ALL RIGHT. GOOD AFTERNOON. I'M DELIGHTED TO WELCOME YOU TO THIS YEAR'S ROBERT WHITNEY NEWCOMB MEMORIAL LECTURESHIP. THIS IS A GREAT PRIVILEGE FOR US. THIS ANNUAL LECTURE IS PART OF A COUPLE OF ACTIVITIES THAT ARE SUPPORTED BY THE ROBERT WHITNEY MEMORIAL FUND. THIS FUND WAS ESTABLISHED AT THE FOUNDATION FOR THE NIH BY DR. NEWCOMB'S FAMILY TO HONOR HIS MEMORY AND WE'RE GRATEFUL, UM, FOR THE OPPORTUNITIES IT GIVES US IN THE NEUROSCIENCES AT NIH AND PARTICULARLY INç NINDS. DR. NEWCOMB BEGAN HIS SCIENTIFIC CAREER AT THE NIH AT A HIGH SCHOOL STUDENT IN KLEE'S LAB AND THAT WAS EXTREMELY INFLUENTIAL AND DICTATED HIS CHOICE TO BECOME A SCIENTIST. SAME WITH ME. THAT'S TYPICAL, A YOUNG STUDENT COMES TO OUR LABS AND THEIR LIVES GET CHANGES. THE FUND FUNDS SOME STUDENTS. SO THE HIGH SCHOOL EXPERIENCE SET HIM OFF TO BE AN UNDERGRADUATE AT THE UNIVERSITY OF COLORADO AND THENv: GRADUATE WORK AT THE UNIVERSITY OF HAWAII WHERE HIS WORK INVOLVED STUDYING INVERTEBRATE NEUROPEPTIDES. HE THEN WENT ON TO A POST DOC WITH RICHARD SCHELLE RESHGS. AN THEN HE BEGINS A CAREER AT NUREX PHARMACEUTICALS AND THERE HIS INTEREST CONTINUES IN NEUROTOXINS THAT AFFECT CHANNEL FUNCTION AND IN THE CAUSES OF STROKE AND HOW TO PREVENT STROKES. IN PARTICULAR HE WAS INTERESTED IN GLUTAMATES IN CAUSING AND PROTECTING AGAINST STROKE. THOSE ARE THE THREE MAJOR INTERESTS THAT THE NEWCOMB MEMORIAL RECOGNIZES AND HONORS THOSEñr IN THE AREA OF NEUROPROTECTION DURING STROKE, NEUROPEPTIDES AND TOXIN'S IN CHANNEL FUNCTION. WE'RE HONORED TO HAVE FAMILY MEMBERS FROM THE NEWCOMB FAMILY. MANY OF THEM CONTRIBUTED TO THE FUND. THEY THOUGHT BASED ON THAT EARLY EXPERIENCE AND BECAUSE OF THE OUTSTANDING WORK WE DO HERE THAT THE NIH WAS AN EXCELLENT PLACE TO FURTHER DR. NEWCOMB'S WORK ON BRAIN RESEARCHi] AND STROKE AND CHANNEL FUNCTION. AND WE WELCOME THE MEMBERS OF THE FAMILY; ROBERT NEWCOMB, HIS WIFE SALLY, AND HIS SISTER GAIL, ARE ARE HERE. GAIL IS THE NIH IS A GOOD PLACE FOR THE NEWCOMB FAMILY. GAIL WORKS HERE. HOW MANY YEARS HAVE YOU BEEN? 19 YEARS AT THE NIH, WOW. UM, SO THAT'S SPECTACULAR. SO AS I SAID, THIS IS AN ANNUAL SERIES AND WE'RE VERY HAPPY TO WELCOME TODAY'S SPEAKER, DIANE LIPSCOMBE. SHE'S A PROFESSOR AT BROWN UNIVERSITY, AND SHE WAS JUST EXPLAINED TO ME -- SHE ALSO HAD ANok EARLY EXPERIENCE IN SCIENCE. AFTER HIGH SCHOOL, SHE BECAME A TECHNICIAN IN SIR JAMES BLACK'S LAB AS THE WELCOME RESEARCH LABORATORY AND THAT CEMENTED HER INTEREST IN SCIENCE AND NEUROSCIENCE. SHE WENT OFF TO GET HER BACHELOR DEGREE AT UNIVERSITY COLLEGE, LONDON. HER Ph.D. ALSO AT THE YOOVRT OF COLLEGE LONDON WHERE HE WAS INTERESTED IN ANY KOE PINIC RECEPTORS AND STUDYING THE ROLE OF THE RECEPTORS AND CARDIAC NEURONS IN THE FLOG. AFTER THAT, SHE BECAME A POST DOC FELLOW AT YALE UNIVERSITY WITH RICHARD CHIN AND THAT'S WHERE HER INTEREST IN CALCIUM CHANNELS STARTS AND CONTINUES TODAY. SHE ROSE THROUGH THE RANKS AT BROWN WHERE SHE'S NOW A PROFESSOR. NEAR AND DEAR TO OURzVw3 HEARTS SHAEND HELPS US RUN THE BROWN NIH DTP PROGRAM. SHE'S DIRECTOR ON THE BROWN SIDE. SHE'S WON NUMEROUS HONORS. THE ONE THAT JUMPS UH OUT AT ME IS THE HARRIOT SHARE DONE AWARD FOR DISTINGUISHED CONTRIBUTION TO TEACHING AT BROWN UNIVERSITY. BROWN, OF COURSE IS ONE OF OUR PREMIER UNIVERSITIES AND TO WINw3 A TEACHING AWARD THERE IS QUITE A HIGH HONOR. SHE'S BEEN VERY ACTIVE IN TRAINING GRADUATE STUDENTS AND POST DOCS. I COUNTED ABOUT 30. AND MANY UNDERGRADUATED AS WELL, I COUNTED ABOUT 40 THAT YOU'VE BEEN INVOLVED IN WITH THEIR THESIS ON HELPING THEM OUT. SO DIANE IS A PERFECT FIT TO THE GOALS OF THE NEWCOMB LECTURESHIP. TODAY SHE'LL TALK TO US ABOUT G PROTEIN SIGNALLING TO NEURONAL CALCIUM CHANNELS DIRECTED BY CELL-SPECIFIC ALTERNATIVE SPLICING. DIANE. >>ok OKAY. UM, DOES THIS MICROPHONE WORK IF I MOVE AWAY? . WONDERFUL. THANK YOU, ALLEN. U(URJ AND I ALSO WANT TO THANK THE COMMITTEE WHO INVITED ME AND ALSO GIVE MY THANKS TO THE NEWCOMB FAMILY FOR MAKING THIS POSSIBLE. IT'S A TREMENDOUSZv HONOR AND IT'S REALLY GREAT PRIVILEGE TO BE HERE TALKING TODAY IN THE NEWCOMB LECTURE. SO IT WAS ACTUALLY REALLY NICE THAT YOU MENTIONED THE MENTORSHIP TEACHING AWARD BECAUSE IF I WAS TO PICK OUT ONE AWARD, THAT WOULD PROBABLY THE ONE I'M2íó3 MOST PROUD OF. IT'S -- I'VE REALLY MENTORED A LOT OF STUDENTS AND A FEW OF THEM ARE HERE BECAUSE OF THE BROWN COLLABORATIVE GRADUATE PROGRAM WITH NIH, AND, UM, ALLEN, YOU MENTIONED THAT THESE BEING TRANSFORMATIVE EXPERIENCES FOR STUDENTS, BUT HAVING THOSE KINDS OF STUDENTS IN MY LAB IS INCREDIBLY TRANSFORMATIVE FOR ME TOO BECAUSE THERE'S NOTHING LIKE A QUESTIONING YOUNG, UM, SKEPTIC STUDENT IN YOUR LAB TO KEEP YOU ON YOUR FEET AND I THINK THAT THAT'S REALLY, UM, UH, WHY I KEEP THESE YOUNG PEOPLE COMING THROUGH THE LAB BECAUSE THEY REALLY INFLUENCE TREMENDOUSLY THE RESEARCH. WHAT I'M GOING TO DO IS TO GIVE KIND OF AN WITH, BUT I FIRST WANT TO ACTUALLY ACKNOWLEDGE THE PEOPLE THAT WHO'S WORK I'LL TALK ABOUT AND THEN GO ON AND GIVE AN OVERVIEW AND THEN TALK A LITTLE BIT MORER SPECIFICALLY ABOUT THE RESEARCH THAT WE'RE IN THE LAB. SO THE KEY PLAYERS IN THE WORK THAT I'LL TALK ABOUT SYLVIA,fá [INDISCERNIBLE] SUMMER ALAN, RACHEL, SIS IS I AND JESSICA ARE REALLY THE KEY PEOPLE WHO WORKED IN MY LABS WHO CONTRIBUTED TO THE WORK THAT I'LL TALK ABOUT. OKAY. SO I THINK MANY OF US