,684 --> 00:16:13,455 RECEPTOR HAS A SHIFT TOWARDS 451 00:16:13,455 --> 00:16:15,590 LOWER SUBCONDUCTANT STATES. 452 00:16:15,590 --> 00:16:18,126 SO THE QUESTIONS THAT THE LAB 453 00:16:18,126 --> 00:16:19,961 HAS BEEN EXPLORING IN THE LAST 454 00:16:19,961 --> 00:16:21,663 FEW YEARS IN AREAS WHERE WE WANT 455 00:16:21,663 --> 00:16:23,598 TO HEAD TOWARDS, FIRST OFF, WE 456 00:16:23,598 --> 00:16:25,066 WANT TO UNDERSTAND THE STRUCTURE 457 00:16:25,066 --> 00:16:29,304 AND DYNAMICS THAT UNDERLIES 458 00:16:29,304 --> 00:16:31,506 FUNDAMENTAL MECHANISM IN THEE 459 00:16:31,506 --> 00:16:35,777 REP TORE FAMTHESERECEPTOR FAMIL. 460 00:16:35,777 --> 00:16:38,647 WE WANT TO REALLY UNDERSTAND 461 00:16:38,647 --> 00:16:40,649 WHAT GOES ON AT THE MOLECULAR 462 00:16:40,649 --> 00:16:40,916 LEVEL. 463 00:16:40,916 --> 00:16:45,053 WE WANT TO UNDERSTAND, WHEN WE 464 00:16:45,053 --> 00:16:46,788 STUDY THESE BY CRYO-EM AT THIS 465 00:16:46,788 --> 00:16:48,256 TIME WE'RE STILL LOOKING AT A 466 00:16:48,256 --> 00:16:50,425 MINIMAL UNIT, PURIFIED 467 00:16:50,425 --> 00:16:51,860 RECEPTORS, BUT THESE RECEPTORS 468 00:16:51,860 --> 00:16:52,527 SELDOM WORK ALONE. 469 00:16:52,527 --> 00:16:55,030 THEY ARE A PART OF SUPER 470 00:16:55,030 --> 00:16:56,498 MOLECULAR COMPLEXS THAT MODULATE 471 00:16:56,498 --> 00:16:57,432 FUNCTION SO WE WANT TO BE ABLE 472 00:16:57,432 --> 00:16:59,167 TO EVENTUALLY ACHIEVE 473 00:16:59,167 --> 00:17:00,335 UNDERSTANDING HOW THESE 474 00:17:00,335 --> 00:17:02,804 RECEPTORS ARE ASSEMBLED IN THEIR 475 00:17:02,804 --> 00:17:05,540 FUNCTIONAL ENTITIES. 476 00:17:05,540 --> 00:17:07,175 AND FINALLY, WE WANT TO BE ABLE 477 00:17:07,175 --> 00:17:09,377 TO LOOK AT THEM IN THEIR NATIVE 478 00:17:09,377 --> 00:17:10,245 CELLULAR ENVIRONMENT IN THIS 479 00:17:10,245 --> 00:17:13,615 CASE IN THE CONTEXT OF A 480 00:17:13,615 --> 00:17:14,683 SYNAPSE, THIS IS A STRETCH GOAL 481 00:17:14,683 --> 00:17:16,017 AT THIS TIME, BUT HOPEFULLY WE 482 00:17:16,017 --> 00:17:17,152 AS A FIELD WILL GET THERE 483 00:17:17,152 --> 00:17:19,621 SOMETIME. 484 00:17:19,621 --> 00:17:20,989 SO WHAT DO WE KNOW ABOUT THE 485 00:17:20,989 --> 00:17:22,491 STRUCTURES OF THESE RECEPTORS? 486 00:17:22,491 --> 00:17:24,593 SO THE PIONEERING WORK IN THE 487 00:17:24,593 --> 00:17:25,961 GLYCINE STRUCTURE FIELD WAS DONE 488 00:17:25,961 --> 00:17:29,164 IN THE LABS OF ERIC GEUL, THIS 489 00:17:29,164 --> 00:17:31,333 WAS A CRYO-EM STRUCTURE GOING 490 00:17:31,333 --> 00:17:35,537 BACK TO 2015 OF ALPHA-1 GLYCINE 491 00:17:35,537 --> 00:17:37,372 RECEPTORS, AND TET, THE GROUP OF 492 00:17:37,372 --> 00:17:41,109 PAUL SCHAEFER FROM AMGEN, THEY 493 00:17:41,109 --> 00:17:42,611 PUBLISHED THIS STRUCTURE OF THE 494 00:17:42,611 --> 00:17:48,183 ALPHA 3 GLYCINE RECEPTORS. 495 00:17:48,183 --> 00:17:50,085 SO TRUNCATING THE INTRACELLULAR 496 00:17:50,085 --> 00:17:53,488 DOMAIN AS YOU CAN IMAGINE, THE 497 00:17:53,488 --> 00:17:55,223 PROTEIN WITH THAT, SO THESE WERE 498 00:17:55,223 --> 00:17:58,693 THE INITIAL EFFORTS AND WITHIN 499 00:17:58,693 --> 00:18:00,362 THESE STRUCTURES THEY WERE ABLE 500 00:18:00,362 --> 00:18:01,763 TO CAPTURE MULTIPLE 501 00:18:01,763 --> 00:18:02,264 CONFORMATIONS. 502 00:18:02,264 --> 00:18:03,164 THERE ARE SOME LINGERING 503 00:18:03,164 --> 00:18:05,767 QUESTIONS THAT WERE EVENTUALLY 504 00:18:05,767 --> 00:18:06,701 ANSWERED WITH STRUCTURED 505 00:18:06,701 --> 00:18:10,939 PUBLISHED BY OUR GROUP, SO THESE 506 00:18:10,939 --> 00:18:12,674 ARE FULL LENGTH RECEPTOR 507 00:18:12,674 --> 00:18:16,211 STRUCTURES, THIS IS IN THE 508 00:18:16,211 --> 00:18:17,879 RECONSTITUTED AND LIPID NANODISK 509 00:18:17,879 --> 00:18:21,283 AND THIS ONE IN SMA POLYMERS. 510 00:18:21,283 --> 00:18:22,984 THESE ARE BOTH FULL LENGTH 511 00:18:22,984 --> 00:18:24,286 STRUCTURES BUT AS YOU CAN SEE WE 512 00:18:24,286 --> 00:18:25,487 HAVEN'T STILL GOTTEN INITIAL 513 00:18:25,487 --> 00:18:26,855 VIEW OF THE INTRACELLULAR 514 00:18:26,855 --> 00:18:29,357 DOMAIN. 515 00:18:29,357 --> 00:18:30,392 AND MORE RECENTLY, LAST COUPLE 516 00:18:30,392 --> 00:18:32,861 OF YEARS, THERE ARE A NUMBER OF 517 00:18:32,861 --> 00:18:36,531 HETEROMERIC GLYCINE RECEPTOR 518 00:18:36,531 --> 00:18:38,033 STRUCTURES AT SOUTHWESTERN AND 519 00:18:38,033 --> 00:18:43,138 THIS IS A STRUCTURE OF RECEPTORS 520 00:18:43,138 --> 00:18:46,374 AND THIS IS FROM OUR LAB. 521 00:18:46,374 --> 00:18:50,045 AT THIS POINT, HOW MANY Bs AND 522 00:18:50,045 --> 00:18:52,480 HOW MANY As CONSTITUTE THESE 523 00:18:52,480 --> 00:18:53,515 RECEPTORS WAS STILL UNCLEAR. 524 00:18:53,515 --> 00:18:56,017 THREE LABS, THREE INDEPENDENT 525 00:18:56,017 --> 00:18:57,218 APPROACHES, THREE EXPRESSION 526 00:18:57,218 --> 00:18:58,887 SYSTEMS, ARRIVING AT THE SAME 527 00:18:58,887 --> 00:19:03,158 CONCLUSION OF A 4 IS TO 1 -- 528 00:19:03,158 --> 00:19:05,093 4 ALPHA AND 1 BETA, I THINK THAT 529 00:19:05,093 --> 00:19:08,263 WAS QUITE REMARKABLE. 530 00:19:08,263 --> 00:19:10,432 ANOTHER POINT, THEY USED GFP SO 531 00:19:10,432 --> 00:19:12,133 THEY REMOVED THE INTRACELLULAR 532 00:19:12,133 --> 00:19:13,602 DOMAIN FROM BETA SUBUNIT AND 533 00:19:13,602 --> 00:19:15,804 REPLACED IT WITH A GFB SO THAT 534 00:19:15,804 --> 00:19:17,772 ALLOWED THEM TO ANNOTATE WHICH 535 00:19:17,772 --> 00:19:18,840 ONE IS AN ALPHA AND BETA 536 00:19:18,840 --> 00:19:19,307 SUBUNIT. 537 00:19:19,307 --> 00:19:23,011 IN THIS CASE, THEY USED AN 538 00:19:23,011 --> 00:19:24,779 ANTIBODY WHICH RECOGNIZES THE 539 00:19:24,779 --> 00:19:26,715 ALPHA SUBUNIT AGAIN USED AS A 540 00:19:26,715 --> 00:19:28,116 MARKER FOR ANNOTATING SUBUNITS 541 00:19:28,116 --> 00:19:31,186 AND IN OUR CASE, WE ACTUALLY 542 00:19:31,186 --> 00:19:33,755 USED GEFFRIN AS A CO-COMPLEX AND 543 00:19:33,755 --> 00:19:35,690 WE WERE ABLE TO IMAGE THIS 544 00:19:35,690 --> 00:19:38,226 WITHOUT ADDITIONAL MARKER AND 545 00:19:38,226 --> 00:19:39,461 PROAT TEEN HAD ENOUGH SIGNATURES 546 00:19:39,461 --> 00:19:41,630 WITHIN IT THAT ALLOWED US TO 547 00:19:41,630 --> 00:19:42,731 SOLVE THE STRUCTURE 548 00:19:42,731 --> 00:19:46,635 INDEPENDENTLY. 549 00:19:46,635 --> 00:19:47,702 SO THERE ARE INTERESTING 550 00:19:47,702 --> 00:19:48,603 QUESTIONS THAT ARE UNIQUE TO 551 00:19:48,603 --> 00:19:54,075 EACH OF THESE SUBTYPES, SO I 552 00:19:54,075 --> 00:19:56,444 WANT TO SHOW YOU THREE VIGNETTES 553 00:19:56,444 --> 00:19:59,481 THAT SHOWS YOU HOW ARE THESE 554 00:19:59,481 --> 00:20:01,216 CAPTURED -- I CHOSE TO TALK 555 00:20:01,216 --> 00:20:03,652 ABOUT TWO ENDOGENOUS MOJ LAY 556 00:20:03,652 --> 00:20:05,253 TORES, ZINC AND PH IN THE 557 00:20:05,253 --> 00:20:05,820 CONTEXT OF PAIN. 558 00:20:05,820 --> 00:20:07,756 I WANT TO SHOW YOU CANNABINOID 559 00:20:07,756 --> 00:20:09,491 MODULATION IN THE ALPHA-1 560 00:20:09,491 --> 00:20:11,059 SUBTYPE AND I WANT TO FINISH OFF 561 00:20:11,059 --> 00:20:14,963 WITH MODULATION OF ALPHA-1 BETA 562 00:20:14,963 --> 00:20:18,033 BY EYE VER ME IVERMECTIN, WHICHA 563 00:20:18,033 --> 00:20:19,067 POTENTIATOR OF THIS CHANNEL, TO 564 00:20:19,067 --> 00:20:22,303 SHOW YOU A GLIMPSE OF HOW WE CAN 565 00:20:22,303 --> 00:20:25,874 SORT OF TACKLE SUBTYPE 566 00:20:25,874 --> 00:20:26,341 SPECIFICITY. 567 00:20:26,341 --> 00:20:27,709 SO LET'S SEE. 568 00:20:27,709 --> 00:20:29,611 AT A FUNDAMENTAL LEVEL, THESE 569 00:20:29,611 --> 00:20:32,480 RECEPTORS WILL CYCLE THROUGH 570 00:20:32,480 --> 00:20:33,648 THESE GATING SCHEMES. 571 00:20:33,648 --> 00:20:35,417 SO THERE'S THE RESTING STATE 572 00:20:35,417 --> 00:20:38,953 GETTING ACTIVATED BY AN AGONIST, 573 00:20:38,953 --> 00:20:40,221 THE CHANNEL IS TRANSIENTLY OPEN, 574 00:20:40,221 --> 00:20:42,457 THEN IT DESENSITIZES SO IT GETS 575 00:20:42,457 --> 00:20:43,324 INTO THE DESENSITIZED STATE. 576 00:20:43,324 --> 00:20:47,829 BUT THIS IS A PRETTY BIG 577 00:20:47,829 --> 00:20:48,430 OVERSIMPLIFICATION BECAUSE AS 578 00:20:48,430 --> 00:20:50,065 YOU CAN SEE IT IN THE SINGLE 579 00:20:50,065 --> 00:20:51,332 CHANNEL, THERE ARE A NUMBER OF 580 00:20:51,332 --> 00:20:52,367 STATES THAT THESE CHANNELS 581 00:20:52,367 --> 00:20:53,702 POPULATE BUT AT LEAST THIS IS A 582 00:20:53,702 --> 00:20:56,438 STARTING MODEL FOR US TO GO. 583 00:20:56,438 --> 00:21:00,608 WHAT DO MODULATORS DO? 584 00:21:00,608 --> 00:21:02,677 THEY ESSENTIALLY SWITCH THE 585 00:21:02,677 --> 00:21:03,378 EQUILIBRIUM BETWEEN THESE 586 00:21:03,378 --> 00:21:03,611 STATES. 587 00:21:03,611 --> 00:21:05,346 SO IF YOU'RE USING A 588 00:21:05,346 --> 00:21:07,415 POTENTIATOR, IT PUSHES THE 589 00:21:07,415 --> 00:21:08,550 EQUILIBRIUM TOWARDS THE OPEN 590 00:21:08,550 --> 00:21:08,983 CONFORMATION. 591 00:21:08,983 --> 00:21:11,352 IF YOU'RE USING AN INHIBITOR, IT 592 00:21:11,352 --> 00:21:12,721 PERHAPS SHIFTS IT TOWARDS A 593 00:21:12,721 --> 00:21:14,723 RESTING STATE, AND THIS IS SORT 594 00:21:14,723 --> 00:21:15,990 OF A THEME THAT YOU WILL SEE IN 595 00:21:15,990 --> 00:21:16,991 THE DATA THAT I'M GOING TO 596 00:21:16,991 --> 00:21:17,425 PRESENT. 597 00:21:17,425 --> 00:21:20,695 SO THIS IS WORK THAT WAS DONE BY 598 00:21:20,695 --> 00:21:22,230 A GRADUATE STUDENT, A VERY 599 00:21:22,230 --> 00:21:23,565 TALENTED GRADUATE STUDENT IN THE 600 00:21:23,565 --> 00:21:26,501 LAB, KAYLA, AND A REALLY 601 00:21:26,501 --> 00:21:30,105 TALENTED POSTDOC, ERIC GIBBS, IN 602 00:21:30,105 --> 00:21:33,742 COLLABORATION WITH PHIL B IGG 603 00:21:33,742 --> 00:21:37,779 IN'S GROUP, ALL WERE DONE IN 604 00:21:37,779 --> 00:21:38,379 PHIL'S GROUP. 