WHO WORK ON CALCIUM AND CALCIUM CHANNELS HAVE A SLIDE LIKE THIS, BUT IT'S REALLY USEFUL TO REMEMBER, UM, THE EXTRAORDINARY NUMBER OF DIFFERENT PROCESSES, CELLAR PROCESS THAT CALCIUM REGULATES AND ACTIVATES. IT'S REALLY PHENOMENAL HOW DIFFERENT THEY ARE AND THAT THERE'S ONE MOLECULE CAN BE THE TRIGGER TO DIRECT ACT VATHS OF VERY, VERY CELLAR FUNCTIONS AND REALLY QUITE DIFFERENT MEMBRANE EXPRESSION, MEMORY FUNCTION, APOPTOSIS, AND OF GREAT INTEREST TO US ALTHOUGH WE RARELY DIRECTLY STUDY THIS PROCESS IS THE ROLE OF CALCIUM IN CONTROLLING TRANSMITTER RELEASE. SO THIS IS A MACHINE, UH, THAT I THINK ENGINEERS WOULD DROOL OVER TO HAVE THAT IS ABLE TO CONVERT DEPOLARIZATION OF THE MEMBRANE INTO A CHEMICAL SIGNAL WITH INCREDIBLE EFFICIENCY. IT'S AN EXTRAORDINARY [INDISCERNIBLE] OF ELECTRICAL SIGNAL INTO A CHEMICAL SIGNAL. THIS IS THE VOLTAGE CHANNEL OR DEPICTION OF IT, AND THIS TOXIN'S THAT ROBERT WAS SO INFLUENTIAL IN IDENTIFYING TARGET A SUBSET OF THESE VOLT GAITED CALCIUM ION CHANNELS. THEY TRANSPORT CALCIUM WITH INCREDIBLE EFFICIENCY TO THE EXCLUSION OF OTHER [INDISCERNIBLE]. THAT'S THE ROLE TO SENSE A CHANGE IN THE MEMBRANE AND POTENTIALLY TRANSPORT -- ACROSS THE THE MEMBRANE OF EXCITABLE CELLS.b THERE ARE TEN GENES IN MAMMALS THAT ENCODE DIFFERENT CALCIUM CHANNELS. I'M GOING TO GET INTO THE DETAILS IN A SECOND. I WANT TO EMPHASIZE THAT AND USE AN ACTION POTENTIAL WAY FORM TO SHOW YOU THAT CALCIUM [INDISCERNIBLE] CHANNELS WHEN ACTIVATED BY<  MEMBRANE DEPOLARIZATION DRIVE CALCIUM ENTRY IN VERY RAPID WAY SYNCHRONIZED WITH THE MEMBRANE DEPOLARIZATION. IN THIS CASE, I'M SHOWING THE INFLUX OF CALCIUM IN RESPONSE TO THIS ACTION POTENTIAL WAY FORM ORIGINATING FROM ACTIVATION OF THESE CALVE THREE T TYPE CURRENTS. THE POINT OF SHOWING THIS AND THEN THIS NEXT RECORDING IS THAT DIFFERENT SUBTYPES OF CALCIUM CHANNELS HAVE DIFFERENT PROPERTIES AND THAT'S GOING BECOME IMPORTANT LATER. THIS IS THE -- UNDER THE SAME CONDITIONS THIS IS A CALCIUM CURB DRIVEN BY ACTIVATION OF THESE CALVE 2.2 ANTICURRENTS, VERY SIMILAR TO THE TYPE THAT ROBERT NEWCOMB WORKED ON AND YOU CAN SEE THAT THEY HAVE DISTINGUISHING FEATURES AND ONE OF THEIC ONES HERE IS THAT THESE CHANNELS DEACTIVATE OR TURN OFF AFTER THE RETURN OF THE MEMBRANE POTENTIAL VERY RAPIDLY COMPARED TO T TYPE CURRENTS THAT TURN OFF VERY SLOWLY. THERE ARE DIFFERENT PROPERTIES THAT DISTINGUISH DIFFERENT SUBSETS OF CALCIUM CHANNELS AND, UM, HERE THEY ALL ARE. SO THEñrd8 MA ç MA MAIL YMMALIAN -- THESE ARE THE T TYPE CURRENTS, THE P TYPE AND R TYPE THAT THE I'LL COME BACK TO AND THE Rd8 TYPE. THEY'RE FOUND IN DIFFERENT CITY TI SHY AND PERFORM DIFFERENT FUNCTIONS. THE SECOND SLIDE I SHOWED WITH THIS REMARKABLE ABILITY FOR CALCIUM TO MEDIATE THE ACTIVATION OF VERY DIFFERENT CELLAR PROCESSES WELL UNDERLYING THE MOLECULAR EXPLANATION FOR THAT IS THAT THESE DIFFERENT CALCIUM CHANNELS COUPLE TO DIFFERENT DOWNSTREAM SIGNALING AND DIFFERENT DOWNSTREAM CELLAR MACHINERY. SO ONE OF THE HUGE ADVANCES IN THE FIELD IT WAS DEVELOPMENT OF SELECTI SELECTIVE TOXINS THAT ALLOWS US WHO WERE LOOKING AT FUNCTION OF DIFFERENT CALCIUM CHANNELS TO BE ABLE TO DISSECT OUT THESE DIFFERENT CURRENT AND THESE DIFFERENT FAMILIES OF CHANNELS EVEN BEFORE CLONING, A LONG TIME BEFORE CLONING, AND SO THIS IS A FIGURE TAKEN FROM A REVIEW THAT ROBERT NEWCOMB WROTE BACK 2000, IN CNS JOB REVIEWS, SHOWING THE VALUE, TREMENDOUS VALUE OF THESE DIFFERENT TOXINS IN DISSECTING OUT THE DIFFERENT CALL VI YUM CHANNELS AND WE STILL USE THESE TODAY. THIS IS A CALCIUM CURRENT HERE. YOU DON'T SEE THE STIMULUS, BUT THERE WOULD BE A STIMULUS ACTIVATING THIS CURRENT. IN RECORDED FROM [INDISCERNIBLE] GRANULE NEURONS, SO HERE'S THE CURRENT IN THE PRESENCE OF A BLOCKER OF THE CALVE ONE FAMILY TL TYPE CAN CALCIUM CHANNELS, SO THEY'RE NOT CONTRIBUTING TO THIS, BUT YOU CAN SEE THAT THE ADDITION OF THESE TWO TOXINS TOGETHER THAT INHIBIT THE CAV 2 OINT 1 AND 2.2 CHANNELS, EACH OF THESE CHANNELS VA BOUT TEN DIFFERENT NAMES. THESE TWO TOXINS TOGETHER INHIBIT PART OF THE [INDISCERNIBLE] BUT NOT ALL OF IT, AND THEN IT'S THE ADDITION OF THIS COMPOUND WHICH ISzV AN R TYPE SELECTIVE ANTAGONIST THAT ROBERT ISOLATED FROM TA RAN CHA LA VENOM. YOU CAN SEE THAT ADDITION OF THIS TOXIN' INHIBITS ALL OF CURRENT SO THIS COMPONENT WE CAN TELL IS DUE TO THE ACTIVATION OF THIS R TYPE CURRENT. SO THE DEVELOPMENT OF SNX 142 WAS REALLY CRITICAL IN BEING ABLE TO IDENTIFY THE CONTRIBUTION OF THIS PARTICULAR CHANNEL THAT WE NOW KNOW IS ENCODED BY At( COMPLETELY SEPARATE GENE FROM A LOT OF CLONING AND ANALYSIS OF THE GENOME. THE OTHER THING WHICH IS VERY NEAR AND DEAR TO WHAT I DO AND I'LL TRANSITION FROM THIS SLIDE TO TALKING ABOUT SPLICING IS THAT AGAIN WAY BACK -- AND IT'S REALLY WONDERFUL TO HAVE THE OPPORTUNITY TO DO THESE LECTURES BECAUSE YOU DIG IN THE LITERATURE AND YOU READ WHAT PEOPLE DID WAY BEFORE YOU HAD ANY OF THESE IDEA PERHAPS LONG TIME AGO ROBERT NEWCOMB AND HIS COLLEAGUES SPECULATED THAT FOR THIS ONE PARTICULAR CLASS OF CHANNEL THAT PROBABLY ALL THESE OTHER VARIANCE THAT THEY REFERRED TO MAY ACTUALLY EXPLAIN WHY THE TOXIN' WASN'T AS EFFECTIVE IN ONE CLASS OF NEURON COMPARED TO ANOTHER CLASS SO THAT IT MIGHT BE THAT THERE'S ANOTHER LAYER, ANOTHER LEVEL OF GRANULATION OF DIVERSITY WITHIN A GIVEN GENE, WITHIN ONE CHANNEL FAMILY AND