605 00:21:38,379 --> 00:21:42,650 SO WE WANTED TO STUDY ZINC 606 00:21:42,650 --> 00:21:48,056 MODULATION IN ALPHA 3 GLYCINE 607 00:21:48,056 --> 00:21:50,525 RECEPTORS. 608 00:21:50,525 --> 00:21:52,594 THEY HAVE A NUMBER OF MODULATORY 609 00:21:52,594 --> 00:21:53,928 EFFECT ON SYNAPTIC RECEPTORS. 610 00:21:53,928 --> 00:21:58,500 THEY GO THROUGH ALL OF THE 611 00:21:58,500 --> 00:21:59,868 MODULATIONS AND ALSO THROUGH 612 00:21:59,868 --> 00:22:02,337 THIS DIFFUSION OF ZINC ACROSS TO 613 00:22:02,337 --> 00:22:03,271 OTHER SYNAPSES. 614 00:22:03,271 --> 00:22:04,873 SO GLYCINE RECEPTOR HAS BEEN NOW 615 00:22:04,873 --> 00:22:07,275 KNOWN TO BE MODULATED BY ZINC, 616 00:22:07,275 --> 00:22:08,877 BUT WHAT IS INTERESTING IS THAT 617 00:22:08,877 --> 00:22:12,347 UNLIKE OTHER RECEPTORS WHERE 618 00:22:12,347 --> 00:22:13,581 THEY INHIBIT -- THE EFFECT OF 619 00:22:13,581 --> 00:22:17,085 GLYCINE ON ALPHA 3 IS BIPHASIC 620 00:22:17,085 --> 00:22:19,320 SO AT VERY LOW CONCENTRATION AND 621 00:22:19,320 --> 00:22:21,589 CLOSE TO 1 TO 10 MICROMOLAR UP 622 00:22:21,589 --> 00:22:24,425 TO 1 TO 10 MICRO, YOU HAVE THIS 623 00:22:24,425 --> 00:22:25,026 POTENTIATION, WHEREAS BEYOND 624 00:22:25,026 --> 00:22:26,294 THAT, YOU START TO SEE THIS 625 00:22:26,294 --> 00:22:26,828 INHIBITION. 626 00:22:26,828 --> 00:22:28,463 SO RIGHT OFF YOU KNOW THAT THERE 627 00:22:28,463 --> 00:22:30,732 ARE TWO BINDING SITES, ONE THAT 628 00:22:30,732 --> 00:22:32,400 IS POTENTIATING HIGH AFFINITY, 629 00:22:32,400 --> 00:22:34,202 THE OTHER ONE IS LOW AFFINITY 630 00:22:34,202 --> 00:22:35,937 INHIBITORY SITES. 631 00:22:35,937 --> 00:22:40,375 SO MU MUTAGENESIS HAVE GIVEN UA 632 00:22:40,375 --> 00:22:41,543 GLIMPSE OF WHERE THEE SITES MAY 633 00:22:41,543 --> 00:22:43,144 BE BUT THERE ARE MANY SITE 634 00:22:43,144 --> 00:22:44,546 OUTSIDE OF THESE THAT ALSO 635 00:22:44,546 --> 00:22:45,547 IMPACT ZINC MODULATION. 636 00:22:45,547 --> 00:22:49,651 AND SO WHAT ARE ALLOSTERIC AND 637 00:22:49,651 --> 00:22:51,052 WHAT ARE REALLY BINDING SITES 638 00:22:51,052 --> 00:22:53,154 ARE STILL QUESTIONS TO BE 639 00:22:53,154 --> 00:22:53,521 ASSESSED. 640 00:22:53,521 --> 00:22:54,989 THE OTHER THING FROM A DISEASE 641 00:22:54,989 --> 00:22:56,391 PHYSIOLOGY THAT MUTATIONS THAT 642 00:22:56,391 --> 00:22:59,360 ELIMINATE ZINC POTENTIATION HAVE 643 00:22:59,360 --> 00:23:00,328 HYPERREFLEXIA PHENOTYPES, 644 00:23:00,328 --> 00:23:02,997 SUGGESTING THAT ZINC MODULATION 645 00:23:02,997 --> 00:23:04,799 MIGHT BE PHYSIOLOGICAL MECHANISM 646 00:23:04,799 --> 00:23:06,968 OF HOW THESE RECEPTORS FUNCTION. 647 00:23:06,968 --> 00:23:09,737 SO TO DO THIS, WHAT WE EXPRESSED 648 00:23:09,737 --> 00:23:12,473 ALPHA 3 FULL LENGTH HUMAN ALPHA 649 00:23:12,473 --> 00:23:13,975 3 GLYCINE RECEPTORS, WE PURIFIED 650 00:23:13,975 --> 00:23:15,944 THEM USING AFFINITY 651 00:23:15,944 --> 00:23:18,713 CHROMATOGRAPHY, WE RECONSTITUTE 652 00:23:18,713 --> 00:23:20,882 THEM IN A BELT OF PEPTIDES THAT 653 00:23:20,882 --> 00:23:22,650 WRAP AROUND THE TRANSMEMBRANE 654 00:23:22,650 --> 00:23:24,485 REGION SO THEN WE IMAGE THESE 655 00:23:24,485 --> 00:23:26,754 AND THEN YOU CAN SEE THESE 656 00:23:26,754 --> 00:23:29,057 IMAGES THAT ARE ALIGNED SHOWING 657 00:23:29,057 --> 00:23:30,091 SIDE VIEWS, TOP VIEWS OF THE 658 00:23:30,091 --> 00:23:31,492 PROTEIN, YOU CAN START TO SEE 659 00:23:31,492 --> 00:23:33,628 SOME HELIC ES. 660 00:23:33,628 --> 00:23:36,297 THIS IS WHERE YOU START TO GET 661 00:23:36,297 --> 00:23:37,765 EXCITED BECAUSE YOU SEE THEM AT 662 00:23:37,765 --> 00:23:38,833 THESE POSITIONS, THEN CAN YOU 663 00:23:38,833 --> 00:23:40,001 TAKE THESE PARTICLES AND 664 00:23:40,001 --> 00:23:41,069 CLASSIFY THEM AND YOU WILL SEE 665 00:23:41,069 --> 00:23:45,707 IN THIS DATASET THAT WITHIN THE 666 00:23:45,707 --> 00:23:47,508 SAME DATASET WE START TO SEE 667 00:23:47,508 --> 00:23:50,078 MULTIPLE CONFORMATIONS THAT ARE 668 00:23:50,078 --> 00:23:54,616 THERE, SO OUR STRATEGY WAS TO 669 00:23:54,616 --> 00:23:56,184 IMAGE THIS LOWER THAN THE EC50 670 00:23:56,184 --> 00:23:59,220 FOR GLYCINE STHOO YOU ARE 671 00:23:59,220 --> 00:24:01,155 CAPTURING SOME NON-ACTIVATED AND 672 00:24:01,155 --> 00:24:02,390 ACTIVATED POPULATION. 673 00:24:02,390 --> 00:24:05,159 SO WHAT WE SAW, WHICH IS WHAT 674 00:24:05,159 --> 00:24:06,494 WE'RE SEEING HERE, SO THIS IS 675 00:24:06,494 --> 00:24:07,996 THE CURRENT TRACES FOR GLYCINE 676 00:24:07,996 --> 00:24:11,900 AND HERE WE IMAGE IT UNDER 100 677 00:24:11,900 --> 00:24:13,067 MICROMOLAR GLYCINE 678 00:24:13,067 --> 00:24:13,835 CONCENTRATIONS, AND ONCE YOU SAW 679 00:24:13,835 --> 00:24:17,005 THE PARTICLES, WE SEE THAT THE 680 00:24:17,005 --> 00:24:18,573 PARTICLES BELONG TO TWO 681 00:24:18,573 --> 00:24:23,778 DIFFERENT CORE FORMAT CONFORMAT, 682 00:24:23,778 --> 00:24:26,114 ROUGHLY AT 50% OF OCCUPANCY INTO 683 00:24:26,114 --> 00:24:26,514 THESE TWO STATES. 684 00:24:26,514 --> 00:24:28,616 SO THIS IS THE MAP OF THOSE TWO 685 00:24:28,616 --> 00:24:29,584 CONFORMATIONS, THIS IS THE MODEL 686 00:24:29,584 --> 00:24:30,785 WE BUILT AND WHAT I'M SHOWING 687 00:24:30,785 --> 00:24:31,986 YOU HERE ARE TWO SUBUNITS. 688 00:24:31,986 --> 00:24:33,421 THIS IS SLICED THROUGH THE 689 00:24:33,421 --> 00:24:35,056 CHANNEL TO SEE THE ION 690 00:24:35,056 --> 00:24:36,357 PERMEATION PATHWAY. 691 00:24:36,357 --> 00:24:38,426 SO THIS IS A RESTING 692 00:24:38,426 --> 00:24:38,793 CONFORMATION. 693 00:24:38,793 --> 00:24:43,131 YOU CAN SEE THAT THE CHANNEL 694 00:24:43,131 --> 00:24:45,366 IS -- SO THE GREEN PATCH RIGHT 695 00:24:45,366 --> 00:24:46,734 THERE IS WHERE AN OCCLUSION 696 00:24:46,734 --> 00:24:47,969 OCCURS OF ION PERMEATION. 697 00:24:47,969 --> 00:24:50,505 I CAN SEE THAT IT A CONSTRICTED 698 00:24:50,505 --> 00:24:51,639 RADII PLOTTED ALONG THE LENGTH 699 00:24:51,639 --> 00:24:52,807 OF THE CHANNEL. 700 00:24:52,807 --> 00:24:57,111 AND THIS IS A RING OF LEUCINE 701 00:24:57,111 --> 00:24:58,780 RESIDUES THAT -- SO THIS FORMS 702 00:24:58,780 --> 00:25:02,917 THE ACTIVATION GATE OF THE 703 00:25:02,917 --> 00:25:03,151 CHANNEL. 704 00:25:03,151 --> 00:25:07,956 IN THE DESENSITIZED CORE 705 00:25:07,956 --> 00:25:09,524 CONFORMATION, SO THIS GATE HERE 706 00:25:09,524 --> 00:25:11,659 IS WIDE OPEN WHEREAS THIS GATE 707 00:25:11,659 --> 00:25:13,227 IS CONSTRICTED, IT'S A RING OF 708 00:25:13,227 --> 00:25:15,096 PROTEIN RESIDUES THAT CAUSES 709 00:25:15,096 --> 00:25:18,166 CONSTRICTION IN THE ACTIVATED -- 710 00:25:18,166 --> 00:25:19,968 ONCE THE CHANNEL IS ACTIVATED. 711 00:25:19,968 --> 00:25:22,070 SO ZOOMING INTO THE BINDING 712 00:25:22,070 --> 00:25:24,505 POCKET RIGHT HERE, THE INTERFACE 713 00:25:24,505 --> 00:25:28,009 BETWEEN THESE TWO SUBUNITS, WE 714 00:25:28,009 --> 00:25:29,210 CAN SEE IN THE RESTING 715 00:25:29,210 --> 00:25:31,045 CONFORMATION OR THE CLOSED 716 00:25:31,045 --> 00:25:34,182 STATE, YOU SEE NO DENSITY, IT'S 717 00:25:34,182 --> 00:25:36,951 AN EMP EMPTY POCKET, WHEREAS HEE 718 00:25:36,951 --> 00:25:38,953 YOU HAVE A NICE DENSITY AND YOU 719 00:25:38,953 --> 00:25:41,356 CAN ACT OUT ALL THE INTERACTING 720 00:25:41,356 --> 00:25:42,056 PARTNERS. 721 00:25:42,056 --> 00:25:43,591 SO WHAT HAPPENS IS GLYCINE BINDS 722 00:25:43,591 --> 00:25:44,993 HERE AND THIS LOOP REFERRED TO 723 00:25:44,993 --> 00:25:47,595 AS LOOP C CLOSES IN ON TO THE 724 00:25:47,595 --> 00:25:49,530 BINDING POCKET, AND CAUSES A 725 00:25:49,530 --> 00:25:51,032 TWISTING MOVEMENT THAT IS 726 00:25:51,032 --> 00:25:53,668 EVENTUALLY CONVEYED TO THE PORE 727 00:25:53,668 --> 00:25:55,370 OF THE CHANNEL SO THAT'S THE 728 00:25:55,370 --> 00:25:56,471 ACTIVATION MECHANISM, A 729 00:25:56,471 --> 00:25:58,206 MECHANISM THAT IS VERY CONSERVED 730 00:25:58,206 --> 00:26:00,608 AMONGST THIS FAMILY OF CHANNELS 731 00:26:00,608 --> 00:26:02,110 SHOWN BY OUR LAB AND SEVERAL 732 00:26:02,110 --> 00:26:04,012 OTHER LABS. 733 00:26:04,012 --> 00:26:05,780 SO THEN WHAT WE DID IS INCREASED 734 00:26:05,780 --> 00:26:07,015 THE CONCENTRATION OF GLYCINE, SO 735 00:26:07,015 --> 00:26:08,883 WE WENT UP TO SATURATING LEVELS, 736 00:26:08,883 --> 00:26:11,085 AND WHAT WE SEE IS THE 737 00:26:11,085 --> 00:26:12,520 EQUILIBRIUM SHIFT TOWARDS THE 738 00:26:12,520 --> 00:26:13,421 ACTIVATED STATE RIGHT HERE, AND 739 00:26:13,421 --> 00:26:15,056 THE STATE IS VERY SIMILAR TO 740 00:26:15,056 --> 00:26:18,993 THAT OF THE STATE CAPTURED HERE. 741 00:26:18,993 --> 00:26:20,361 SO HERE IS A MOVIE THAT 742 00:26:20,361 --> 00:26:21,896 SUMMARIZES THE MOVEMENTS THAT WE 743 00:26:21,896 --> 00:26:23,598 SEE UPON GLYCINE ACTIVATION. 744 00:26:23,598 --> 00:26:25,099 SO WHAT YOU ARE SEEING HERE IS 745 00:26:25,099 --> 00:26:28,236 THE GLYCINE POCKET, YOU SEE THIS 746 00:26:28,236 --> 00:26:30,238 INWARD MOVEMENT THAT I TALKED 747 00:26:30,238 --> 00:26:31,672 ABOUT OF LOOP C AND THAT'S A 748 00:26:31,672 --> 00:26:33,307 LITTLE GLYCINE DENSITY AT THE 749 00:26:33,307 --> 00:26:35,276 BACK END, AND ONCE GLYCINE 750 00:26:35,276 --> 00:26:36,878 BINDS, SO GLYCINE BINDS IN THIS 751 00:26:36,878 --> 00:26:38,746 POCKET RIGHT HERE, SO THESE ARE 752 00:26:38,746 --> 00:26:49,190 THE INTERACTING RESIDUES. 753 00:26:52,060 --> 00:26:54,395 AND THAT'S THE INWARD MOVEMENT 754 00:26:54,395 --> 00:26:55,396 OVER THE GLYCINE. 