THAT THE EXPRESSION [INDISCERNIBLE] THAT ALL SEEM TO ACTkO RATHER SIMILAR TO ONE CLASS OF CHANNEL, UM, MIGHT ACTUALLY ALSO HAVE DIFFERENT ACTIVITIES. OKAY. AND SO THIS IS WHAT I WORK ON AND IT WAS REALLY QUITE WONDERFUL TO SEE ALL OF THIS WORK THAT WENT AHEAD OF WHAT WE DID. SO. UM, SO THIS IS THE GENE TREE THAT I SHOWED YOU BEFORE, BUT I'VE ADDED THESE ARROWS HERE, AND THIS BECOMES A LITTLE TERRIFYING BECAUSE, UH, WE NOW KNOW THAT THERE ARE PROBABLY LITERALLY HUNDREDS OR THOUSANDS OF DIFFERENT FORMS OF CALCIUM CHANNELS THAT CAN ARISE FROM THESE TEN DIFFERENT GENES. NOW, THERE'S THERE INCREDIBLE LAYER OF COMPLEXITY THAT IS INTERESTING BUT THAT WE HAVE TO FIND TOOLS TO BE ABLE TO STUDY. I'M GOING;HáO FOCUS ON THIS GENE, WHICH IS VERY SIMILAR TO THE GENE THAT UNDERLIES THE R TYPE CHANNELS THAT ROBERT NEWCOMB WORKED ON BUT IT'S A SISTER GENE THAT ENCODES THE N TYPE CHANNELS. ALL OF THESE THREE CHANNELS ARE EXPRESSED AT PRESYNAPTIC TERMINALS. SO THIS IS THE CALVE 2.2 PROTEIN ÷í ENCODES N TYPE CURRENTS. AND THEY CONTROL RELEASE AT MANY PRESYNAPTIC TERMINALS AND THIS CHANNEL IS INHIBIT BID THE DIFFERENT TYPE OF TOXIN. SO THIS IS, UM, GIVING YOU A SENSE OF THE LEVEL OF DIVERSITY THAT WE KNOW FOR SURE EXISTS IN THIS GENE FITTING ENCODES THE CALCIUM CHANNEL. SO THIS IS AN ILLUSTRATION OF THE GENE, THE GENE HAS YET A DIFFERENT NAME, BUT THAT'S IRRELEVANT RIGHT NOW. SO THE BLACK BARS SHOW d8 EXONS THAT ARE ALWAYS FOUND THROUGHOUT THE CALCIUM CHANNEL FOUND THROUGHOUT THE NERVOUS SYSTEM. THERE'S THIS BACKBONE STRUCTURE. IN ADDITION, THERE ARE THESE EXONS THAT ARE ALTERNATIVE EXPRESSED. THEY MAY NOT BE INCLUDED IN THE FINAL PRO SEEN PRODUCT OR THE mRNA. IT'S PRETTY SIMPLE TO SEEyM THAT IF THERE ARE FOUR DIFFERENT SITES OF THESE ALTERNATIVELY EXPRESSED EXON SEQUENCES, EXONS, THEN YOU CAN GENERATE 16 DIFFERENT POSSIBLE mRNA ISOFORMS AND PERHAPS 16 DIFFERENT PROTEINS. -- FROM A SINGLE GENE. THERE'S LIKELY TO BE A LARGE NUMBER OF OTHER EXONS WE HAVEN'T IDENTIFIED UP HERE IN THE FIVE PRIME END AND WE KNOW A LOT ABOUT THE THREE PRIME END BECAUSE OF -- AT LEAST IT USED TOç BE THREE PRIME BIAS OF THE DATABASES. THAT'S BEING [INDISCERNIBLE] NOW SO WE'VE GOT A LITTLE BIT MORE INFORMATION UP NEAR THE FIVE  A LOT PRIME END, BUT WE in ABOUT THESE ALTERNATIVE SITES OF SPLICING. WE KNOW THEY'RE TISSUE-SPECIFIC AND WE KNOW FOR AT LEAST THREE OF THESE SITES WHICH SPLICING SITES ARE CONTROLS THEIR EXPRESSIONS. NOT SURPRISINGLY AS WELL THEY'RE FOUND IN OUTSIDE OF THE TRANCE MEMBRANE SPAN IN PART OF THE PROTEIN, ALTHOUGH THERE ARE ALTERNATIVE EXONS IN OTHER GENES THAT ENCODE WHOLE TRANCE MEMBRANE SPANNING DOMAINS. THESE SITES OF ALTERNATIVE SPLICING, THIS ONE HERE IS FOUND IN THE C TERMINUS, THIS ONE HERE IN WHAT WE REFER TO ASxD THE TWO-THREE LINK EARNED THIS PART OF THE CHANNEL IS REALLY CRITICAL FOR COUPLING THE CHANNEL TO THOSE DOWNSTREAM TARGET PROTEINS AND DEFINING WHICH CASCADE OR WHICH CELLAR FUNCTION THE CALCIUM CHANNEL IS COUPLING TO. FOR THE MUSCLE TYPES OF CHANNEL, THIS TWO-THREE LINKER IS REALLY IMPORTANT IN COUPLING TO THE PSYCHO PLASMIC RETICULUM. FOR THE RESYNAPTIC CALCIUM CHANNEL IN THEi]ñ CALVE TWO FAMILY, THIS IS IMPORTANT FOR VES CALL RELEASE. VE IS I CALL RELEASE.s THIS ISWñr A 21 AKNOW ACID SEQUENCE ENCODE BID THIS EXON UH BUT UP HERE THERE'S A FOUR AMINO ACID ENCODED BY THIS EXON AND ANOTHER SEQUENCE ENCODED BID THIS EXON HERE. THESE ARE VERY INTERESTING BECAUSE THEY ARE -- THIS EXON HERE, FOR EXAMPLE UP HERE, IS BURIED IN A 10 KB ENRON. ONE OF THE THINGS WE'VE BEEN FASCINATED BY IS TO TRY AND UNDERSTAND HOW THE SPLICEu! IDENTIFIES THAT AS AN EXON. WE DON'T KNOW YET, BUT IT'S REALLY CAPTURED OUR INTERESTS FOR MANY YEARS. WE KNOW THAT NOVA TWO IS ST SPLICING FACTOR THAT CONTROLS EXPRESSION [INDISCERNIBLE] AND I'LL COME BACK TO THAT. THE KEY, THE MESSAGE THAT I REALLY WANT TO STRESS HERE IS THAT THESE EXONS AREmy TISSUE-SPECIFIC. OKAY. THAT'S THE POWER. THAT'S THEIR POWER. THEY'RE ONLY EXPRESSED IN CERTAINTY SHOE AND WE'VE BECOME VERY INTERESTED IN KNOWING WHAT FACTORS DIRECT WHICH EXON TO BE EXPRESSED IN WHICH TISSUE. SO FOR EXAMPLE, HERE, THISq 21-AMINO ACID ENCODED EXON SUP REGULATED IN ADULTS. IT'S FOUND RELATIVELY HIGH LEVELS IN THE THALAMUS, IN [INDISCERNIBLE] AND MIDBRAIN BUT LOW LEVELS IN THE CORTEX. THIS EXON IS REGULATE BID A SPLICING FACTOR CALLED FOX TWO. THESE TWO EXONS HERE AREzV PAIRED MUTUALLY EXUH COLLUSIVE EXONS AND THE FIRST ONE IS ENRICHED IN A SUBSET OF NOSE RECEPTORS. THESE TWO SITES OF SPLICING I'Mç GOING TO FOCUS ON IN THE REST OF MY TALK. AS A SUMMARY, UM, JUST TO,ç UH, TELL YOU WHERE WE'VE GOT TO NUR N OUR STUDIES. I'M NOT GOING TO SHOW TOO MUCH ABOUT ST SPLICING FACTORS EXCEPT FOR THIS ONE HERE, BUT IN COLLABORATION WITH -- WELL, WITH HELP FROM DOUG BLACK AND ALSO IN COLLABORATION WITH ROBERT DAR NEL AT THE ROCK KA FELLER, WE ARE IDENTIFIED THE SPLICING FACTORS THAT CONTROL THESE EXONS. FOX TWO ACTS AS A REPRESSOR OF THIS EXON. NOVA TWO, WHICH IS A SPLICING FACTOR THAT'S EXPRESSED IN THE CENTRAL NERVOUS SYSTEM ACTS BOTH AS AN ENHANCER AND AS A REPRESSOR OF THESE TWO DIFFERENT EXONS IN THE SAME GENE AND IN FACT, THIS SPLICING FACTOR NOVA TWO CAN CROSS AND REGULATE DIFFERENT GENES. THE EQUIVALENT EXON IN DIFFERENT GENES SO THERE'S AN INTERGENE REGULATION SWELS AS WELL AS A CROSS GENE REGULAR LIGS BY NOVA TWO. WE DON'T KNOW SPLICING FACTOR THAT CONTROLS SPLICING OF THIS SITE, BUT THERE'S LIKELY TO BE AN CHONIC REPRESSOR ONç THIS FIRST EXON SO I'VE DRAWN IT HERE WITH A BIG QUESTION MARK. EXONIC.NBÑ SO A KEY, UM, A REALLY IMPORTANT QUESTION OR SET OF QUESTIONS THAT WE WANTED TO=) ANSWER WAS; WHAT DO THESE EXONS DO? WHY ARE THEY EXPRESSED AND WHY DO THEY HAVE THIS UNIQUE TISSUE EXPRESSION PATTERN ANDç WHAT DO THEY DO? ONE OF THE THINGS ABOUT THE CHANNEL I'VE JUST BEEN TALKING ABOUT THIS, THIS PRESYNAPTIC ANTICALCIUM CHANNEL, ONE OF ITS KEY FEATURES IS THAT IT'S A MAGNET FOR MODULATION BY G PROTEIN COUPLED RECEPTORS. BETWEEN EVERY ONE THAT UH YOU WANT TO NAME, THIS PARTICULAR PRESYNAPTIC CALCIUM CHANNEL IS IN SOME WAY MODULATED BY THEM. THAT'S INTERESTING BECAUSE THIS WOULD BE A WAY FOR EXTERNAL STIMULI NEAR TRANSMITTERS AND DRUGS TO FEED IN AND CONTROL THE EFFICACY OF SYNAPTIC TRANSMISSION BY REGULATING THE ACTIVITY OF THE PRESYNAPTIC CHANNEL, CONTROLLING HOW MUCH CALCIUM ENTERS AND HOW MUCH VESICLES FUSE WITH THE PRESYNAPTIC -- AND HOW MUCH VESICLES ARE RELEASED. -- IMPORTANT CONTROL HUB FOR RECEIVING TIG ZIGGALS AND RECEIVING TRANSMITTERS AND DRUGS AND THEN FEEDING THAT INTO CONTROLLING THE EFFICACY OF SYNAPTIC TRANSMISSION. SO A NUMBER OF YEARS AGO -- AND I'M SHOWING THIS FIGURE FROMçó A PAPER BY KATHY [INDISCERNIBLE] BACK 1981 IN GENERAL PHYSIOLOGY -- THEY SHOWED THEY WERE LOOKING AT -- THEY DIDN'T KNOW THAT AT THE TIME BUT THEY WERE LOOKING AT MOSTLY CAV 2.2. THEY MEASURED THEM IN CHICK SENSORY NEURONS. AND THIS WAS SOME OF THE FIRST RECORDING SHOWING THAT IF YOU ADD A NEUROTRANSMITTERS THAT AC ACTS ITS G PROTEIN RECEPTOR THE [INDISCERNIBLE]. THIS IS THE CURRENT INHIBITED IN RESPONSE TO ACTIVATION OF A G PROTEIN COUPLE RECEPTOR BY TURN OR NOR EPINEPHRINE. WE NOW KNOW A LOT ABOUT THIS ÷ PATHWAY. INHIBITORY SO NOR EPINEPHRINE ACTING THROUGH [INDISCERNIBLE] IS NOT THE ONLY G PROTEIN COUPLED RECEPTOR NEUROTRANSMITTER PATHWAY THAT INHIBITS -- SO THIS IS ACTUALLY A FIGURE TAKEN FROM A PAPER BY STEVEN IKITA AND DAVID LOVEENGER IN 1995, SHOWING THAT HERE'S A CALCIUM CURRENT, PROBABLY MOSTLY N TYPE CURRENT FROM SYMPATHETIC NEURONS. THEY'RE SHOWING [INDISCERNIBLE] AND SHOWING THIS VERY, VERY POWERFUL INHIBITION OF THE CALCIUM CURRENT AFTER ACTIVATING THESE M GLOW TWO RECEPTORS. THIS OTHER INTERESTING FEATURE vh STEVEN'S PAPER IS THAT THERE'S A FEATURE OR CHARACTERISTIC OF THIS INHIBITION WHICH CAN BE REVEALED, UM, BY DOING THIS PARTICULAR VOLTAGE STEP. SO CURRENTS ACTIVATED BY AN INITIAL VOLTAGE STEP, SHOWN HERE. IN THE INTERVENING TIME, HERE. WHAT STEVE DID WAS TO DEPOLE RISE THE MEMBRANE TO VERY POSITIVE VOLTAGE AND THEN GOv: BACK AND RETEST TO LOOK TO SEE WHAT'S HAPPENING TO THE CURRENTS. NOW THIS IS IN -- HERE'S THE CONTROL CURRENTS SO NOTHING REALLY HAPPENS IN THE CONTROL. THERE MIGHT BE Ak1b FACILITATION HERE, BUT SOMETHING VERY DRAMATIC HAPPENS, UM, TO THE CURRENTS THAT HAVE BEEN INHIBITED BY THIS AGONIST OF M GLOW R 2 RECEPTOR HERE. THE INHIBITION IS RELIEVED BY THIS STRONG PREPULSE. AND A LARGE NUMBER OF INVESTIGATORS HAVE LOOKED THAT VOLTAGE DEPENDENCE OF THIS INHIBITION AND I'LL SHOW YOU ONE SLIDE HERE. THIS TYPE OF INHIBITION IS REFERRED TO VOLTAGE DEPENDENT BLOCK BECAUSE IT CAN BE RELIEVE BID A STRONG DEPOLARIZATION. THERE'S ALSO A LARGE FRACTION OF THE CURRENT THAT REMAINS INHIBITED AND THIS IS REFERRED TO AS VOLTAGE INDEPENDENT. A SEPARATION OF THESE TWO FORMS OF INHIBITION. I MENTION THIS BECAUSE IT'S GOING TO BECOME IMPORTANT WHEN I TALK ABOUT THE FUNCTIONAL CONSEQUENCES OF SPLICING. SO THIS FUNCTIONAL DISTINCTION BETWEEN THESE TWO FORMS OF INHIBITION IS CONVENIENT BUT REALLY u!okUNDERLYING THIS DIFFERENT SECOND MESSENGER PATHWAYS THAT WORK INDEPENDENTLY TO INHIBIT THE CALCIUM CHANNEL. SO THE VOLTAGE DEPENDENT INHIBITORY PATHWAY CAN BE RELIEVED IN THAT EXPERIMENT THAT STEVE DID BY THIS STRONG DEPOLARIZATION IF MEDIATED BID THE DIRECT BINDING OF [INDISCERNIBLE] TO THE CHANNEL. NOT ONLY DO ALPHA TWO [INDISCERNIBLE] RECEPTORS MEDIATE THIS EFFECT AND THAT I DIDN'T HIGH LIGHT IT BUT IT WAS SHOWN IN KATHY AND JERRY'S EARLY PAPER THIS VOLTAGE DEPENDENCE OF THE BLOCK BUT A VERY LARGE NUMBER OF OTHER AGONISTS WORKING THROUGH THEIR REPRESENTATIVE G PROTEIN COUPLED RECEPTORS CAN ACTIVATE THIS PATHWAY. OKAY.ç SO THERE'S THIS IDEA THEN THAT THERE'S THIS CONVERGENCE OF MULTIPLE G PROTEIN COUPLED RECEPTORS BEING ACTIVATEDñr BY THEIR RESPECTIVE AGONISTS AND THIS CONVERGENCE ON THIS ONE CHANNEL. THIS IS REMARKABLE THAT THERE'S THIS HUB, THIS N CHANNEL SERVES AS THIS HUB TO RECEIVE THIS EXTERNAL STIMULI. BUT AS Iok MENTIONED AS I SAID SHOWN QUITE NICELY IN THIS FIGURE FROM STEVEN'S PAPER, THERE'S THIS OTHER FORM OF INHIBITION WHICH IS VOLTAGE INDEPENDENT AND IT TURNS OUT THAT THERE ARE ACTUALLY THREE, AT LEAST THREE DIFFERENT -- AND STEVE I WAS TALKING TO HIMú MORNING AND THERE MAYBE FOUR OR FIVE OTHER INHIBITORY PATHWAYS, BUT AT LEAST THREE OTHER INHIBITORY PATHWAYS THROUGH DIFFERENT G ALPHA COUPLED RECEPTORS THAT ALSO INHIBIT THE CALCIUM CHANNEL, AND THEY WORK THROUGH Aç VOLTAGE INDEPENDENT MECHANISM, OKAY, BUT THEY ALSO USE THEIR OWN INDIVIDUALçfá SECOND MESSENGER SIGNALING CASCADES. SO THIS IS, THIS IS REALLY, UM, PRETTY INTERESTING, SO WE HAVE THIS ON