755 00:26:55,396 --> 00:26:59,000 AND THIS IS AN IRIS-LIKE 756 00:26:59,000 --> 00:27:00,435 MOVEMENT OF THE CHANNEL PORE, SO 757 00:27:00,435 --> 00:27:02,103 THIS ALLOWS FOR IONS NOT TO PASS 758 00:27:02,103 --> 00:27:04,372 THROUGH. 759 00:27:04,372 --> 00:27:07,341 SO HOW IS THIS BASIC MECHANISM 760 00:27:07,341 --> 00:27:10,578 OF GATING OR GLYCINE ACTIVATION, 761 00:27:10,578 --> 00:27:12,346 AFFECTED BY ZINC AT LOW 762 00:27:12,346 --> 00:27:12,880 CONCENTRATION? 763 00:27:12,880 --> 00:27:14,849 SO HERE IS THE ZINC POTENTIATION 764 00:27:14,849 --> 00:27:17,685 THAT YOU CAN SEE AT LOW 765 00:27:17,685 --> 00:27:19,420 CONCENTRATION, IT TAPERS OUT AND 766 00:27:19,420 --> 00:27:20,955 STARTS TO MOVE INTO THE 767 00:27:20,955 --> 00:27:22,657 INHIBITION MODE RIGHT HERE AT 768 00:27:22,657 --> 00:27:23,124 HIGHER CONCENTRATION. 769 00:27:23,124 --> 00:27:24,826 SO THEN WE IMAGE THIS IN THE 770 00:27:24,826 --> 00:27:26,427 SAME CONDITION WHAT WE ARE 771 00:27:26,427 --> 00:27:27,862 LOOKING AT, SO THIS IS IN THE 772 00:27:27,862 --> 00:27:29,864 ABSENCE OF GLYCINE, AGAIN, YOU 773 00:27:29,864 --> 00:27:31,065 HAVE THESE DISTRIBUTION, AND 774 00:27:31,065 --> 00:27:33,668 THEN WHEN WE ADD ONE MICROMOLAR 775 00:27:33,668 --> 00:27:35,670 OF GLYCINE WHICH IS WHEN YOU'RE 776 00:27:35,670 --> 00:27:37,705 POTENTIATING, WE ARE SIMPLY 777 00:27:37,705 --> 00:27:38,673 ELIMINATING THIS CLOSED 778 00:27:38,673 --> 00:27:39,073 CONFORMATION. 779 00:27:39,073 --> 00:27:40,208 YOU REALLY HAVE ALL OF THE 780 00:27:40,208 --> 00:27:41,409 PARTICLES UP HERE TO BELONG TO 781 00:27:41,409 --> 00:27:42,577 THE DESENSITIZED STATE RIGHT 782 00:27:42,577 --> 00:27:44,579 HERE. 783 00:27:44,579 --> 00:27:46,914 THEN IF WE GO TOWARDS THE HIGHER 784 00:27:46,914 --> 00:27:47,982 CONCENTRATION OF GLYCINE, WE 785 00:27:47,982 --> 00:27:50,184 SHIFT THE EQUILIBRIUM TOWARDS 786 00:27:50,184 --> 00:27:51,285 THE CLOSED CONFORMATION SO YOU 787 00:27:51,285 --> 00:27:52,887 CAN SEE THESE STATES ARE VERY 788 00:27:52,887 --> 00:27:54,355 SIMILAR TO GLYCINE-BOUND STATES 789 00:27:54,355 --> 00:27:56,324 THAT WE SAW BEFORE. 790 00:27:56,324 --> 00:27:58,359 THE ZINC'S ROLE HAS BEEN IN 791 00:27:58,359 --> 00:27:59,560 SHIFTING THIS EQUILIBRIUM. 792 00:27:59,560 --> 00:28:02,230 SO WE ASKED THE QUESTION WHAT IS 793 00:28:02,230 --> 00:28:03,798 DRIVING THIS SHIFT IN 794 00:28:03,798 --> 00:28:05,800 EQUILIBRIUM BETWEEN THESE 795 00:28:05,800 --> 00:28:06,100 POPULATION. 796 00:28:06,100 --> 00:28:07,368 SO TO SUMMARIZE ACTUALLY, SO 797 00:28:07,368 --> 00:28:09,537 HERE WE HAVE THE TWO, WE HAVE 798 00:28:09,537 --> 00:28:10,838 IDENTIFIED TWO ZINC BINDING 799 00:28:10,838 --> 00:28:11,639 SITES. 800 00:28:11,639 --> 00:28:15,776 THE POTENTIATING OR THE HIGH 801 00:28:15,776 --> 00:28:17,378 AFFINITY ZINC SITES THAT ARE 802 00:28:17,378 --> 00:28:18,846 OCCUPIED AT LOW CONCENTRATIONS 803 00:28:18,846 --> 00:28:20,481 OF GLYCINE IS RIGHT AT THE 804 00:28:20,481 --> 00:28:22,016 INTERFACE BETWEEN THE 805 00:28:22,016 --> 00:28:24,886 INTRACELLULAR AND TRANSMEMBRANE 806 00:28:24,886 --> 00:28:25,753 DOMAIN AND YOU WILL SEE IN THE 807 00:28:25,753 --> 00:28:27,889 NEXT SLIDES THAT THIS POSITION 808 00:28:27,889 --> 00:28:29,423 IS OPTIMALLY PLACED TO INTERACT 809 00:28:29,423 --> 00:28:31,492 OR REGULATE THE MOVEMENT OF LOOP 810 00:28:31,492 --> 00:28:31,926 C. 811 00:28:31,926 --> 00:28:34,428 SO THIS ALLOWS FOR LOOP C 812 00:28:34,428 --> 00:28:36,197 MOVEMENT TO HAPPEN THAT 813 00:28:36,197 --> 00:28:37,965 FACILITATES GLYCINE MEDIATED 814 00:28:37,965 --> 00:28:38,432 ACTIVATION. 815 00:28:38,432 --> 00:28:40,568 AND THE SECOND BINDING SITE FOR 816 00:28:40,568 --> 00:28:42,436 GLYCINE, WHICH IS THE LOW 817 00:28:42,436 --> 00:28:43,804 AFFINITY INHIBITORY SITE IS AT 818 00:28:43,804 --> 00:28:45,306 THE BACK END OF THE POCKET. 819 00:28:45,306 --> 00:28:48,009 SO THIS IS THE HIGH AFFINITY 820 00:28:48,009 --> 00:28:50,311 BINDING SITE, WE CAN CLEARLY SEE 821 00:28:50,311 --> 00:28:51,746 AT THIS INTERFACE. 822 00:28:51,746 --> 00:28:54,148 THERE ARE FOUR AMINO ACIDS THAT 823 00:28:54,148 --> 00:28:56,751 COORDINATE THE ZINC TO AS PER 824 00:28:56,751 --> 00:28:58,753 TATE GLUED MATE AND THEN THE WHO 825 00:28:58,753 --> 00:29:00,054 HISTIDINES SO THEY BELONG TO 826 00:29:00,054 --> 00:29:02,723 DIFFERENT BETA STRANDS HERE AND 827 00:29:02,723 --> 00:29:05,426 THE M4 HELIX, SO AT AN OPTIMAL 828 00:29:05,426 --> 00:29:06,994 POSITION TO COUPLE MOVEMENTS IN 829 00:29:06,994 --> 00:29:09,564 THE EXTRACELLULAR AND 830 00:29:09,564 --> 00:29:10,031 TRANSMEMBRANE DOMAINS. 831 00:29:10,031 --> 00:29:11,365 AND THIS IS THE INHIBITORY SITE 832 00:29:11,365 --> 00:29:12,533 THAT WE SEE AT THE BACK END OF 833 00:29:12,533 --> 00:29:14,035 THIS POCKET. 834 00:29:14,035 --> 00:29:15,836 UNLIKE THE HIGH AFFINITY SITE 835 00:29:15,836 --> 00:29:18,539 WHICH HAS A TETRACOORDINATION, 836 00:29:18,539 --> 00:29:19,807 THE LOW AFFINITY SITE IS 837 00:29:19,807 --> 00:29:21,876 COORDINATED BY A HISTIDINE AND A 838 00:29:21,876 --> 00:29:25,680 GLUTE PLATE ANGLUTAMATE AND THEO 839 00:29:25,680 --> 00:29:26,647 LIKELY THROUGH WATER MOLECULES. 840 00:29:26,647 --> 00:29:28,649 SO THIS IS A SITE WHOSE 841 00:29:28,649 --> 00:29:31,152 ACCESSIBILITY IS NOT -- DOES NOT 842 00:29:31,152 --> 00:29:32,119 UNDERLIE LOW AFFINITY, IT'S JUST 843 00:29:32,119 --> 00:29:33,888 THAT THE COORDINATION IS WEAKER 844 00:29:33,888 --> 00:29:36,090 THAN THE HIGH AFFINITY SITES. 845 00:29:36,090 --> 00:29:42,330 SO HERE IS A MOVIE THAT 846 00:29:42,330 --> 00:29:43,764 HIGHLIGHT THE ZINC SITES IN BOTH 847 00:29:43,764 --> 00:29:45,833 THE CLOSED AND THE DESENSITIZED 848 00:29:45,833 --> 00:29:48,502 CONFORMATION. 849 00:29:48,502 --> 00:29:50,104 SO THAT IS A ZINC COORDINATION 850 00:29:50,104 --> 00:30:00,581 FOR THE HIGH AFFINITY SITE. 851 00:30:11,626 --> 00:30:14,629 SO THE TRACES IN ORANGE ARE THE 852 00:30:14,629 --> 00:30:15,429 RESTING CONFORMATION AND THE 853 00:30:15,429 --> 00:30:16,497 BLUE IS THE ACTIVATED STATE. 854 00:30:16,497 --> 00:30:18,032 SO THERE IS A SLIGHT 855 00:30:18,032 --> 00:30:19,400 REARRANGEMENT BETWEEN THE TWO 856 00:30:19,400 --> 00:30:22,136 STATES THAT SEEM TO SUGGEST THAT 857 00:30:22,136 --> 00:30:24,138 ZINC IN THE ACTIVATED STATE IS 858 00:30:24,138 --> 00:30:29,010 BETTER COORDINATED, AND THIS IS 859 00:30:29,010 --> 00:30:30,845 EVENTUALLY TRANSLATED TO LOOP C 860 00:30:30,845 --> 00:30:32,480 MOVING INWARD AND THIS IS THE 861 00:30:32,480 --> 00:30:33,014 BACK END. 862 00:30:33,014 --> 00:30:34,815 SO THIS WAS A TRICKY SITE TO 863 00:30:34,815 --> 00:30:36,484 UNDERSTAND WHY THIS WOULD LEAD 864 00:30:36,484 --> 00:30:38,386 TO AN INHIBITION AT THIS POCKET. 865 00:30:38,386 --> 00:30:40,288 IT DOESN'T SEEM TO BE VERY CLOSE 866 00:30:40,288 --> 00:30:42,023 TO -- IT'S BEHIND THE BINDING 867 00:30:42,023 --> 00:30:43,357 POCKET AND CANNOT DIRECTLY 868 00:30:43,357 --> 00:30:44,892 UNFLEUNS THE BINDING SITE, BUT 869 00:30:44,892 --> 00:30:46,694 DIGGING DEEPER INTO THE 870 00:30:46,694 --> 00:30:48,095 LITERATURE, IT WAS CLEAR THAT 871 00:30:48,095 --> 00:30:51,866 THIS ARGININE AND GLUTE MADE, 872 00:30:51,866 --> 00:30:54,068 THEY MAKE AN INTERACTION VERY 873 00:30:54,068 --> 00:30:55,436 CRUCIAL FOR GLYCINE ACTIVATION 874 00:30:55,436 --> 00:30:56,904 AND MUTATION TO THESE RESIDUES 875 00:30:56,904 --> 00:30:59,640 HAVE BEEN ASSOCIATED WITH 876 00:30:59,640 --> 00:30:59,974 HYPERREPLEXIA. 877 00:30:59,974 --> 00:31:02,476 SO THIS INTERACTION NEEDS TO BE 878 00:31:02,476 --> 00:31:04,245 IN PLACE FOR GLYCINE MEDIATED 879 00:31:04,245 --> 00:31:05,513 ACTIVATION, AND WE THINK THAT 880 00:31:05,513 --> 00:31:07,948 HAVING A ZINC IN THIS 881 00:31:07,948 --> 00:31:09,450 COORDINATION TRIAD IS WEAKENING 882 00:31:09,450 --> 00:31:12,119 THE INTERACTION BETWEEN ARGININE 883 00:31:12,119 --> 00:31:14,522 AND GLUTAMATE, AND THAT MIGHT 884 00:31:14,522 --> 00:31:15,389 MEDIATE INHIBITION. 885 00:31:15,389 --> 00:31:19,894 SO WE COLLABORATED WITH PHIL 886 00:31:19,894 --> 00:31:21,429 BIGGINS GROUP, THEY EVALUATED 887 00:31:21,429 --> 00:31:23,397 THE STABILITY OF THESE 888 00:31:23,397 --> 00:31:25,466 INTERACTIONS BY SIMULATIONS AND 889 00:31:25,466 --> 00:31:27,568 THEY FIND THAT ZINC THROUGHOUT 890 00:31:27,568 --> 00:31:30,071 THE -- IN THE POTENTIATED SITE 891 00:31:30,071 --> 00:31:30,938 STAYS PRETTY CLOSE. 892 00:31:30,938 --> 00:31:32,340 THESE RESIDUES INTERCHANGE IN 893 00:31:32,340 --> 00:31:34,575 TERMS OF THEIR INTERACTIONS, BUT 894 00:31:34,575 --> 00:31:36,477 THE ZINC ITSELF STAYS FIRMLY 895 00:31:36,477 --> 00:31:37,778 BOUND HERE. 896 00:31:37,778 --> 00:31:39,947 THEY ALSO SAW THAT THE GLUTAMATE 897 00:31:39,947 --> 00:31:41,349 WAS AN IMPORTANT INTERACTING 898 00:31:41,349 --> 00:31:43,184 RESIDUE FOR THE INHIBITORY SITE, 899 00:31:43,184 --> 00:31:45,419 AND AGAIN, STABLE INTERACTIONS 900 00:31:45,419 --> 00:31:47,788 WHEN ORDERED FOR THE GLUTAMATE 901 00:31:47,788 --> 00:31:51,959 AND ZINC RIGHT HERE. 902 00:31:51,959 --> 00:31:53,327 SO AGAIN COMES TO OUR PROPOSED 903 00:31:53,327 --> 00:31:55,329 MECHANISM THAT YOU HAVE THIS 904 00:31:55,329 --> 00:31:56,364 INTERFACE INTERACTION THAT 905 00:31:56,364 --> 00:31:57,732 MEDIATES LOOP C MOVEMENT AND 906 00:31:57,732 --> 00:32:00,134 WHICH MIGHT BE STABILIZING 907 00:32:00,134 --> 00:32:03,537 GLYCINE IN THE POCKET AND 908 00:32:03,537 --> 00:32:05,306 INHIBITORY SITE MIGHT BE 909 00:32:05,306 --> 00:32:07,408 IMPACTING GLUED GLEUT MATE 910 00:32:07,408 --> 00:32:08,342 ARGININE INTERACTION. 