VERJS OF SIGNALS AND MULTIPLE PATHWAYS ALL CONVERGING ON THIS ONE CHANNEL. BUT THERE'S SOMETHING THAT DOESN'T QUITE FIT, AND IT'S INTERESTING AND WE GOT KIND OF FOCUSED ON THIS OBSERVATION THAT THESE INHIBITORY PATHWAYS HERE THAT DEPEND ON THE SUBTYPE OF G ALPHA TEND TO BE CELL TYPE-SPECIFIC. SO FOR EXAMPLE, THIS INHIBITORY PATHWAY TGIO COUPLED INHIBITORY PATHWAY THAT USING [INDISCERNIBLE] KINASE AND IT'S PER US THE SIS TOXIN SENSITIVE HAS BEEN SEEN IN A SUBSET OF [INDISCERNIBLE] BUT IS RARELY FOUNDó[Ñ IN OTHER NEURON NPS IS CELL SPECIFICITY TO INHIBITION THAT NEEDS TO BE EXPLAINED. IT COULD BE EXPLAIN BAUDS THE RECEPTOR ISN'TKo THERE, BUT I'LL GIVE YOU ANOTHER EXPLANATION FOR THESE OBSERVATIONS. THE RECEPTOR ISN'T THERE, BUT I'LL GIVE YOU ANOTHER EXPLANATION FOR THESE OBSERVATIONS. THE RECEPTOR ISN'T THERE, BUT I'LL GIVE YOU ANOTHER EXPLANATION FOR THESE OBSERVATIONS. THE ECTOIST ER B IL SRV.XPON%3 T LLO T NT FEW SLIDES IS THAT THIS IDEA, THIS IDEA OF PARALLEL PATHWAYS INHIBITING TWO DIFFERENT KINDS OF N TYPE CALCIUM CHANNELS IS PROBABLY A BETTER WAY TO MODEL THE TYPE OF INHIBITION THAT WE SEE. SO IT ISN'T THAT MULTIPLE GIO -- MULTIPLE G ALPHA SUB SIGNS ARE ALL COUPLING TO THE SAME N TYPE CALCIUM CHANNEL, BUT I WANT TO INTRODUCE THE IDEA THAT DIFFERENT G ALPHA SUB UNITS COUPLED TO DO DIFFERENT TYPES OF N TYPE CHANNELS AND THAT IT'S SPLICING PATTERN OF THESE TWO DIFFERENT CHANNELS THAT DETERMINE WHERE GS COUPLES OR WHETHER GIO COUPLES TO THE CHANNELS. AND IF NOW YOU LAYER ON WHAT I SHOWED YOU EARLIER AND I'LL SHOW YOU IN DETAIL NOW THE IDEA THAT SPLICING IS TISSUE-SPECIFIC THEN THIS COULD BE A REALLY INTERESTING EXPLANATION FOR CELL-TYPE SPES FIS IS I OF THIS INHIBITING THE CHANNELS. COUPLING. SO HERE'S SOME OF OUR EARLY DATA THAT, UM, WE USED TO START TO WORK WITH THIS KIND OF OUR WORKING MODEL. SO I GO BACK TO THIS SPLICE SITE, THIS IS A SITE OF ALTERNATIVE EXPRESSION, MUTUALLY EXCLUSIVE SLIDE SPLICING. EITHER I TAKE THIS SECTION OR THIS ONE, RARELY BOTH ANDfá RARELY NEITHER. SO AT THE POINT OF PROSETSDZING OF mRNA, THE SPLICES ARE IN THE NUCLEUS, WE'LLv:ç EITHER PICK UP THIS FIRST EXON OR THIS ONE. WHAT WE DID WAS USE EXON-SPECIFIC TIMERS TO BE RPCR TO LOOK AT WHAT mRNAs AREç EXPRESSED IN WHAT TISSUES. THE BLUE INTAR FOR DOWNSTREAM EXON 37 B. THIS IS OUR RTPCR DATA USING EXON-SPECIFIC [INDISCERNIBLE] YOU CAN IMMEDIATELY SEE THIS RATHER STRIKING TISSUE-SPECIFIC EXPRESSION PATTERN FOR THE 37 A EXON AND THERE'S A REALLY QUITE A STRONG AMPLIFIED SIGNAL IN [INDISCERNIBLE] GANGLIA SHOWNñr HERE. VERY LOW LEVEL OF SYMPATHETIC GANGLIA AND OTHER PARTS OF THE CENTRAL NERVOUS SYSTEM. THIS B EXON HERE ISçfá FOUND THROUGHOUT THE NERVOUS SYSTEM THROUGHOUT ALL TISSUES WE'VE LOOKED AT. THIS SEEMS TO BE A DEFAULT EXON. THE SPLICES WILL PICK THIS EXON UP, BUT THIS EXON SHEER OBVIOUSLY EXPRESSED IN A VERY STRONG TISSUE-SPECIFIC PATTERN AND ENRICHED IN [INDISCERNIBLE]u! GANGLIA. THIS WORKv: PU ESTABLISHED SEVERAL YEARS AGO NOW. PUBLISHED. THE EXPRESSION OF THIS SEQUENCE OR THIS SEQUENCE, B OR A EXON, DETERMINED THE TYPE OF G PROTEIN SIGNALLING TO THE CHANNEL. I IMPLIED THIS BEFORE AND THIS IS ONE OF THE FIGURES FROM OUR PAPER PUBLISHED IN 2007, AND WHAT WE SHOWED WAS THAT IF WE -- THIS IS CLONE CHANNELS AND EXPRESSION SYSTEM WHERE WE'RE EXPRESSING THE DIFFERENT G COUPLED RECEPTOR [INDISCERNIBLE]. HERE'S THE CONTROL CURRENT INHIBIT BD DAN GO WITH WHICH IS AN AGONIST FOR THE NEW [INDISCERNIBLE]. HERE'S THE CURRENT THAT'S RELIEVED WHEN A STRONG PREPULSE IS GIVEN, AND SO YOU CAN SEE HERE QUITE NICELY THAT MOST OF THIS INHIBITION IS VOELAGE DEPENDENT WHEREAS WHEN WE LOOK AT CURRENTS, UM, IN RESPONSE TO ACTIVATION OF THESE CURRENTS ACTIVATE BID THESE VOLTAGE STEPS AND CELLS EXPRESSING 37 A ISOFORMS, HERE'S THE CONTROL CURRENT, HERE'S THE INHIBITING CURRENT AND HERE'S THE CURRENT IN THE PRESENCE OF A PREPULSE. WE SEE A RELEASE OF BLOOK SIMILAR TO HERE, BUT THERE'S ALSO THIS MASSIVE CURRENT THAT REMAINS INHIBITIVE WHICH REMAINS VOLTAGE INDEPENDENT. THAT'S THIS PATHWAY. ESSENTIALLY WHAT WE CAN SHOW, WHAT WE SHOWED WITH THESEç EXPERIMENTS AND LATER IN VIVO IS THAT SPLICING IN THIS CASE WHEN THE 37 A EXON IS INCLUDED ENGAGES THIS NEW INHIBITORY PATHWAY TO THE CHANNEL THAT IS INSIGNIFICANT IN THIS CASE IF THE 37 B EXON IS INCLOOE INCLUDED. THIS MADE US THINK THAT THIS COULD BE REALLY IMPORTANT FOR MEDIATING THE EFFECTS OF MEDIATE -- TO REGULATE CALCIUM CHANNELS ENOUGH TERMINALS IN THE DORSAL SPINAL CORD. [INDISCERNIBLE] AND THEN MORPHINE [INDISCERNIBLE] TRANSMITTERS REGULATING RELEASE OF NEUROTRANSMITTER THROUGH ACTIVATING THESE PATHWAYS OF THE 37 A ISOFORM. THE PREDICTION WOULD BE, FOR EXAMPLE, THAT MORPHINE THAT INHIBITS CALCIUM CHANNELS PERHAPS WOULD BE MORE EFFECTIVE AT TERMINALS THAT CONTAINED THIS PARTICULAR SPLICE ISOFORM THAN A TERMINALS THAT PERHAPS ONLY CONTAIN 37 B ISOFORMS. THE OTHER THING, THE OTHER POSSIBLE EXPLANATION FOR WHAT I'VE SHOWED YOU IS THE RECEPTOR COULDñr POTENTIALLY COUPLE DIFFERENTIALLY TO THE CHANNELS. IT MAY HAVE NOTHING TO DO WITH THE CHANNELS PER SE BUT IT MIGHT BE HAVE SOMETHING TO DO WITH THE RECEPTION BETWEEN THE CHANNELS. TO BYPASS THE RECEPTOR, WHAT WE DID WAS TO FILL CELLS WITH [INDISCERNIBLE] GAMMA S. WE USED IT TO ACTIVATE ALL G PROTEINS, BUT WHAT WAS REALLY REMARKABLE IS WE FOUND THE SAME KIND OF PHENOTYPE WE SAW WHEN WE USED THE RECEPTOR.