911 00:32:08,342 --> 00:32:12,980 SO TO FURTHER LOOK INTO OW MUCH 912 00:32:12,980 --> 00:32:15,683 THIS PROPOSAL -- THIS IDEA 913 00:32:15,683 --> 00:32:19,120 HOLDS, SO PHIL BIGGINS GROUP 914 00:32:19,120 --> 00:32:21,455 LOOKED AT THE INTERACTION DURING 915 00:32:21,455 --> 00:32:23,891 THE SIMULATION THAT COWARDS 916 00:32:23,891 --> 00:32:25,326 NAITS THOSE WITH NO ZINC VERSUS 917 00:32:25,326 --> 00:32:27,128 THOSE WITH ZINC. 918 00:32:27,128 --> 00:32:29,029 THE INTERACTION IS VERY STABLE 919 00:32:29,029 --> 00:32:33,267 WHEN YOU ADD ZINC AND -- AND SO 920 00:32:33,267 --> 00:32:34,502 WE THINK THIS COULD BE A 921 00:32:34,502 --> 00:32:36,003 STARTING MECHANISM. 922 00:32:36,003 --> 00:32:38,205 WE ALSO -- STRUCTURES, THESE ARE 923 00:32:38,205 --> 00:32:39,974 STATIC IMAGES, SO THESE 924 00:32:39,974 --> 00:32:41,709 MECHANISMS NEED TO BE VALIDATED. 925 00:32:41,709 --> 00:32:44,578 IN THIS CASE, SO THIS IS LOOKING 926 00:32:44,578 --> 00:32:46,380 AT ZINC MODULATION, SO THIS 927 00:32:46,380 --> 00:32:48,482 IS -- IN BLUE IS THE 928 00:32:48,482 --> 00:32:49,383 POTENTIATION WINDOW AND ORANGE 929 00:32:49,383 --> 00:32:50,684 IS THE INHIBITION. 930 00:32:50,684 --> 00:32:52,887 WE TESTED THE HYPOTHESIS BY 931 00:32:52,887 --> 00:32:54,355 MAKING MUTATION ESSENTIALLY 932 00:32:54,355 --> 00:32:56,624 DELETING THIS END OF M4, WHERE 933 00:32:56,624 --> 00:32:58,793 THERE ARE MULTIPLE RESIDUES THAT 934 00:32:58,793 --> 00:32:59,460 WERE PARTICIPATING IN THIS 935 00:32:59,460 --> 00:33:00,694 COORDINATION AND WE SEE THAT 936 00:33:00,694 --> 00:33:01,996 ZINC POTENTIATION COMPLETELY 937 00:33:01,996 --> 00:33:04,999 GOES AWAY. 938 00:33:04,999 --> 00:33:06,967 SIMILARLY WE MAKE A MUTATION TO 939 00:33:06,967 --> 00:33:08,335 THIS GLUTAMATE, AND WE SEE THAT 940 00:33:08,335 --> 00:33:09,937 THE INHIBITION GETS SHIFTED TO 941 00:33:09,937 --> 00:33:13,207 MUCH HIGHER CONCENTRATION, SO 942 00:33:13,207 --> 00:33:14,675 THIS SORT OF GAVE US SOME IDEA 943 00:33:14,675 --> 00:33:16,544 ON WHAT'S HAPPENING WITH ZINC 944 00:33:16,544 --> 00:33:18,078 MODULATION. 945 00:33:18,078 --> 00:33:21,715 SO ANOTHER MODULATORY FACTOR 946 00:33:21,715 --> 00:33:24,852 THAT IS IMPORTANT IN PAIN 947 00:33:24,852 --> 00:33:26,487 SENSATION IS PH. 948 00:33:26,487 --> 00:33:29,156 DURING NEUROPATHIC PAIN AND 949 00:33:29,156 --> 00:33:29,590 HYPOXIA. 950 00:33:29,590 --> 00:33:34,261 SO YOU HAVE THE EXTRACELLULAR PH 951 00:33:34,261 --> 00:33:35,696 MOVES TOWARDS ACIDIC DIRECTION, 952 00:33:35,696 --> 00:33:39,733 EVEN TO SOME EXTENT TO ONE PH 953 00:33:39,733 --> 00:33:42,436 UNIT, AND OTHER STUDIES HAVE 954 00:33:42,436 --> 00:33:45,039 SHOWN GLYCINE SENSITIVITY IS 955 00:33:45,039 --> 00:33:47,641 RIGHT-SHIFTED. 956 00:33:47,641 --> 00:33:49,810 ALSO ITS ACTIVITY TO PARTIAL 957 00:33:49,810 --> 00:33:51,479 AGONISM IS DECREASED, ALTHOUGH 958 00:33:51,479 --> 00:33:53,614 IT IS VERY INTERESTING TO NOTE 959 00:33:53,614 --> 00:33:55,716 THAT WHAT WE ARE ESSENTIALLY 960 00:33:55,716 --> 00:33:57,718 SEEING IS A REDUCED GLYCINERGIC 961 00:33:57,718 --> 00:34:01,322 RESPONSE BASED ON THE PEAK 962 00:34:01,322 --> 00:34:03,390 AMPLITUDE, AND AT THE SINGLE 963 00:34:03,390 --> 00:34:04,825 CHANNEL LEVEL UNDER ACIDIC 964 00:34:04,825 --> 00:34:05,960 CONDITIONS, YOU HAVE THESE VERY 965 00:34:05,960 --> 00:34:07,862 SHORT LIVED CLOSURES, SO THE 966 00:34:07,862 --> 00:34:10,898 CHANNEL TENDS TO MOVE INTO THE 967 00:34:10,898 --> 00:34:12,032 CLOSED CONFORMATION, MULTIPLE 968 00:34:12,032 --> 00:34:13,834 TYPES OF CLOSED CONFORMATION SO 969 00:34:13,834 --> 00:34:15,769 YOU HAVE ONE COMING FROM REDUCED 970 00:34:15,769 --> 00:34:17,171 AMPLITUDE BUT ALSO THESE FAST 971 00:34:17,171 --> 00:34:19,507 FLICKERS THAT ARE CONTRIBUTING 972 00:34:19,507 --> 00:34:21,141 TO LOWER OPEN PROBABILITY. 973 00:34:21,141 --> 00:34:27,014 SO WE THEN LOOKED AT HOW ALPHA E 974 00:34:27,014 --> 00:34:27,615 CONDITIONS. 975 00:34:27,615 --> 00:34:32,253 SO HERE IS ELECTROPHYSIOLOGY. 976 00:34:32,253 --> 00:34:34,722 BUT LIKE OTHER -- YOU DIMINISH 977 00:34:34,722 --> 00:34:35,856 THE EFFECT OF THIS MODULATOR. 978 00:34:35,856 --> 00:34:40,261 SO WHAT WE NOTICED IS UNDER 979 00:34:40,261 --> 00:34:43,097 ACIDIC PH, THIS IS AT NORMAL 980 00:34:43,097 --> 00:34:45,599 CONDITIONS, PH -- LOWER PH 981 00:34:45,599 --> 00:34:47,701 POPULATED A NUMBER OF STATES 982 00:34:47,701 --> 00:34:49,270 THAT HAD DISTINCT FEATURES THAT 983 00:34:49,270 --> 00:34:51,005 WERE DIFFERENT FROM THAT OF 984 00:34:51,005 --> 00:34:52,773 THESE CONFORMATIONS. 985 00:34:52,773 --> 00:34:54,575 SO THE SHIFT WAS -- THE FIRST 986 00:34:54,575 --> 00:34:56,277 THING WE NOTICED WAS A SHIFT 987 00:34:56,277 --> 00:34:58,913 TOWARDS MORE CLOSED AND MORE 988 00:34:58,913 --> 00:35:00,447 CONDUCTING CONFORMATIONS, AND 989 00:35:00,447 --> 00:35:03,384 ALSO APPEARANCE OF A NEW 990 00:35:03,384 --> 00:35:06,253 INTERMEDIATE STATE. 991 00:35:06,253 --> 00:35:09,023 SO ALL OF THESE STATES SHIFT 992 00:35:09,023 --> 00:35:10,591 TOWARDS A DESENSITIZED STATE. 993 00:35:10,591 --> 00:35:13,694 SO HERE IS A MOVIE THAT SHOWS 994 00:35:13,694 --> 00:35:15,362 YOU WHAT THESE CHANGES ARE SO 995 00:35:15,362 --> 00:35:17,431 THE CLOSE CONFORMATION THAT WE 996 00:35:17,431 --> 00:35:19,266 STABILIZED HAD A DIFFERENT 997 00:35:19,266 --> 00:35:22,269 ORIENTATION FOR LOOP C, IT ALSO 998 00:35:22,269 --> 00:35:23,003 INTERESTING HAS GLYCINE IN THE 999 00:35:23,003 --> 00:35:24,872 POCKET SO YOU HAVE GLYCINE YET 1000 00:35:24,872 --> 00:35:26,740 THE CHANNEL IS NOT ACTIVATED. 1001 00:35:26,740 --> 00:35:30,544 AND YOU CAN SEE SOME AUTOMATIC 1002 00:35:30,544 --> 00:35:31,545 DIFFERENCES, AGAIN, YOU HAVE 1003 00:35:31,545 --> 00:35:33,314 GLYCINE IN THE POCKET, THE 1004 00:35:33,314 --> 00:35:34,748 CHANNEL YOU WILL SEE -- SO THIS 1005 00:35:34,748 --> 00:35:36,483 IS MOVING TOWARDS GLYCINE 1006 00:35:36,483 --> 00:35:46,093 ACTIVATED STATE. 1007 00:35:46,093 --> 00:35:47,895 AND HERE IS A DESENSITIZED 1008 00:35:47,895 --> 00:35:49,330 CONFORMATION, HERE IS THE IPT 1009 00:35:49,330 --> 00:35:50,931 IMMEDIATE CONFORMATION, AND THEN 1010 00:35:50,931 --> 00:35:52,766 MOVING TO THE CLOSED 1011 00:35:52,766 --> 00:35:53,100 CONFORMATION. 1012 00:35:53,100 --> 00:35:55,669 SO WE HAVE THE PORE OF THE 1013 00:35:55,669 --> 00:35:57,237 CHANNEL IS STABILIZED IN THREE 1014 00:35:57,237 --> 00:36:00,708 DISTINCT STATES THAT CONTRIBUTES 1015 00:36:00,708 --> 00:36:02,676 TO NON-CONDUCTING STATES AND WE 1016 00:36:02,676 --> 00:36:03,844 HYPOTHESIZE THAT THIS 1017 00:36:03,844 --> 00:36:05,145 INTERMEDIATE STATE MIGHT BE 1018 00:36:05,145 --> 00:36:07,014 CORRESPONDING TO THE 1019 00:36:07,014 --> 00:36:07,815 FLICKERING -- FREQUENTLY 1020 00:36:07,815 --> 00:36:08,549 FLICKERING STATES THAT WAS 1021 00:36:08,549 --> 00:36:11,785 OBSERVED IN ELECTROPHYSIOLOGY. 1022 00:36:11,785 --> 00:36:13,387 SO SWITCHING GEARS, I WANT TO 1023 00:36:13,387 --> 00:36:14,855 TELL YOU -- SO THESE WERE THE 1024 00:36:14,855 --> 00:36:15,923 ENDOGENOUS REGULATORS OF THE 1025 00:36:15,923 --> 00:36:16,357 CHANNELS. 1026 00:36:16,357 --> 00:36:19,994 WHAT ABOUT SOME EXOGENOUS 1027 00:36:19,994 --> 00:36:21,996 MODULATORS, ESPECIALLY LIPID 1028 00:36:21,996 --> 00:36:22,363 MODULATORS. 1029 00:36:22,363 --> 00:36:25,165 SO THIS WORK WAS DONE BY ARVIN 1030 00:36:25,165 --> 00:36:26,500 KUMAR, A POSTDOC IN THE LAB 1031 00:36:26,500 --> 00:36:32,539 ALONG WITH KAYLA KINDIG, 1032 00:36:32,539 --> 00:36:34,575 AFRODITI AND PHIL BIGGIN'S 1033 00:36:34,575 --> 00:36:34,975 GROUP. 1034 00:36:34,975 --> 00:36:36,543 ONE THING THAT REALLY POPS OUT 1035 00:36:36,543 --> 00:36:37,911 IS THESE CAVITIES THAT I SHOWED 1036 00:36:37,911 --> 00:36:39,780 IN THE FIRST SLADE WHICH ARE 1037 00:36:39,780 --> 00:36:41,949 SITES FOR BINDING OF VARIOUS 1038 00:36:41,949 --> 00:36:43,884 MODULATORS, SO THESE CAVITIES 1039 00:36:43,884 --> 00:36:45,653 CHANGE VOLUMES BETWEEN CLOSED 1040 00:36:45,653 --> 00:36:48,055 AND DESENSITIZED CONFORMATION 1041 00:36:48,055 --> 00:36:49,657 THAT MIGHT EXPLAIN FOR SOME OF 1042 00:36:49,657 --> 00:36:52,092 THE STATE-DEPENDENT BINDING OF 1043 00:36:52,092 --> 00:36:52,793 THESE LIGANDS. 1044 00:36:52,793 --> 00:36:55,562 SO HERE I WANT TO SHOW YOU, SO 1045 00:36:55,562 --> 00:36:56,764 AGAIN, THESE ARE THE CALFITIES 1046 00:36:56,764 --> 00:37:02,603 WHERE ACAVITIESWHERE A NUMBER OE 1047 00:37:02,603 --> 00:37:04,772 MODULATORS BIND, SO I WANT TO 1048 00:37:04,772 --> 00:37:05,839 HIGHLIGHT ONE HERE IN THIS CASE 1049 00:37:05,839 --> 00:37:08,075 THE GLYCINE RECEPTORS ARE 1050 00:37:08,075 --> 00:37:11,045 MODULATED BY ENDOCANNABINOIDS, 1051 00:37:11,045 --> 00:37:18,318 THESE ARE POLYUNSATURATED -- POE 1052 00:37:18,318 --> 00:37:19,920 10 SHAITS GLYCINE 1 CURRENTS, 1053 00:37:19,920 --> 00:37:22,089 AND ALSO THIS SORT OF AN EFFECT 1054 00:37:22,089 --> 00:37:27,795 IS MIMICKED BY THC OR DELTA-9 1055 00:37:27,795 --> 00:37:28,729 TETRAHYDROCANNABINOL WHICH IS 1056 00:37:28,729 --> 00:37:30,464 THE PSYCHOACTIVE INGREDIENT IN 1057 00:37:30,464 --> 00:37:31,198 THE CANNABIS PLANT. 1058 00:37:31,198 --> 00:37:34,835 SO YOU CAN SEE AGAIN 1059 00:37:34,835 --> 00:37:36,036 POTENTIATION, IT HAS PREVIOUSLY 1060 00:37:36,036 --> 00:37:38,172 BEEN PROPOSED THAT T HC BINDS 1061 00:37:38,172 --> 00:37:40,507 WITHIN THESE TRANSMEMBRANE 1062 00:37:40,507 --> 00:37:42,342 HELIXES TO BRING ABOUT THIS 1063 00:37:42,342 --> 00:37:43,143 POTENTIATION. 1064 00:37:43,143 --> 00:37:45,212 SO AGAIN, SO THERE IS ANOTHER 1065 00:37:45,212 --> 00:37:47,147 MODULATOR THAT I'LL TALK ABOUT 1066 00:37:47,147 --> 00:37:48,348 IS IVERMECTIN, WHICH BINDS INTO 1067 00:37:48,348 --> 00:37:50,250 A SITE THAT IS BETWEEN THE TWO 1068 00:37:50,250 --> 00:37:52,086 SUBUNITS. 1069 00:37:52,086 --> 00:37:55,122 SO THC HAS THIS VERY NICE AND 1070 00:37:55,122 --> 00:37:58,092 STRONG POTENTIATION THAT YOU CAN 1071 00:37:58,092 --> 00:37:59,960 SEE AND THIS POTENTIATION IS 1072 00:37:59,960 --> 00:38:01,829 FULLY DIVERSIBLE AND YOU CAN 1073 00:38:01,829 --> 00:38:03,197 RECAPTURE THE ACTIVITY. 1074 00:38:03,197 --> 00:38:05,833 LIKE OTHER ALLOSTERIC MODULATORS 1075 00:38:05,833 --> 00:38:07,334 AT SATURATING CONCENTRATION YOU 1076 00:38:07,334 --> 00:38:09,703 HAVE THIS POTENTIATION EFFECT GO 1077 00:38:09,703 --> 00:38:10,037 AWAY. 1078 00:38:10,037 --> 00:38:12,372 SO WE SAW THE NUMBER OF 1079 00:38:12,372 --> 00:38:13,841 STRUCTURES IN THE PRESENCE OF 1080 00:38:13,841 --> 00:38:15,442 VARYING CONCENTRATIONS OF 1081 00:38:15,442 --> 00:38:19,213 GLYCINE, SO UNLIKE IN THE ALPHA 1082 00:38:19,213 --> 00:38:21,982 3, WHERE AT SUBSATURATING 1083 00:38:21,982 --> 00:38:23,584 CONCENTRATIONS WHERE AT 1084 00:38:23,584 --> 00:38:25,586 MICROMOLAR WE CAPTURED -- THE 1085 00:38:25,586 --> 00:38:27,054 SENSITIVITY TO GLYCINE OF 1086 00:38:27,054 --> 00:38:30,557 ALPHA-1 IS HIGHER SO AS YOU CAN 1087 00:38:30,557 --> 00:38:33,360 SEE, AT 100 MICROMOLAR WE SIMPLY 1088 00:38:33,360 --> 00:38:34,428 HAVE ONE DESENSITIZED 1089 00:38:34,428 --> 00:38:35,395 CONFORMATION. 1090 00:38:35,395 --> 00:38:36,930 SO IT WAS A CHALLENGING TASK FOR 1091 00:38:36,930 --> 00:38:39,967 US TO CAPTURE AN OPEN LIFE STATE 1092 00:38:39,967 --> 00:38:42,569 IN THIS CASE, OR PARTIALLY 1093 00:38:42,569 --> 00:38:44,371 ACTIVATED STATE SO IN THIS CASE, 1094 00:38:44,371 --> 00:38:46,874 WHEN WE USE THC SO HERE WE ARE 1095 00:38:46,874 --> 00:38:48,542 REPEATING THIS ENTIRE TITRATION, 1096 00:38:48,542 --> 00:38:50,644 BUT IN THE PRESENCE OF 32 1097 00:38:50,644 --> 00:38:53,580 MICROMOLAR THC, WE START TO 1098 00:38:53,580 --> 00:38:55,215 POPULATE STATES, THERE ARE SOME 1099 00:38:55,215 --> 00:38:57,985 IN BETWEEN THE RESTING AND THE 1100 00:38:57,985 --> 00:38:58,685 DESENSITIZED CONFORMATION, AND 1101 00:38:58,685 --> 00:39:00,821 IN THIS PURSUIT, WE ACTUALLY 1102 00:39:00,821 --> 00:39:02,556 CAPTURED AN OPEN -- THE 1103 00:39:02,556 --> 00:39:04,124 TRANSIENT OPEN CONFORMATION. 1104 00:39:04,124 --> 00:39:06,493 SO THIS IS A STATE THAT WE HAVE 1105 00:39:06,493 --> 00:39:08,195 SEEN BEFORE, IT'S DESENSITIZED, 1106 00:39:08,195 --> 00:39:10,164 HERE IS THE DESENSITIZATION GATE 1107 00:39:10,164 --> 00:39:12,566 CLOSED IN AND MOLECULAR DYNAMIC 1108 00:39:12,566 --> 00:39:13,834 SIMULATIONS WHICH ALLOW YOU TO 1109 00:39:13,834 --> 00:39:16,937 LOOK AT PERMEATION EVENTS ACROSS 1110 00:39:16,937 --> 00:39:19,406 THE MEMBRANE, SO YOU CAN SEE 1111 00:39:19,406 --> 00:39:21,141 THAT HERE THIS WHITE PATCH 1112 00:39:21,141 --> 00:39:22,810 SUGGESTED THERE ARE A FEWER 1113 00:39:22,810 --> 00:39:24,444 NUMBER OF IONS PASSING THROUGH 1114 00:39:24,444 --> 00:39:25,879 THIS DESENSITIZED GATE OF THE 1115 00:39:25,879 --> 00:39:26,113 CHANNEL. 1116 00:39:26,113 --> 00:39:28,582 SO THIS IS IN THE PRESENCE OF 1117 00:39:28,582 --> 00:39:31,285 THC THE CHANNELS OPENING WIDENS 1118 00:39:31,285 --> 00:39:32,686 UP AND THEN YOU START TO SEE A 1119 00:39:32,686 --> 00:39:33,520 MORE PERMEATING CHANNEL. 1120 00:39:33,520 --> 00:39:35,823 SO AGAIN, THE SHIFT IN 1121 00:39:35,823 --> 00:39:37,191 EQUILIBRIUM TOWARDS A MORE 1122 00:39:37,191 --> 00:39:39,660 CONDUCTING CONFORMATION. 1123 00:39:39,660 --> 00:39:41,461 SO WHERE IS THC BINDING HERE? 1124 00:39:41,461 --> 00:39:43,096 WE HAVE AN ADDITIONAL DENSITY 1125 00:39:43,096 --> 00:39:45,999 THAT WE SEE LIKELY CORRESPONDS 1126 00:39:45,999 --> 00:39:48,869 TO THE HTC MOLECULE, THE SAME 1127 00:39:48,869 --> 00:39:50,103 SITE THAT WAS PREVIOUSLY 1128 00:39:50,103 --> 00:39:52,139 PROPOSED, AND IT WAS PROPOSED 1129 00:39:52,139 --> 00:39:55,576 THAT THC INTERACTS WITH THE -- 1130 00:39:55,576 --> 00:40:01,281 RESIDUE IN THE TM3 OR THE THIRD 1131 00:40:01,281 --> 00:40:01,849 HELIX. 1132 00:40:01,849 --> 00:40:02,649 THIS HAS BEEN SHOWN BEFORE AND 1133 00:40:02,649 --> 00:40:04,051 ALSO WE CAN SEE IT IN OUR AND 1134 00:40:04,051 --> 00:40:06,653 THAT WHEN YOU GET RID OF THIS 1135 00:40:06,653 --> 00:40:08,889 CHAIN THEN YOU LOWER THC 1136 00:40:08,889 --> 00:40:09,223 POTENTIATION. 1137 00:40:09,223 --> 00:40:12,626 SO WHEN WE COMPARE THOUGHT THC, 1138 00:40:12,626 --> 00:40:14,228 WE NOTICE THAT YOU HAVE THE 1139 00:40:14,228 --> 00:40:15,262 BINDING POCKET BUT THE 1140 00:40:15,262 --> 00:40:17,564 STRUCTURAL CHANGES ARE ALSO SEEN 1141 00:40:17,564 --> 00:40:19,466 ALONG OTHER TM HEE LISTS SUCH 1142 00:40:19,466 --> 00:40:20,634 THAT IT WAS SORT OF GOING 1143 00:40:20,634 --> 00:40:23,036 TOWARDS THE DESENSITIZED 1144 00:40:23,036 --> 00:40:24,238 CONFORMATION. 1145 00:40:24,238 --> 00:40:27,541 PARTICULARLY NOTEWORTHY ARE 1146 00:40:27,541 --> 00:40:28,909 THESE REORIENTATIONS OF THESE 1147 00:40:28,909 --> 00:40:30,444 SITE CHAINS WHICH EVENTUALLY 1148 00:40:30,444 --> 00:40:31,945 PHYSICALLY COUPLE TO THE 1149 00:40:31,945 --> 00:40:32,980 DESENSITIZED GATE. 1150 00:40:32,980 --> 00:40:36,283 SO WE MAKE NOTATIONS TO EACH OF 1151 00:40:36,283 --> 00:40:38,118 THESE POSITIONS AND WE NOTICE 1152 00:40:38,118 --> 00:40:40,387 THAT THC POTENTIATION GOES AWAY 1153 00:40:40,387 --> 00:40:42,990 AND IN SOME CASES PRETTY 1154 00:40:42,990 --> 00:40:45,058 DRAMATIC THAT THERE IS NO 1155 00:40:45,058 --> 00:40:45,993 POTENTIATION TO THC. 1156 00:40:45,993 --> 00:40:47,661 SO THE MECHANISM THAT WE ARE 1157 00:40:47,661 --> 00:40:51,932 PROPOSING IS THC BINDING RIGHT 1158 00:40:51,932 --> 00:40:54,067 HERE, EE VENGSLY -- AUTOMATIC 1159 00:40:54,067 --> 00:40:56,036 RESIDUES TO COMMUNICATE TO THE 1160 00:40:56,036 --> 00:40:57,137 DESENSITIZATION GATE WHICH 1161 00:40:57,137 --> 00:40:57,871 STABILIZES THE OPEN 1162 00:40:57,871 --> 00:41:00,207 CONFORMATION. 1163 00:41:00,207 --> 00:41:02,743 SO -- AND PHIL BIGGINS' GROUP, 1164 00:41:02,743 --> 00:41:04,778 THEY TOOK THE THC COORDINATES, 1165 00:41:04,778 --> 00:41:06,880 THEY DOCUMENTED ON THE GLYCINE 1166 00:41:06,880 --> 00:41:07,881 ALONE STATE AND THEY LOOKED AT 1167 00:41:07,881 --> 00:41:09,683 THE STABILITY AND YOU CAN SEE 1168 00:41:09,683 --> 00:41:12,185 THREE OUT OF FOUR OR FIVE 1169 00:41:12,185 --> 00:41:14,421 SUBUNITS HOLDS ON TO THC AND IN 1170 00:41:14,421 --> 00:41:16,556 FACT THE CONFORMATIONAL CHANGES 1171 00:41:16,556 --> 00:41:17,925 THE PROTEIN UNDERGOES WERE 1172 00:41:17,925 --> 00:41:19,626 SIMILAR TO WHAT WE HAD OBSERVED 1173 00:41:19,626 --> 00:41:21,662 UNDER THE CRYO-EM STRUCTURES, TO 1174 00:41:21,662 --> 00:41:23,931 THE POINT THAT THC DOCKED 1175 00:41:23,931 --> 00:41:25,599 GLYCINE STRUCTURES MOVED FROM 1176 00:41:25,599 --> 00:41:26,600 THESE INITIAL COORDINATE TO 1177 00:41:26,600 --> 00:41:28,335 START OPENING IT UP AT THE 1178 00:41:28,335 --> 00:41:30,437 DESENSITIZED COP FORMATION. 1179 00:41:30,437 --> 00:41:33,006 COULD BE FORMATION. 1180 00:41:33,006 --> 00:41:33,340 CONFORMATION. 1181 00:41:33,340 --> 00:41:34,975 SO TO THE LAST PART I WANT TO 1182 00:41:34,975 --> 00:41:36,410 TELL YOU ABOUT ANOTHER MODULATOR 1183 00:41:36,410 --> 00:41:38,278 BUT IN THE CONTEXT OF A 1184 00:41:38,278 --> 00:41:39,579 HETEROAMERICA GLYCINE RECEPTOR. 1185 00:41:39,579 --> 00:41:41,181 SO THIS WAS A CHALLENGE, SO THIS 1186 00:41:41,181 --> 00:41:43,650 WORK WAS DONE BY ERIC GIBBS AND 1187 00:41:43,650 --> 00:41:46,453 EMILY KRUEGER, AN MD PH.D. 1188 00:41:46,453 --> 00:41:47,521 STUDENT IN THE LAB. 1189 00:41:47,521 --> 00:41:48,922 SO AS WE WERE WORKING ON THIS 1190 00:41:48,922 --> 00:41:54,361 FOR SEVERAL YEARS, WE NAIVELY 1191 00:41:54,361 --> 00:41:56,964 THOUGHT WE COULD USE GEF RIN 1192 00:41:56,964 --> 00:41:58,365 WHICH BINDS TO THE DOMAIN. 1193 00:41:58,365 --> 00:42:00,867 WE COULD CAPTURE A COMPLEX IN 1194 00:42:00,867 --> 00:42:02,269 THE PRESENCE OF SCAFFOLDING 1195 00:42:02,269 --> 00:42:05,005 PROTEIN BUT ALSO GEF RIN WOULD 1196 00:42:05,005 --> 00:42:06,807 ACT AS A IF I DURABLE MARKER 1197 00:42:06,807 --> 00:42:08,075 ALLOWING FOR THE IDENTIFICATION 1198 00:42:08,075 --> 00:42:09,910 OF BETA SUBUNIT IN THESE. 1199 00:42:09,910 --> 00:42:11,979 SO WE TOOK A BIT OF A SHORTCUT, 1200 00:42:11,979 --> 00:42:15,415 WE USED DOMAIN E, A DIMERIZATION 1201 00:42:15,415 --> 00:42:16,750 DOMAIN THAT HAS PREVIOUSLY BEEN 1202 00:42:16,750 --> 00:42:20,387 SHOWN TO BIND TO THE BETA 1203 00:42:20,387 --> 00:42:22,489 PEPTIDE REGION RIGHT HERE IN THE 1204 00:42:22,489 --> 00:42:25,792 INTRACELLULAR DOMAIN. 1205 00:42:25,792 --> 00:42:27,361 SO WE WORKED OUT A SYSTEM TO 1206 00:42:27,361 --> 00:42:28,495 EXPRESS ALL THE THREE PROTEIN 1207 00:42:28,495 --> 00:42:33,233 AND YOU CAN SEE WE GOT DOWN TO A 1208 00:42:33,233 --> 00:42:34,434 PRETTY DECENT BEAUTIFICATION 1209 00:42:34,434 --> 00:42:34,968 STRATEGY. 