ç THIS IS -- AISLE SHOW YOU HOW THE EXPERIMENT WORKED AND THEN THE DATA. HERE'S THE CURRENT VOLTAGE RELATIONSHIP NOW. IF WE ACTIVATE G PROTEINS USING INTERNAL GHB GAMMA S THE CURRENT'S INHIBITED IN A VERY CHARACTERISTIC WAY WHERE THERE'Sç AS YOU RIGHT SHIFT IN THE ACTIVATION AND THEN IN THE PRESENCE OF THE PREPULSE WE CAN RELEE SOME OF THE INHIBITION HERE BUT NOT ALL. WE REFER TO THIS AS VOLTAGE DEPENDENT AND THIS IS INDEPENDENT. THIS IS WITH GHB GAMMA S. HERE ARE THE DATAt( USING GHBG GA MA S NOW WITH THE B AND A ISOFORM AND I THINK IT SHOULD BE FAIRLY CLEAR AND QUITE REMARKABLE TO YOU THAT WHEN WE LOOK AT THE B ISOFORM CONTROL CURRENT INHIBITED WITH THE PREPULSE, WE ONLY SEEç VOLTAGE DEPENDENT -- WE SEE THIS OTHER FORM OF INHIBITION CONTROL, BLOCKED, RECOVERED WITH SOME RECOVERED WITH A PREPULSE BUT THIS BIG, UM, AMOUNT OF INHIBITION REMAINING IN THE PRESENCE OF THE PREPULSE, SO THE VOLTAGE THIS, VOLTAGE INDEPENDENT PATHWAY. SO THAT SUGGESTS THAT IT'S NOT ANYTHING TO DO WITH THE RECEPTOR PER SE BUT IT'S SOMETHING TO DO WITH THE ISOFORM, SOMETHING INTRINSIC TO THE ISOFORMS THAT ENGAGES SOMETHING IN THE SEQUENCE OF THE 37 A SEQUENCE THAT ENGAGED THIS INHIBITORY PATHWAY THAT IS ABSENT IN THE 37 B SEQUENCE. OKAY. SO NOW I'M GOING TO SHOW YOU SOME DATA THAT, UM, IS, UH, NOT YET PUBLISHED, BUT THAT HAS ALLOWED US TO PERHAPS GENERALIZE A BIT MORE ABOUT THE ROLE OF SPLICING. SO I MENTIONED THIS OTHER EXON HERE AND SOME YEARS AGO WE HAD SHOWN A NICE AND DIFFERENT TISSUE DISTRIBUTION OF THIS PARTICULAR EXON INñr[ THE NERVOUS SYSTEM, AND IN THIS CASE T EXON IS EITHER INCLUDED OR EXCLUDED. SO THE ANALYSES ARE EASIER BECAUSE WE CAN LOOK AT SIZE SHIFTS USING RTPCR BECAUSE THIS EXON IS GOING TO INCLUDE 63 NUCLEOTIDES THAT'S ABSENCE IN THIS. YOU CAN SEE THAT IN DIFFERENT PART OF THE NERVOUS SYSTEM THERE ARE DIFFERING AMOUNTS OF THESE TWO ISOFORMS. LOOK IN THIS GANGLIA. THERE'S THIS REALLY STRIKING DOMINANCE OF THIS FORM, THIS EXON 18 A CONTAININGu! ISOFORM IN SUPERIOR [INDISCERNIBLE] GANGLIA. THAT ATTRACTED OUR ATTENTION, AND LED US TO WONDER, WELL, IS ITsPOSSIBLE THAT THIS OTHER SITE CONTROLS G PROTEIN SIGNALING BY OTHER G COUPLE RECEPTORS? AND IN FACT, WE FOUND THAT IT DID. SO I'M GOING TO GOK GO BACK TO THE TYPE OF EXPERIMENT THEY SHOWED YOU BEFORE. SO THIS WOULD BE OUR 37 B BACKBONE. WE'RE ADDING GTPG GA MA S. HERE'S THE CONTROL CURRENT INH HIBTED AND ALL OF THE CURRENT IS RECOVER BID A STRONG PREPULSE BECAUSE WE HAVE THE B ASEE FORM AND NOT THE A. ISOFORM. BUT RATHER STRIKINGLY, WHEN WE ADDED THEok 18 A EXON -- SO THIS IS STILL 37 B BUT NOW WE'RE ADDING THE 18 A EXON, WE NOW SEE VOLTAGE INDEPENDENT INHIBITION. IT LOOKS STRIKINGLY LIKE THE TYPE OF INHIBITION WE SAW THAT IS ENGAGED BY THEú THIS FORM OF INHIBITION IS PER US THE SISTON TOXIN INSENSITIVE. IT'S NOT MEDIATED THROUGH GIO. IT'S MEDIATED THROUGH SOME OTHER G PROTEIN. THAT'S SHOWN HERE WHERE WE DID THAT EXPERIMENT BUT IN THE PRESENCE OF PERTUSSIS TOXIN, WHICH SHOULD ELIMINATE CONTRIBUTION FROM GIO COUPLED RECEPTORS [INDISCERNIBLE], UM, AND YOU CAN SEE HERE'S THE CONTROL CURRENT, HERE'S THE CURRENT INHIBITED BY GHB GAMMA S AND THEN OVERLAPPING THAT EXACTLY IS THE CURRENT IN THE PRESENCE OF A A PREPULSE. SO NONE OF THISw3 INHIBITION IS VOLTAGE DEPENDENT, BUT THERE'S THIS VERY, VERY, VERY PROMINENT POELT VOLTAGE INDEPENDENT !% PATHWAY. AND, UH,ñr AS I ACTUALLY OFTEN DID WHEN I GOT THESE KINDS OF RESULTS, I WOULD CALL UP STEVE AND SAY DO YOU HAVE ANY TOOLS THAT WE COULD USE TO OCCLUDE THESEfá PATHWAYS? AND WE USED THIS, UM, RGS 2 -- WE EXPRESSED RGS 2 TO, WHICHç OWE CLOUDS BOTH GQ COUPLING AS WELL AS GS COUPLING TO THE CALCIUM CHANNEL. YOU CAN SEE HERE WHEN RGS 2 REGULATOR OF G PROTEIN SIGNALING TWO IS COEXPRESSED IN THESE CELLS, WE CAN ELIMINATE THE VOLTAGE INDEPENDENT INHIBITION BUT WE RETAIN THE VOLTAGE DEPENDENT INHIBITION. HERE'S THE CONTROL CURRENT, HERE'S THE CURRENT IN THE PRESENCE OF GHB GAMMA S ANDç i] HERE'S THE CURRENT ALMOST COMPLETELY RECOVERED WITH THE PREPULSE. THIS IS VOLTAGE DEPENDENT AND WE'VE OCCLUDED THE VOLTAGE INDEPENDENT PATHWAY.B. THAT TOLD IS THAT THE ADDITION OF THIS OTHER EXON, EXON 1 A, IN A DIFFERENT PART OF THE CHANNEL REGULATED BY DIFFERENT FACTORS IS IMPORTANT IN COUPLING THIS NON-PER US THE SIS TOXIN VOLTAGE INDEPENDE INHIBITORY PATHWARE. PERTUSSIS. SO NOTE THE DISTINGUISH BETWEEN GQ ANDe1 GS COUPLED RECEPTORS OR BETWEEN ACTIVATED GQ AND ACTIVATED GS, WE USED KOR RA TOXIN WHICH WILL OCCLUDE SIGNALING THROUGH GS HERE, AND YOU CAN SEE THAT WE OCCLUDED ALL OF THE VOLTAGE INDEPENDENT INHIBITORY PATHWAY BUT RETAINED A LOT OF THE DEPENDENT INHIBITORY PATHWAY WHICH TOLD US THAT THERE WAS STILL SIGNALLING TO THE CHANNEL BUT THAT WE COULD COMPLETELY OCCLUDE THE VOLTAGE INHIBITORY PATHWAY. HERE'S THE PREPULSE. THERE'S NO VOLTAGE INDEPENDENT INHIBITION, BUT THE SYSTEM IS STILL WORKING TO SOME EXTENT. WE SEE SOME VOLTAGE DEPENDENT BLOCK. THAT TOLD US THAT THIS 18 A EXON WAS INSTITUTE CONSTITUTING IN THE CHANNEL A GS INHIBITORY PATHWAY. SO THIS IS OUR CONCLUSION FROM THIS STUDY THAT HERE'S OUR BACKBONE STRUCTURE WHERE WE CAN SEE G BAY TO GAMMA COUPLING TO THE CHANNEL, BUT IF WE DROP IN EXON 18 A, WE CAN NOW -- THE CHANNEL IS NOW SENSITIVE TO