1210 00:42:34,968 --> 00:42:37,104 WE COULD ALSO SEE IN GEL 1211 00:42:37,104 --> 00:42:39,072 FILTRATION THAT WE HAVE A NICE 1212 00:42:39,072 --> 00:42:40,273 SHIFT THAT INDICATES HIGH 1213 00:42:40,273 --> 00:42:43,377 MOLECULAR WA WEIGHT AND WE CAN O 1214 00:42:43,377 --> 00:42:46,580 ESTIMATE THE WEIGHT BY MASS FLOW 1215 00:42:46,580 --> 00:42:49,182 TOM TRI. 1216 00:42:49,182 --> 00:42:50,350 IN THE BEGINNING WE START TODAY 1217 00:42:50,350 --> 00:42:51,885 SEE SOME ADDITIONAL DENSITIES WE 1218 00:42:51,885 --> 00:42:53,153 HAD NOT SEEN IN THE 1219 00:42:53,153 --> 00:42:54,154 INTRACELLULAR DOMAIN BEFORE, BUT 1220 00:42:54,154 --> 00:42:55,889 AS WE STARTED TO PROCESS IT, WE 1221 00:42:55,889 --> 00:42:57,424 REALIZED THAT THE INTRACELLULAR 1222 00:42:57,424 --> 00:43:00,594 DOMAIN IS SO FLEXIBLE THAT IT 1223 00:43:00,594 --> 00:43:02,696 SWINGS LIKELY WITH THE BIG BULKY 1224 00:43:02,696 --> 00:43:04,131 GEPHYRIN AND WE REALLY COULDN'T 1225 00:43:04,131 --> 00:43:06,333 CAPTURE MUCH, BUT IN THE PROCESS 1226 00:43:06,333 --> 00:43:09,469 WE WERE ABLE TO GET SOME 1227 00:43:09,469 --> 00:43:10,704 GLIMPSES OF THE INTRACELLULAR 1228 00:43:10,704 --> 00:43:12,205 DOMAIN THAT I'LL SHOW YOU. 1229 00:43:12,205 --> 00:43:14,908 BUT WHAT WAS PRETTY NEAT IS THAT 1230 00:43:14,908 --> 00:43:16,777 THE ALPHA AND THE BETA SUBUNITS 1231 00:43:16,777 --> 00:43:18,311 HAVE DIFFERENT GLYCOSYLATION 1232 00:43:18,311 --> 00:43:22,382 SITES, SO THESE HAVE -- THESE 1233 00:43:22,382 --> 00:43:23,917 EXTENSIONS THAT YOU SEE IN THE 1234 00:43:23,917 --> 00:43:26,119 BETA SUBUNIT ALSO HAS AN END 1235 00:43:26,119 --> 00:43:27,888 TERMINAL EXTENSION AND THE 1236 00:43:27,888 --> 00:43:32,392 M4 LENS AT ALPHA AND BETA 1237 00:43:32,392 --> 00:43:35,462 SUBUNIT ARE DIFFERENT -- TO 1238 00:43:35,462 --> 00:43:37,364 ASSIGN THE SUBUNITS, THE ALPHA 1239 00:43:37,364 --> 00:43:39,966 AND BETA SUBUNITS. 1240 00:43:39,966 --> 00:43:43,904 SO HERE IS A MOVIE THAT GOES 1241 00:43:43,904 --> 00:43:44,805 THROUGH MULTIPLE CONTOURING SO 1242 00:43:44,805 --> 00:43:48,809 THAT YOU CAN SEE, SO THIS IS THE 1243 00:43:48,809 --> 00:43:50,277 ALPHA SUBUNITS ARE IN LIGHT 1244 00:43:50,277 --> 00:43:51,812 PURPLE AND THE VIOLET IS THE 1245 00:43:51,812 --> 00:43:53,480 BETA SUBUNIT. 1246 00:43:53,480 --> 00:43:54,981 AND SO THIS PARTICULAR 1247 00:43:54,981 --> 00:43:56,683 CONFORMATION WAS CAPTURED IN THE 1248 00:43:56,683 --> 00:43:59,219 PRESENCE OF STRICK NEEN WHICH IS 1249 00:43:59,219 --> 00:44:02,389 A HIGHLY TOXIC INHIBITOR OF 1250 00:44:02,389 --> 00:44:08,962 GLYCINE RECEPTORS DERIVED -- 1251 00:44:08,962 --> 00:44:10,430 HERE IS THE CLOSED CHANNEL AND 1252 00:44:10,430 --> 00:44:12,032 YOU CAN SEE THE SITE CHAINS THAT 1253 00:44:12,032 --> 00:44:14,935 FORM THE ACTIVATION AND THE 1254 00:44:14,935 --> 00:44:15,502 DESENSITIZATION GATE RIGHT 1255 00:44:15,502 --> 00:44:24,277 THERE, THIS IS THE PROLINE. 1256 00:44:24,277 --> 00:44:25,512 SO AS WE WERE LOOKING THROUGH 1257 00:44:25,512 --> 00:44:27,814 THESE STRUCTURES, WE NOTICED 1258 00:44:27,814 --> 00:44:30,350 THAT THE DENSITY FOR THE 1259 00:44:30,350 --> 00:44:30,784 INTRACELLULAR DOMAIN 1260 00:44:30,784 --> 00:44:31,852 INTERESTINGLY IS NOT PLACED AT 1261 00:44:31,852 --> 00:44:35,522 THE CENTER OF THE PORE AXIS BUT 1262 00:44:35,522 --> 00:44:36,957 SHIFTED AWAY FROM THE BETA 1263 00:44:36,957 --> 00:44:39,392 SUBUNIT. 1264 00:44:39,392 --> 00:44:41,027 WE SOLVED CONFORMATIONS OF 1265 00:44:41,027 --> 00:44:42,562 MULTIPLE -- I MEAN, WITH 1266 00:44:42,562 --> 00:44:44,064 MULTIPLE MODULATORS IN THIS AND 1267 00:44:44,064 --> 00:44:45,799 WE REALIZED THAT THE 1268 00:44:45,799 --> 00:44:47,834 INTRACELLULAR DOMAIN, THE ROLE 1269 00:44:47,834 --> 00:44:49,703 TO PLAY BESIDES ALL THE OTHER 1270 00:44:49,703 --> 00:44:53,006 ROLES AT THE GATING PART OF IT, 1271 00:44:53,006 --> 00:44:54,508 IT INFLUENCES THE OVERALL 1272 00:44:54,508 --> 00:44:56,143 MOVEMENT OF THE TRANSMEMBRANE 1273 00:44:56,143 --> 00:44:56,776 HEE LISTS. 1274 00:44:56,776 --> 00:44:58,111 SO THIS IS THE INITIAL VIEW, YOU 1275 00:44:58,111 --> 00:44:59,412 CAN SEE THE BETA SUBUNIT AND 1276 00:44:59,412 --> 00:45:00,780 THIS IS PART OF THE 1277 00:45:00,780 --> 00:45:01,882 INTRACELLULAR DOMAIN SO WE ARE 1278 00:45:01,882 --> 00:45:03,016 REALLY WORKING VERY HARD TO 1279 00:45:03,016 --> 00:45:05,452 TEASE THIS OUT AND ACTUALLY 1280 00:45:05,452 --> 00:45:08,588 CAPTURE MORE OF THIS DOMAIN. 1281 00:45:08,588 --> 00:45:11,358 BUT HERE IS THE POSITIONAL 1282 00:45:11,358 --> 00:45:12,626 DIFFERENCE THIS, IS THE BULK OF 1283 00:45:12,626 --> 00:45:14,261 THE INTRACELLULAR DOMAIN AND IF 1284 00:45:14,261 --> 00:45:15,862 YOU LOOK AT THE MOVEMENTS WITHIN 1285 00:45:15,862 --> 00:45:18,365 THE TRANSMEMBRANE HELIXES, THE 1286 00:45:18,365 --> 00:45:19,966 EXTENT OF MOVEMENTS OF HELIXES 1287 00:45:19,966 --> 00:45:22,202 THAT ARE AWAY FROM THIS BULKY 1288 00:45:22,202 --> 00:45:23,236 INTRACELLULAR DOMAIN ARE MUCH 1289 00:45:23,236 --> 00:45:25,105 MORE THAN THE MOVEMENTS THAT YOU 1290 00:45:25,105 --> 00:45:26,640 SEE IN THE HEE LISTS THAT ARE 1291 00:45:26,640 --> 00:45:27,674 RIGHT BENEATH IT. 1292 00:45:27,674 --> 00:45:30,343 AND THE CONSEQUENCE OF THAT IS 1293 00:45:30,343 --> 00:45:32,045 AN ASYMMETRIC PORE MOVEMENT. 1294 00:45:32,045 --> 00:45:35,282 AND ALSO WE THINK THAT THE AN 1295 00:45:35,282 --> 00:45:36,750 IONIC RECEPTORS HAVE A DIFFERENT 1296 00:45:36,750 --> 00:45:38,818 MOLD OF GATING WITHIN THE 1297 00:45:38,818 --> 00:45:39,553 INTRACELLULAR DOMAIN. 1298 00:45:39,553 --> 00:45:41,154 THIS IS BECAUSE PREVIOUS WORK 1299 00:45:41,154 --> 00:45:42,389 FROM OUR LAB AND OTHERS HAVE 1300 00:45:42,389 --> 00:45:44,958 SHOWN THIS IS FROM SEROTONIN 1301 00:45:44,958 --> 00:45:46,359 RECEPTORS AS A CHANNEL GATES, 1302 00:45:46,359 --> 00:45:48,595 YOU HAVE THE OPENING OF THESE 1303 00:45:48,595 --> 00:45:49,896 INTRACELLULAR DOMAIN LEADING TO 1304 00:45:49,896 --> 00:45:52,866 CREATION OF THESE PORTALS 1305 00:45:52,866 --> 00:45:54,301 THROUGH WHICH IONS GO THROUGH. 1306 00:45:54,301 --> 00:45:55,769 IN THIS CASE IT'S NOT A DIRECT 1307 00:45:55,769 --> 00:45:57,270 PORTAL AND THIS IS A PRETTY 1308 00:45:57,270 --> 00:45:58,338 INTERESTING ARCHITECTURE THAT WE 1309 00:45:58,338 --> 00:46:00,273 WANT TO DIG FURTHER INTO. 1310 00:46:00,273 --> 00:46:03,276 AND SO THIS IS THE ASYMMETRY OF 1311 00:46:03,276 --> 00:46:04,444 THE PORE THAT I'M TALKING ABOUT. 1312 00:46:04,444 --> 00:46:08,081 SO HAVING AN ICD RIGHT HERE, 1313 00:46:08,081 --> 00:46:10,350 THIS IS NO MORE A SYMMETRICAL 1314 00:46:10,350 --> 00:46:15,288 MOVEMENT OF THE PORE HE HELIXES. 1315 00:46:15,288 --> 00:46:18,625 THERE WERE DIFFERENCES IN THE 1316 00:46:18,625 --> 00:46:23,163 LIPID DENSITIES, PREVIOUSLY IT 1317 00:46:23,163 --> 00:46:24,798 HAS BEEN SHOWN THAT CERTAIN 1318 00:46:24,798 --> 00:46:26,333 MODULATORS DO NOT BIND TO THE 1319 00:46:26,333 --> 00:46:27,634 BETA SUBUNIT. 1320 00:46:27,634 --> 00:46:28,768 AND IVERMECTIN WAS PROPOSED TO 1321 00:46:28,768 --> 00:46:31,538 BE ONE OF THEM SO IVERMECTIN 1322 00:46:31,538 --> 00:46:33,807 IS -- IT'S A PRETTY STRONG 1323 00:46:33,807 --> 00:46:35,175 POTENTIATION OF -- POTENTIATOR 1324 00:46:35,175 --> 00:46:37,577 OF GLYCINE RECEPTORS, POE 10 1325 00:46:37,577 --> 00:46:39,312 SHAITS LOW GLYCINE 1326 00:46:39,312 --> 00:46:40,180 CONCENTRATIONS, UNIQUE IN THAT 1327 00:46:40,180 --> 00:46:41,348 AT HIGHER CONCENTRATION IT CAN 1328 00:46:41,348 --> 00:46:42,482 ACT AS AN ACTIVATOR. 1329 00:46:42,482 --> 00:46:43,650 SO IT'S KNOWN THAT IT BINDS 1330 00:46:43,650 --> 00:46:46,186 WITHIN THE TRANSMEMBRANE DOMAIN 1331 00:46:46,186 --> 00:46:48,788 PREVIOUSLY AND THE LAB HAS SHOWN 1332 00:46:48,788 --> 00:46:49,122 THIS. 1333 00:46:49,122 --> 00:46:51,157 SO THIS IS WHERE IVERMECTIN 1334 00:46:51,157 --> 00:46:51,358 BINDS. 1335 00:46:51,358 --> 00:46:52,025 IT BINDS AT THE INTERFACE 1336 00:46:52,025 --> 00:46:53,593 BETWEEN THE TWO SUBUNITS AND 1337 00:46:53,593 --> 00:46:55,662 DIRECTLY CONTACTS M2 WHICH FORMS 1338 00:46:55,662 --> 00:46:57,831 THE PORE OF THE CHANNEL. 1339 00:46:57,831 --> 00:47:01,835 AND THEN IT SORT OF ACTIVATES 1340 00:47:01,835 --> 00:47:02,469 THE CHANNEL. 1341 00:47:02,469 --> 00:47:05,105 SO IT WAS PREVIOUSLY SUGGESTED 1342 00:47:05,105 --> 00:47:06,706 THAT SO AS IT INSERTS, THIS IS 1343 00:47:06,706 --> 00:47:07,574 LOOKING AT IT FROM THE TOP, 1344 00:47:07,574 --> 00:47:10,343 THERE IS AN ALANINE RES DUAL IN 1345 00:47:10,343 --> 00:47:13,213 THE ALPHA SUBUNIT WHEREAS THIS 1346 00:47:13,213 --> 00:47:15,148 RESIDUE IS ISOLEUCINE IN THE 1347 00:47:15,148 --> 00:47:15,615 BETA SUBUNIT. 1348 00:47:15,615 --> 00:47:18,084 SO BECAUSE OF THIS BULKINESS, IT 1349 00:47:18,084 --> 00:47:19,386 WAS PROPOSED THAT IT LIKELY 1350 00:47:19,386 --> 00:47:21,121 DOESN'T BIND THIS BETA SUBUNIT, 1351 00:47:21,121 --> 00:47:25,058 AND AGAIN, I THINK A NICE 1352 00:47:25,058 --> 00:47:26,860 FEATURE ABOUT THIS IS THAT 1353 00:47:26,860 --> 00:47:31,064 IVERMECTIN IS AN ANTIPARASITIC 1354 00:47:31,064 --> 00:47:32,198 DRUG AND IN NEMATODES WHERE 1355 00:47:32,198 --> 00:47:35,735 THERE IS A CHANNEL THAT IS 1356 00:47:35,735 --> 00:47:38,138 GLUTAMATE GATED, CHLORIDE 1357 00:47:38,138 --> 00:47:39,572 CHANNEL ACTIVATED VERY STRONGLY 1358 00:47:39,572 --> 00:47:41,474 BY IVERMECTIN AND THIS RIGHT 1359 00:47:41,474 --> 00:47:42,609 HERE IS A GLYCINE, SO YOU MAKE 1360 00:47:42,609 --> 00:47:44,077 THIS RESIDUE SMALLER, YOU HAVE A 1361 00:47:44,077 --> 00:47:45,679 GREATER POTENTIATION WITH 1362 00:47:45,679 --> 00:47:46,012 IVERMECTIN. 1363 00:47:46,012 --> 00:47:49,482 SO AGAIN, BUT WHAT WE FOUND IS 1364 00:47:49,482 --> 00:47:50,450 THAT IVERMECTIN BINDS INTO ALL 1365 00:47:50,450 --> 00:47:53,186 OF THE INTERFACES, BUT HAS A 1366 00:47:53,186 --> 00:47:56,256 UNIQUE ORIENTATION IN THE BETA 1367 00:47:56,256 --> 00:47:57,257 SUBUNIT INTERFACE BECAUSE OF 1368 00:47:57,257 --> 00:48:00,593 THIS PUSHING OVER IT AND VEGING 1369 00:48:00,593 --> 00:48:02,028 OUT THE IVERMECTIN MOLECULE. 1370 00:48:02,028 --> 00:48:05,465 SO HERE IS A TOP VIEW THAT YOU 1371 00:48:05,465 --> 00:48:07,500 CAN SEE, THIS IS IN THE ALPHA 1372 00:48:07,500 --> 00:48:10,904 VERSUS BETA SUBUNIT. 1373 00:48:10,904 --> 00:48:13,873 THIS BINDING MOLD HAS A WEAKER 1374 00:48:13,873 --> 00:48:15,108 AFFINITY FOR GLYCINE. 1375 00:48:15,108 --> 00:48:17,677 AND SO WE THEN ASKED, OKAY, IF 1376 00:48:17,677 --> 00:48:20,146 THIS IS THE CASE, IF IVERMECTIN 1377 00:48:20,146 --> 00:48:22,282 IS A LIGAND THAT HAS ITS OWN 1378 00:48:22,282 --> 00:48:23,683 ACTIVATION PATHWAY, SO INSTEAD 1379 00:48:23,683 --> 00:48:25,318 OF ACTIVATION ORIGINATING AT THE 1380 00:48:25,318 --> 00:48:26,720 LEVEL OF THE EXTRACELLULAR 1381 00:48:26,720 --> 00:48:28,355 DOMAIN MOVING INTO THE 1382 00:48:28,355 --> 00:48:29,022 TRANSMEMBRANE REGION, NOW YOU 1383 00:48:29,022 --> 00:48:31,858 HAVE A PATH THAT GOES FROM 1384 00:48:31,858 --> 00:48:33,493 TRANSMEMBRANE DOMAIN, SO CAN WE 1385 00:48:33,493 --> 00:48:37,330 USE THIS TO UNDERSTAND WHETHER 1386 00:48:37,330 --> 00:48:39,165 THERE ARE BINDING AFFINITY 1387 00:48:39,165 --> 00:48:41,000 DIFFERENCES, ARE THERE -- I 1388 00:48:41,000 --> 00:48:43,002 MEAN, CAN WE CAPTURE 1389 00:48:43,002 --> 00:48:43,737 INTERMEDIATES BECAUSE WE'VE BEEN 1390 00:48:43,737 --> 00:48:45,739 ONLY TO GET THESE STATES EITHER 1391 00:48:45,739 --> 00:48:48,308 CLOSED, OPEN OR DESENSITIZED. 1392 00:48:48,308 --> 00:48:50,643 SO WHAT WE DECIDED TO DO IS WE 1393 00:48:50,643 --> 00:48:57,851 ADDED -- THEN WE ADD IVERMECTIN 1394 00:48:57,851 --> 00:49:00,587 TO BASICALLY LOOK AT BINDING 1395 00:49:00,587 --> 00:49:02,489 MODES AND ALSO LOOK AT 1396 00:49:02,489 --> 00:49:03,556 INTERMEDIATES IF POSSIBLE. 1397 00:49:03,556 --> 00:49:08,628 SO IN THIS PURSUIT, SO WE HAVE 1398 00:49:08,628 --> 00:49:15,869 IVERMECTIN WITH STRYCHNINE, THIS 1399 00:49:15,869 --> 00:49:17,904 IS THE BENCHMARKING, THIS IS IN 1400 00:49:17,904 --> 00:49:19,939 THE PRESENCE OF GLYCINE 1401 00:49:19,939 --> 00:49:22,742 IVERMECTIN, YOU SEE THESE 1402 00:49:22,742 --> 00:49:26,146 BINDING MODES, YOU CAN SEE THIS 1403 00:49:26,146 --> 00:49:26,913 DISTINCT MODE HERE. 1404 00:49:26,913 --> 00:49:30,116 SO AS YOU START TO DECREASE 1405 00:49:30,116 --> 00:49:31,718 IVERMECTIN CONCENTRATION, WE 1406 00:49:31,718 --> 00:49:34,754 START TO LOSE ON THE OCCUPANCY 1407 00:49:34,754 --> 00:49:37,390 FIRST IN THE BETA INTERFACE BETA 1408 00:49:37,390 --> 00:49:39,092 ALPHA INTERFACE, THEN IN ALPHA 1409 00:49:39,092 --> 00:49:42,429 BETA, AND THEN EVENTUALLY IN THE 1410 00:49:42,429 --> 00:49:43,797 ALPHA-ALPHA INTERFACES, 1411 00:49:43,797 --> 00:49:46,332 SUGGESTING HIGH AFFINITY BINDING 1412 00:49:46,332 --> 00:49:47,600 MOLD WHICH SHIFTS DOWN AS WE GO 1413 00:49:47,600 --> 00:49:48,968 TO BINDING INTERFACES. 1414 00:49:48,968 --> 00:49:51,538 AND THEN WE CAN ALSO LOOK AT THE 1415 00:49:51,538 --> 00:49:52,672 CONFORMATION SO THIS IS SIMPLY 1416 00:49:52,672 --> 00:49:53,873 LOOKING AT PROJECTION, THIS IS 1417 00:49:53,873 --> 00:49:57,143 RESTING, THIS IS ACTIVATED 1418 00:49:57,143 --> 00:49:58,778 STATE, AND THESE HEE LISTS AND 1419 00:49:58,778 --> 00:50:00,146 STRANDS MEANS THAT THEY LOOK 1420 00:50:00,146 --> 00:50:01,614 MORE LIKE STRICK NEEN AS THEY 1421 00:50:01,614 --> 00:50:03,216 BECOME MORE RED AND THICKER, 1422 00:50:03,216 --> 00:50:06,953 THEY LOOK MORE LIKE THE OPEN 1423 00:50:06,953 --> 00:50:11,124 CONFORMATION. 1424 00:50:11,124 --> 00:50:13,126 AS YOU INCREASE CONCENTRATIONS, 1425 00:50:13,126 --> 00:50:15,128 YOU START TO MOVE TOWARDS THE 1426 00:50:15,128 --> 00:50:16,463 ACTIVATED STATE RIGHT HERE. 1427 00:50:16,463 --> 00:50:18,064 BUT WHAT'S INTERESTING IS MOST 1428 00:50:18,064 --> 00:50:19,866 OF THE CONFORMATIONS WITHIN THE 1429 00:50:19,866 --> 00:50:21,201 TRANSMEMBRANE DOMAIN HAS ALREADY 1430 00:50:21,201 --> 00:50:25,672 HAPPENED SO EVEN WITH PARTIAL 1431 00:50:25,672 --> 00:50:26,606 IVERMECTIN OCCUPANCIES, YOU HAVE 1432 00:50:26,606 --> 00:50:27,674 A FULLY ACTIVATED STATE. 1433 00:50:27,674 --> 00:50:29,175 BUT THE RESPONSE IN THE 1434 00:50:29,175 --> 00:50:31,544 EXTRACELLULAR DOMAIN IS MORE 1435 00:50:31,544 --> 00:50:32,479 NUANCED AS YOU CAN SEE SOME OF 1436 00:50:32,479 --> 00:50:33,780 THESE REGIONS ARE ACTIVATED, 1437 00:50:33,780 --> 00:50:35,582 SOME OF THEM ARE NOT, LIKELY 1438 00:50:35,582 --> 00:50:36,449 SUGGESTING INTERMEDIATES WITHIN 1439 00:50:36,449 --> 00:50:40,987 THE GATING PATHWAY. 1440 00:50:40,987 --> 00:50:42,255 SO HERE IS A MOVIE THAT 1441 00:50:42,255 --> 00:50:42,689 SUMMARIZES THIS. 1442 00:50:42,689 --> 00:50:44,290 SO YOU HAVE THIS MOVEMENT, THERE 1443 00:50:44,290 --> 00:50:46,459 IS NO IVERMECTIN OCCUPANCY YET. 1444 00:50:46,459 --> 00:50:48,328 YOU CAN SEE THERE'S NOT MUCH 1445 00:50:48,328 --> 00:50:49,762 CHANGE, THEN AS YOU MOVE, YOU 1446 00:50:49,762 --> 00:50:51,764 CAN SEE THE FIRST IVERMECTIN 1447 00:50:51,764 --> 00:50:53,032 COMES IN AND THEN AS YOU START 1448 00:50:53,032 --> 00:50:55,602 TO SEE HIGHER BINDING AFFINITY 1449 00:50:55,602 --> 00:50:57,203 MODES, THEN THE CHANNEL MOVES TO 1450 00:50:57,203 --> 00:50:57,604 IT. 1451 00:50:57,604 --> 00:50:59,939 SO IF YOU ALIGN THEM, YOU EITHER 1452 00:50:59,939 --> 00:51:01,741 SEE THEY'RE ACTIVATED OR THEY'RE 1453 00:51:01,741 --> 00:51:05,945 NOT, SO THERE IS FULL 1454 00:51:05,945 --> 00:51:09,282 COOPERATIVITY IN THIS CASE. 1455 00:51:09,282 --> 00:51:10,817 SO THIS IS STRYCHNINE AND YOU 1456 00:51:10,817 --> 00:51:13,853 CAN ALSO SEE AS YOU INCREASE 1457 00:51:13,853 --> 00:51:14,687 IVERMECTIN STRYCHNINES, 1458 00:51:14,687 --> 00:51:16,656 ORIENTATION STARTS TO CHANGE AND 1459 00:51:16,656 --> 00:51:20,293 MOVE TOWARDS LOW AFFINITY MODES. 1460 00:51:20,293 --> 00:51:21,961 SO AGAIN, THESE ARE SOME OF THE 1461 00:51:21,961 --> 00:51:23,763 DIFFERENT, WE BELIEVE, GATING 1462 00:51:23,763 --> 00:51:25,798 INTERMEDIATES IN THIS ACTIVATION 1463 00:51:25,798 --> 00:51:26,266 PATHWAY. 1464 00:51:26,266 --> 00:51:27,600 ANOTHER WAY TO REPRESENT HERE, 1465 00:51:27,600 --> 00:51:29,802 THIS IS JUST ALONG THE LENGTH OF 1466 00:51:29,802 --> 00:51:31,638 THE SEQUENCE, SO CHANGES HERE IN 1467 00:51:31,638 --> 00:51:32,939 THE TRANSMEMBRANE DOMAIN ARE 1468 00:51:32,939 --> 00:51:34,707 ALMOST ALL OR NONE WHEREAS HERE 1469 00:51:34,707 --> 00:51:37,443 YOU SEE VERY MANY GREY 1470 00:51:37,443 --> 00:51:38,545 INTERMEDIATES WITHIN THE 1471 00:51:38,545 --> 00:51:39,212 EXTRACELLULAR DOMAIN. 1472 00:51:39,212 --> 00:51:43,216 SO THIS IS SORT OF -- CONCLUDES 1473 00:51:43,216 --> 00:51:45,051 WHAT I WANTED TO SAY HERE. 1474 00:51:45,051 --> 00:51:46,853 WE ARE JUST STARTING TO 1475 00:51:46,853 --> 00:51:49,055 UNDERSTAND THE BASIS FOR THESE 1476 00:51:49,055 --> 00:51:50,490 SUBTYPE SPECIFICITY, WE ARE 1477 00:51:50,490 --> 00:51:51,724 STILL SCRATCHING THE TIP OF THE 1478 00:51:51,724 --> 00:51:52,559 ICEBERG HERE. 1479 00:51:52,559 --> 00:51:55,662 AND HOPEFULLY WE CAN USE THESE 1480 00:51:55,662 --> 00:51:58,865 UNDERSTANDING TO LOOK AT OTHER 1481 00:51:58,865 --> 00:51:59,198 MODULATORS. 1482 00:51:59,198 --> 00:52:00,934 SO A GENERAL SUMMARY IS THAT 1483 00:52:00,934 --> 00:52:04,571 THESE CRYO-EM STRUCTURES REVEAL 1484 00:52:04,571 --> 00:52:06,873 THESE INTRICATE MOVEMENT THAT 1485 00:52:06,873 --> 00:52:08,007 UNDERLIE FUNCTIONAL BEHAVIORS 1486 00:52:08,007 --> 00:52:09,776 AND MODULATIONS, AND WE CAN 1487 00:52:09,776 --> 00:52:12,912 START TO TEASE OUT ASYNCHRONOUS 1488 00:52:12,912 --> 00:52:14,380 MOVEMENTS THAT ARE PART OF THESE 1489 00:52:14,380 --> 00:52:17,150 INDIVIDUAL DOMAINS, AND I THINK 1490 00:52:17,150 --> 00:52:18,685 THE BIG PART OF -- WE WANT TO 1491 00:52:18,685 --> 00:52:19,852 UNDERSTAND MODULATION, WE WANT 1492 00:52:19,852 --> 00:52:21,487 TO UNDERSTAND HOW THEY ARE 1493 00:52:21,487 --> 00:52:24,090 MODULATED BY LIPIDS AND LIPID 1494 00:52:24,090 --> 00:52:26,092 MODULATORS AND COMPOUNDS THAT 1495 00:52:26,092 --> 00:52:29,762 HAVE GOOD THERAPEUTIC PROFILES. 1496 00:52:29,762 --> 00:52:31,230 SO WITH THAT, I WANT TO 1497 00:52:31,230 --> 00:52:32,198 ACKNOWLEDGE THE PEOPLE WHO HAVE 1498 00:52:32,198 --> 00:52:34,334 DONE ALL OF THIS WORK, 1499 00:52:34,334 --> 00:52:35,935 PARTICULARLY ALL THE PAST 1500 00:52:35,935 --> 00:52:37,437 MEMBERS WHO HAVE MOVED ON AND 1501 00:52:37,437 --> 00:52:39,038 THIS IS OUR CURRENT TEAM AND 1502 00:52:39,038 --> 00:52:41,307 HAVE ACKNOWLEDGED INDIVIDUAL 1503 00:52:41,307 --> 00:52:43,142 PEOPLE AS WE WENT THROUGH THIS 1504 00:52:43,142 --> 00:52:48,147 IS THE WORK OF -- THAT I DIDN'T 1505 00:52:48,147 --> 00:52:50,016 TALK ABOUT, CRYO-EM ACCESS, WE 1506 00:52:50,016 --> 00:52:51,150 ARE FORTUNATE TO HAVE LOCAL 1507 00:52:51,150 --> 00:52:53,353 ACCESS BUT WE RETEENLY USE 1508 00:52:53,353 --> 00:52:58,558 NATIONAL SENATONATIONAL SENATOR, 1509 00:52:58,558 --> 00:53:00,293 THANK YOU FOR LISTENING AND 1510 00:53:00,293 --> 00:53:04,564 HAPPY TO TAKE QUESTIONS. 