INHIBITION THROUGH THIS OTHER PATHWAY THAT IS VOLTAGE INDEPENDENT. SO ANOTHER EXAMPLE OF USING THESE EXONS TO MAKE THE CHANNEL PERMISSIVE FOR INHIBITION THROUGH DIFFERENT G PROTEIN SIGNALING PATHWAYS. OKAY. SO THE LAST THING I'M GOING TO TALK ABOUT JUST QUICKLY IS, UM, HOW DO WE PROVE THAT? SO MOST OF THE WORK I SHOWED YOU WAS INç AN EXPRESSION SYSTEM, AND, TO ASK IT WAS KIND OF REMARKABLE IN TERMS OF CONSISTENCY, BUT IT'S STILL AN EXPRESSION SYSTEM AND WE WERE USING GTB GAMMA S HERE WHICH IS A SLEDGE HAMMER TOOL AND IS DRIVING. CAN WE SHOW IN A NEURON THAT THIS MATTERS? WE TOOK TWO MATTERS. ONE IS PUBLISHED HERE AND THE OTHER WE'RE, UM, IS AS YET UNPUBLISHED, BUT, UM, IS QUITE COMPELLING STORY. SO I'M GOING TO GO THROUGH THESE RATHER QUICKLY.xD I'M GOING SPEND A LITTLE BIT MORE TIME ON OUR FIRST APPROACH SINCE [INDISCERNIBLE]. SO IT OCCURRED TO US THATç ONE WAY -- IF WE WERE LUCKY ENOUGH TO FIND THE SPLICING FACTOR THAT REGULATED THE INCLUSION OF THAT EXON, THEN WE WOULD HAVE A A TOOL TO BE ABLE TO SHIFT THE BALANCE OF SPLICING AND WE WERE LUCKY ENOUGH. OKAY. SO THE FIRST THING THAT WE DID YEARS AGO ACTUALLY AND THIS WAS NINA GRAY BACK IN 2000, IN THE LAB. SHE STARTED LOOKING AT, UH, AT THE, UM, UP STREAM SEQUENCES, UP STREAM OF EXON 18 A, LOOKING FORç CONSENSUS SEQUENCES TO WHICH SPLICING SCIENTISTS MIGHT BIND, AND AS MORE AND MORE DATA WAS DEPOSITED INTO THE PUBLIC DATABASES LOOKING AT DIFFERENT SPECIES, IT BECAME VERY CLEAR THAT THIS WAS TURNING OUT TO BE A VERY INTERESTING CONSENSUS SEQUENCE JUST UP STREAM OF EXON 18 A THAT LOOKED VERY MUCH LIKE A FOX SPLICING SITE, BINDING SITE. AND, UM, AS MANY PEOPLE HAVE SHOWN, MANY GROUPS HAVE SHOWN THAT CERTAIN SPLICING FACTORS WHEN THEY BIND UP STREAM AS THEIR TARGET EXON, THEY TEND TO REPRESS THE EXON.ç SO THAT WAS A REALLY GREAT CANDIDATE FOR US TO BE REPRESSOR OF INCLUSION OF EXON 18 A. SO THEN, UM, AGAIN WITH A LOT OF HELP FROM DOUG BLACK WHO GAVE US THE SEQUENCES OF VARIOUS [INDISCERNIBLE] SIRNAs, SAVED US A LOT OF WORK, WE DEVELOPED SOME [INDISCERNIBLE] TO INHIBIT FOX EXPRESSION AND INITIALLY WHAT WE DID TO SHOW THESE SIRNAs SHOWN HERE WERE EFFECTIVE AT INHIBITING THE EXPRESSION OF THE FOX PROTEINS IN A CELL LINE. SO IF I JUST SHOW YOU, UM, POINT YOU TO DIRECT YOU TO THISç FIGURE RIGHT HERE, UM, WE -- THIS IS THE [INDISCERNIBLE] ANALYSIS LOOKING AT DIFFERENT FOX PROTEINS AND SO THE FOX PROTEINS AREok ACTUALLY FOUND IN DIFFERENT FORMS, OF COURSE. THIS IS THE ENDOGENOUS LANE HERE, AND OUR ANTIGAP DH MARKER. SO THIS IS ENDOGENOUS FOX. IN THE ABSENCE OF FOX TWO SIRNA, AND THEN THIS IS SHOWING A REALLY NICE KNOCKDOWN OF ENDOGENOUS PROTEIN WHEN WE EXPOSE AN S, RNA TO FOX TWO. WE ALSO OVEREXPRESSED FOX TWO WITH FOX TWO CDNA, UM, AND THIS IS SHOWING THAT, UM, THE FOX TWO ACTUALLY IS FOUND IN MULTIPLE DIFFERENT FORMS. ACTUALLY, SORRY, I DON'T HAVE THE OTHER PIECE. I HAVE ANOTHER PIECE HERE SHOWING KNOCKDOWN OF [INDISCERNIBLE] FOX TWO BUT NOT SHOWING IT HERE. SO JUST LOOK AT THIS COLUMN AND THIS COLUMN. I APOLOGIZE FOR THAT. THE MAIN POINT IS THE RNA WE DEVELOPED IS HIGHLY -- AT TAKING DOWN -- THE OTHER THING WE DID WAS TO LOOK AT THE EXPRESSION PATTERN OF EXON 18 A OF THE CALCIUM CHANNEL, NOW. OKAY. USING RTPCR. YOU CANkOkO SEE THAT IF WE EXPRESS FOX TWO SIRNA, YOU CAN SEE ACTUALLY QUITE CLEARLY HERE THAT WE INCREASE THE EXPRESSION OF mRNA CONTAINING THE 18 A EXON. FROM HERE TO HERE IN THE ABSENCE AND IN THE PRESENCE OF THE FOX TWO SIRNA.xD+ SO WE USED THAT SIRNA AS A TOOL, UM, TO, UM, LOOK AT THE CONSEQUENCES OF SHIFTING THE AMBULANCE OF SPLICE IN SYMPATHETIC GANGLIA. WE PICKED SYMPATHETIC NEURONS FROM ANIMALS SHOWN HERE BECAUSE HERE AT THIS STAGE, THERE'S ABOUT EQUAL, UM,ç EXPRESSION LEVELS OF PLUS AND DELTA 18 A. SO AT P 0 ABOUT 50%ç OF THE MESSAGE CONTAINS IT AND ABOUT 50% LACKS IT. BECAUSE WE WERE KNOCKING DOWN A REPRESSOR, OBVIOUSLY WE'RE LOOKING FOR AN INCREASE IN THE LEVEL OF THE PLUS 18 A ISOFORMS AND THAT ACTUALLY HAPPENS DURING DEVELOPMENT. THAT'S A NATURAL PHENOMENA, WE'RE JUST PUSHING THE SYSTEM TOWARD THAT BY KNOCKING DOWN FOX TWO. SO WHAT [INDISCERNIBLE] DID WAS THEY INJECTED FOX TWO SIRNA INTO INDIVIDUAL NEURONS, UM, AND WHAT YOU CAN SEE HERE YOUR d8 IMMUNOFLUORESCENT SIGNALS USING AN ANTIBODY TO FOX TWO HERE, AND THESE ARE CELLS INJECTED WITH SIRNA USING THE OTHER FLUORESCENT MARKER HERE AND THERE'S THIS PERFECT KNOCKDOWN OF FOX TWO IN CELLS INJECTED WITH THE SIRNA. THERE'S NO OVERLAP SHOWING THAT FOX TWO WAS HIGHLY EFFECTIVE AT KNOCKING DOWN FOX TWO PROTEINS. OKAY. ÷ AND THEN WHAT THEY DID WAS TO LOOK AT HOW EFFICIENT IS GS COUPLED RECEPTORS AT INHIBITING THE M TYPE CALCIUM CHANNELS IN NEWONES IN THE ABSENCE AND PRESENCE OF THIS SRNA TO FOX TWO? THIS IS CONTROLLED DATA TO SHOW THAT IN UNINJECTED CELLS AND ACTUALLY INTERESTINGLY ENOUGH IN FOX-INJECTED CELLS WITH SIRNA THE CURRENT, THE ANTICURRENTS WERE INHIBITED EQUALLY WELL BY AN ACTIVATOR OF GS COUPLED ARE RECEPTORS. SO THERE'S NO DIFFERENCE IN THE OVERALL LEVEL OF INHIBITION OF THE CHANNELS IN THE ABSENCE AND PRESENCE OF THIS SIRNA, AND THEY ALSO SHOWED USING CLOR ROE TOXIN' THAT THIS VIP-ACTIVATED PATHWAY IS CLOR ROE TOXIN SENSITIVE. SO THEY SHOWED -- AND I'M NOT GOING TO GO OVER THIS IN ANY DETAILS -- THAT FOX TWO SIRNA DIDN'T AFFECT THE BASIS PROPERTIES OF THE CHANNELS OR