1511 00:53:04,564 --> 00:53:06,232 >> I ENCOURAGE PEOPLE TO TAKE 1512 00:53:06,232 --> 00:53:07,033 THE MICROPHONES. 1513 00:53:07,033 --> 00:53:08,701 THERE ARE NO QUESTIONS ONLINE 1514 00:53:08,701 --> 00:53:09,002 YET. 1515 00:53:09,002 --> 00:53:11,170 WE DO HAVE A ROBUST AUDIENCE. 1516 00:53:11,170 --> 00:53:14,741 THE CME CODE, 57663. 1517 00:53:14,741 --> 00:53:23,182 PLEASE USE THE MICS. 1518 00:53:23,182 --> 00:53:25,585 >> BEAUTIFUL TALK. 1519 00:53:25,585 --> 00:53:28,454 I WAS VERY INTRIGUED BY SORT OF 1520 00:53:28,454 --> 00:53:30,156 THE CHANGE IN THE CAVITY VOLUMES 1521 00:53:30,156 --> 00:53:32,759 THAT YOU ALLUDED TO. 1522 00:53:32,759 --> 00:53:35,428 AND I'M IMAGINING THAT AS THESE 1523 00:53:35,428 --> 00:53:37,163 PROTEINS IN GENERAL UNDERGO 1524 00:53:37,163 --> 00:53:38,631 CONFORMATIONAL CHANGES NOT ONLY 1525 00:53:38,631 --> 00:53:40,233 DO YOU CHANGE THE VOLUMES OF 1526 00:53:40,233 --> 00:53:42,869 THESE CAVITIES, YOU ALSO 1527 00:53:42,869 --> 00:53:44,470 POTENTIALLY CHANGE THE SURFACE 1528 00:53:44,470 --> 00:53:46,239 CHEMISTRY OF THESE CALFITIES. 1529 00:53:46,239 --> 00:53:48,441 SO IN PRINCIPLE, THAT MEANS THAT 1530 00:53:48,441 --> 00:53:49,809 OPENS UP THE POSSIBILITY OF 1531 00:53:49,809 --> 00:53:52,178 THESE PROTEINS BEING REGULATED 1532 00:53:52,178 --> 00:53:54,447 BY PHOSPHOLIPIDS THAT ARE IN THE 1533 00:53:54,447 --> 00:53:54,781 MEMBRANE. 1534 00:53:54,781 --> 00:53:56,916 IS THERE ANY WAY TO TEASE THIS 1535 00:53:56,916 --> 00:53:58,284 APART, DO YOU THINK THIS IS JUST 1536 00:53:58,284 --> 00:53:58,985 A FANTASY? 1537 00:53:58,985 --> 00:54:01,521 CAN YOU SORT OF EDUCATE ME ON 1538 00:54:01,521 --> 00:54:01,721 THIS? 1539 00:54:01,721 --> 00:54:03,990 >> I MEAN, ABSOLUTELY 1540 00:54:03,990 --> 00:54:05,024 FASCINATING QUESTION. 1541 00:54:05,024 --> 00:54:07,060 AND ALSO A CHALLENGE FOR A 1542 00:54:07,060 --> 00:54:07,593 STRUCTURAL BIOLOGIST. 1543 00:54:07,593 --> 00:54:09,729 THIS IS OUR FIELD AND WE 1544 00:54:09,729 --> 00:54:11,230 STRUGGLE TO TEASE THIS APART, 1545 00:54:11,230 --> 00:54:11,431 RIGHT? 1546 00:54:11,431 --> 00:54:13,199 SO THESE, I MEAN, WHAT YOU POINT 1547 00:54:13,199 --> 00:54:14,167 OUT IS VERY IMPORTANT. 1548 00:54:14,167 --> 00:54:16,035 IT'S NOT JUST THE VOLUME, IT'S 1549 00:54:16,035 --> 00:54:16,836 THE CHEMISTRY OF THIS. 1550 00:54:16,836 --> 00:54:19,472 SO SOME OF THESE POCKETS ARE 1551 00:54:19,472 --> 00:54:20,773 HYDROPHOBIC AS THEY MOVE OUT AND 1552 00:54:20,773 --> 00:54:25,311 REORIENT THE HELICES, AND ONCE 1553 00:54:25,311 --> 00:54:27,513 THE CHANNEL AT A NORMAL GATING 1554 00:54:27,513 --> 00:54:29,015 CONDITIONS AS THEY OPEN AND 1555 00:54:29,015 --> 00:54:30,950 THESE CAVITIES OPEN UP, IT'S NOT 1556 00:54:30,950 --> 00:54:31,284 IN VACUUM. 1557 00:54:31,284 --> 00:54:32,585 YOU HAVE LIPIDS GETTING THERE 1558 00:54:32,585 --> 00:54:35,321 AND THOSE LIPIDS ARE NATURAL 1559 00:54:35,321 --> 00:54:36,789 REGULATORS OF CHANNEL FUNCTION. 1560 00:54:36,789 --> 00:54:39,392 AND NOW YOU PUT A MODULATOR THAT 1561 00:54:39,392 --> 00:54:41,027 LOOKS LIKE SOME OF THESE LIPIDS, 1562 00:54:41,027 --> 00:54:42,395 HOW DO YOU DISTINGUISH BETWEEN 1563 00:54:42,395 --> 00:54:43,963 THAT? 1564 00:54:43,963 --> 00:54:45,698 SO I THINK THAT'S A CHALLENGE 1565 00:54:45,698 --> 00:54:48,868 AND ALSO WHEN YOU WORK WITH THE 1566 00:54:48,868 --> 00:54:49,969 MINIMALISTIC SYSTEM, WE REALLY 1567 00:54:49,969 --> 00:54:51,771 DON'T HAVE THOSE LIPIDS, THEN WE 1568 00:54:51,771 --> 00:54:54,340 PUT THE MODULATOR IN, SO THEIR 1569 00:54:54,340 --> 00:54:55,208 AFFINITYS ARE PROBABLY 1570 00:54:55,208 --> 00:54:55,475 DIFFERENT. 1571 00:54:55,475 --> 00:54:59,545 SO I THINK THOSE ARE SOME OF THE 1572 00:54:59,545 --> 00:55:02,715 CHALLENGES, CAN WE USE MASS SPEC 1573 00:55:02,715 --> 00:55:04,650 TO START IDENTIFYING WHAT THOSE 1574 00:55:04,650 --> 00:55:06,452 LIPIDS ARE SO THEN CAN YOU START 1575 00:55:06,452 --> 00:55:07,487 INCORPORATING THEM INTO OUR 1576 00:55:07,487 --> 00:55:08,221 STRUCTURAL STUDIES. 1577 00:55:08,221 --> 00:55:11,190 BUT I THINK INTERESTING AND 1578 00:55:11,190 --> 00:55:13,693 CHALLENGING █PURSUIT THERE. 1579 00:55:13,693 --> 00:55:15,294 >> A COUPLE QUESTIONS ARE COMING 1580 00:55:15,294 --> 00:55:16,062 IN ONLINE. 1581 00:55:16,062 --> 00:55:16,929 OKAY. 1582 00:55:16,929 --> 00:55:20,266 SO DO YOU THINK THE PH 1583 00:55:20,266 --> 00:55:21,934 DEPENDENCE OF ZINC ACTIVATION ON 1584 00:55:21,934 --> 00:55:23,903 THE GLYCINE RECEPTOR IS DUE TO 1585 00:55:23,903 --> 00:55:26,272 THE EYE ONIZATION STATE OF 1586 00:55:26,272 --> 00:55:28,841 HISTIDINE WHICH COORDINATES TO 1587 00:55:28,841 --> 00:55:30,610 THE HIGH AFFINITY ZINC STATE AND 1588 00:55:30,610 --> 00:55:33,946 TO THE LOW AFFINITY ZINC STATE? 1589 00:55:33,946 --> 00:55:37,483 >> SO GREAT QUESTION. 1590 00:55:37,483 --> 00:55:39,652 SO ABOUT THE PH EFFECTS, AND THE 1591 00:55:39,652 --> 00:55:41,687 IONIZABLE RESIDUES AND WHAT 1592 00:55:41,687 --> 00:55:42,755 IS -- WHAT ARE THE RESIDUES THAT 1593 00:55:42,755 --> 00:55:44,257 ARE MEDIATING THIS, IT'S UNCLEAR 1594 00:55:44,257 --> 00:55:46,425 AT THIS POINT. 1595 00:55:46,425 --> 00:55:47,527 BUT THERE ARE SEVERAL RESIDUES 1596 00:55:47,527 --> 00:55:49,962 THAT HAVE BEEN IMPLICATED IN PH 1597 00:55:49,962 --> 00:55:51,430 DEPENDENCE. 1598 00:55:51,430 --> 00:55:53,099 SO HISTIDINES IN THE 1599 00:55:53,099 --> 00:55:54,834 POTENTIATING SITES ARE LIKELY TO 1600 00:55:54,834 --> 00:55:56,936 BE SOME OF THEM BECAUSE WHEN YOU 1601 00:55:56,936 --> 00:55:59,105 HAVE ACIDIC PH ZINC MODULATION 1602 00:55:59,105 --> 00:55:59,872 GOES AWAY. 1603 00:55:59,872 --> 00:56:04,076 ZINC POTENTIATION GOES AWAY. 1604 00:56:04,076 --> 00:56:05,912 SO IT IS LIKELY THOSE HISTIDINES 1605 00:56:05,912 --> 00:56:06,913 PLAY A ROLE. 1606 00:56:06,913 --> 00:56:12,218 ALSO INTERESTINGLY, SOME -- 1607 00:56:12,218 --> 00:56:14,954 THREE ANINES HAVE BEEN 1608 00:56:14,954 --> 00:56:17,056 IMPLICATED, THAT THEY ELIMINATE 1609 00:56:17,056 --> 00:56:19,759 PH -- OR ACIDIC PH INHIBITION. 1610 00:56:19,759 --> 00:56:21,027 IT'S UNCLEAR WHAT THEY'RE DOING 1611 00:56:21,027 --> 00:56:22,762 THERE BUT IT'S ALSO POSSIBLE 1612 00:56:22,762 --> 00:56:24,397 THAT IT'S AN ALLOSTERIC EFFECT 1613 00:56:24,397 --> 00:56:26,299 THAT YOU MAKE A MUTATION, IT IS 1614 00:56:26,299 --> 00:56:28,968 CHANGING THE WAY THE HISTIDINE 1615 00:56:28,968 --> 00:56:30,736 IS SEEN IN OTHER INTERACTIONTION 1616 00:56:30,736 --> 00:56:31,504 AND IT COULD BE POSSIBLE BECAUSE 1617 00:56:31,504 --> 00:56:32,038 OF THAT. 1618 00:56:32,038 --> 00:56:33,940 BUT FOR SURE THE POTENTIATING 1619 00:56:33,940 --> 00:56:36,442 RESIDUES HAVE A ROLE DIRECTLY. 1620 00:56:36,442 --> 00:56:38,277 >> ANOTHER QUESTION. 1621 00:56:38,277 --> 00:56:41,080 IS THE BINDING CONSTANT KD KNOWN 1622 00:56:41,080 --> 00:56:45,651 FOR THE ZINC OR OTHER LIGANDS? 1623 00:56:45,651 --> 00:56:47,353 >> YEAH, SO THE BINDING -- THE 1624 00:56:47,353 --> 00:56:48,554 BUY LYE GANDZ THAT I WAS TALKING 1625 00:56:48,554 --> 00:56:51,023 ABOUT, AGAIN, THE KDs VARY 1626 00:56:51,023 --> 00:56:54,427 QUITE A BIT, SO FOR THE ZINC, AT 1627 00:56:54,427 --> 00:56:56,629 THE LOWER -- SO FOR SURE THERE 1628 00:56:56,629 --> 00:56:58,464 ARE LOW MICROMOLARS FOR THE 1629 00:56:58,464 --> 00:57:02,101 POTENTIATING SITES, AND HIGHER 1630 00:57:02,101 --> 00:57:04,003 FOR THE INHIBITORY SITE. 1631 00:57:04,003 --> 00:57:06,439 AGAIN IT DEPENDS ON THE TYPE OR 1632 00:57:06,439 --> 00:57:08,608 SUBTYPE THAT YOU'RE LOOKING AT, 1633 00:57:08,608 --> 00:57:10,710 THE ALPHA 3s AND THE 300 1634 00:57:10,710 --> 00:57:11,944 MICROMOLAR RANGE FOR THE 1635 00:57:11,944 --> 00:57:14,046 INHIBITORY ONE. 1636 00:57:14,046 --> 00:57:15,815 >> PROFOUND CLOSING COMMENTS? 1637 00:57:15,815 --> 00:57:17,884 >> ONE LAST QUESTION. 1638 00:57:17,884 --> 00:57:19,552 SO DO HEAVY METALS BIND TO THE 1639 00:57:19,552 --> 00:57:20,119 ZINC SITES? 1640 00:57:20,119 --> 00:57:20,853 >> ALSO, YES. 1641 00:57:20,853 --> 00:57:22,889 >> DOES THAT -- DOES THAT HAVE 1642 00:57:22,889 --> 00:57:26,092 ANYTHING TO DO WITH LEAD OR 1643 00:57:26,092 --> 00:57:27,059 MERCURY NEUROTOXICITY? 1644 00:57:27,059 --> 00:57:27,426 >> YEAH. 1645 00:57:27,426 --> 00:57:29,262 I MEAN, IT IS POSSIBLE. 1646 00:57:29,262 --> 00:57:32,231 AGAIN, THE SITE IS PRIME FOR 1647 00:57:32,231 --> 00:57:33,366 BINDING OTHER -- AND IT'S KNOWN 1648 00:57:33,366 --> 00:57:35,534 THAT SOME OF THESE HAVE, BUT IN 1649 00:57:35,534 --> 00:57:39,038 TERMS OF WHETHER THEY LEAD TO 1650 00:57:39,038 --> 00:57:40,740 NEUROTOXICITY AND THAT KIND OF 1651 00:57:40,740 --> 00:57:46,946 THING UNCLEAR, BUT IT IS 1652 00:57:46,946 --> 00:57:48,514 POSSIBLE, IT'S NOT EXCLUSIVE TO 1653 00:57:48,514 --> 00:57:51,117 ZINC, BUT ITS DIRECT ROLE, NOT 1654 00:57:51,117 --> 00:57:51,317 SURE. 1655 00:57:51,317 --> 00:57:52,084 >> FANTASTIC. 1656 00:57:52,084 --> 00:57:54,654 IF THERE ARE NO OTHER QUESTIONS, 1657 00:57:54,654 --> 00:57:56,656 THEN LET'S THANK SUDHA FOR A 1658 00:57:56,656 --> 00:57:57,156 FANTASTIC TALK. 1659 00:57:57,156 --> 00:57:59,892 [APPLAUSE] 1660 00:57:59,892 --> 00:58:01,594 OH, AND DON'T FORGET THERE'S A 1661 00:58:01,594 --> 00:58:02,228 RECEPTION OUTSIDE. 1662 00:58:02,228 --> 00:58:12,538 SO PLEASE JOIN US.