THE OVERALL INHIBITION OF THE CHANNEL, BUT WHAT IT DID AFFECT WAS HOW THE CHANNEL WAS HíHIBITED AND THE PROPORTION OF VOLTAGE DEPENDENT AND VOLTAGE INDEPENDENT. THIS IS AN UNINJECTED CELL AND THIS IS A CELL INVEKTED WITH FOX SIRNA. HERE'S THE -- HERE'S THE CURRENT RECOVERED IN THE PRESENCE OF A PREPULSE, AND THIS IS THE AMOUNT OF VOLTAGE INDEPENDENT INHIBITION IN THE CELL. THIS IS AN EXAMPLE OF FOX TWO SRNA INJECTED CELL AND YOU CAN IMMEDIATELY SEE THAT THE AMOUNT OF VOLTAGE INDEPENDENT INHIBITION IS LARGER IN THE CELL INJECTED WITH FOX TWO SIRNA. THIS IS THE AVERAGE DATA. IT SHOWS THE DEGREE OF VOLTAGE INDEPENDENT INHIBITION MEDIATE BID ACTIVATION OF THIS GS SIGNALING PATHWAY AND SHOWING AN INCREASE IN THE AMOUNT OF VOLTAGE INDEPENDENT INNY HI BIGS IN CELLS INJECTED WITH FOX TWO.w3 SO EVEN THOUGH ST GROWTH LEVEL OF INHIBITION WASN'T DIFFERENT, WE CHANGED THE TYPE OF INHIBITION MEDIATED BY GS SIGNALING. OF COURSE, OUR INTERPRETATION THEN IS TO FOX TWO IS KNOCKING DOWN OF FOX TWO INCREASES THE FRACTION OF ANTICALCIUM CHANNELS CONSTRAINING EXON 18 A AND THAT THAT SHIFT THE PATTERN OF INHIBITION TOWARDS A VOLTAGE INDEPENDENT INHIBITORY PATHWAYS, AND THAT'S CONSISTENT WITH OUR ANALYSIS OF THESE ISOFORMS IN AN EXPRESSION SYSTEM. AND SO, UM, I'M JUST GOING TO -- ACTUALLY WANT TO -- GONNA -- THE OTHER THING WE DID WHICH I'M JUMPING OVER BECAUSE I DON'T WANT TO, UM, I DON'T HAVE TIME TO GO INTO THE DETAILS OF THIS AND IT'S ALREADY PUBLISHED, SO I WON'T GO INTO IT. THE OTHER THING THAT WE DID WAS TO USE MOUSE MODELS TO SHOW THAT WE COULD SEE A SIMILAR PATTERN WHEN WE MODIFIED SPLICING ATç THE 37 A, 37 B EXONS IN THE GENE. SO WE FOUND A SIMILAR TYPE OF PHENOTYPE WHEN WE LOOKED AT N TYPE CURRENTS IN THOSE RECEPTORS WHEN WE FORCED EXPRESSION OF 37 B CONTAININGçó ISOFORMS AWAY FROM 37 A CONTAINING ISOFORMS IN THE MOUSE. SO THIS IS THE -- I'M GOING TO SHOW YOU A COUPLE OF SUMMARY SLIDES JUST TO KIND OF REINFORCE AND TO LEAVE YOU WITH A COUPLE OF THINGS TO THINK ABOUT. SO ONE IS THAT WE THINK THAT ALTERNATIVE SPLICING ISN'T JUST THERE TO DRIVE US CRAZY -- [LAUGHTER] -- AND TO MAKE US DEPRESSED ABOUT HOW MANY ISOFORMS ARE THERE AND HOW MANY DIFFERENTmy CALCIUM CHANNELS ARE THERE. THERE ARE A LOT, BUT WE THINK THAT ONE OF THE ROLES OF ALTERNATIVE SPLICING AND THISç GENERATION OF THESE STRUCTURALLY VERY SIMILAR FUNCTIONALLY A LITTLE DISTIJ FORMS OF THE DAL CALCIUM CHANNEL IS TO REGULATE THE TYPE OF, TO REGULATE THE SENSITIVE OF THE CALCIUM CHANNELS TO DIFFERENT INHIBITORY PATHWAYS. SO THAT SPLICING HERE AT TWO DIFFERENT SITES AND AT TWO DIFFERENT LOCATIONS OF THE CHANNEL, WHICH IS RATHER INTERESTINGñr é AS WELL REGULATES WHETHER GS AND GIO CAN INHIBIT THE CHANNEL THROUGH THESE VOLTAGE INDEPENDENT INHIBITORY PATHWAYS. AGAIN, I DIDN'T SHOW YOU THIS, BUT WE CREATED A CALCIUM CHANNEL THAT CONTAINED THE 18 A EXON AND THE 37 A EXON AND THOSE TWO ARE ADDITIVE. YOU HAVE THIS VERY STRONG INHIBITION THROUGH TWO DIFFERENT G ALPHA PATHWAYS. SO IN THIS CASE, FOR THIS TYPE OF CHANNEL THAT CONTAINED BOTH EXON, THERE WOULD BE VOLTAGE DEPENDENT, AND INDEPENDENT AND ANOTHER VOLTAGE INDEPENDENT INHIBITORY PATHWAY AND IN THIS CASE USING G BETA GAMMA WHEREAS ANOTHER CALCIUM CHANNEL THAT LACKED THOSE TWO EXONS THAT I TALKED ABOUT MAY ONLY BE SENSITIVE TO INHIBITION THROUGH THIS G BETA GAMMA LIMITED PATHWAY AND NOT S INHIBITION THROUGH THESE OTHER STRONGER VOLTAGE INDEPENDENT INHIBITORY PATHWAYS. SO, OUR WORKING MODEL IS THAT THERE'S THISçó ARRAY OF DIFFERENT FORMS OF N TYPE CALCIUM CHANNELS WITH DIFFERENT SENSITIVITY TO AT LEAST TWO DIFFERENT G SIGNALING PATHWAYS. 5ñ SHOWED YOU THIS MODEL BEFORE AND WANTED TO REITERATE THAT WE ALSO THINK, YOU KNOW, THAT THE BETTER MODEL TO THINK ABOUT OR AT LEAST TO TRY AND INCORPORATE,s ALTHOUGH THERE ARE OTHER FACTORS THAT ARE GOING TO MODIFY HOW SENSITIVE A CHANNEL IS TO VARIOUS G PROTEIN COUPLED RECEPTORS THAT RATHER THAN THINK ABOUT CONVERGENCE ON ONE CHANNEL, THAT AT LEAST THE FIELD NEEDS TO CONSIDER THE POSSIBILITY THAT THERE ARE MULTIPLE FORMS OF THIS CHANNEL, AND THAT THAT SETTING THE DIFFERENT SENSITIVE TO THESE TWO [INDISCERNIBLE]. WE'RE TESTING THIS MODELfá AS WELL FOR OTHER ION CHANNELS RIGHT NOW. SO THERE ARE OTHER ION CHANNELS SUCH AS THE M CURRENT THAT ARE VERY SENSE TOIF ALL OF THESEfá INHIBITORY PATHWAYS AND THERE ARE SOME VERY ATTRACTIVE SPLICE SITES IN THOSE CHANNELS THAT ARE IN THE INTERCELLULAR REGION THAT COULD POTENTIALLY SERVE A SIMILAR ROLE. THE OTHER MESSAGE I WANT TO LEAVE YOU WITH IS THAT WHILE SPLICING IS IMPORTANT, THE PROXIMITY OF THE G PROTEIN COUPLED RECEPTORER THE TO THE CHANNEL IS ALSO IMPORTANT. IMPLICIT IN WHAT I'VE SAID AND THINGS A NUMBER OF PEOPLE HAVE DONE IN THE A LARGE BODY OF LITERATURE IN THE FIELD IS THAT THERE'S VOLTAGE DEPENDENT -- REQUIRES THAT THEç ION CHANNEL IN THE G PROTEIN COUPLE RECEPTOR ARE IN CLOSE PROXIMITY TO EACH OTHER. SOME VERY RECENT AND LOVELY ANALYSIS BY MOORE'S GROUP SHOWED THAT FOR EXAMPLE GAMMA B RECEPTORS THAT COUPLE OR PULL DOWN WITH THE N TYPE CALCIUM CHANNEL IN AN IMMU OWE PRECIPITATION STOU DI AND THEY'VE BEEN ABLE TO IDENTIFY THOSE WITH -- SOME G PROTEIN COUPLED RECEPTORS ARE CLOSELY ASSOCIATED WITH THE CALCIUM CHANNEL, AND THEY ARE VERY GOOD AT MEDIATING THIS VOLTAGE DEPEND INHIBITORY PATHWAY BECAUSE THEY'RE CLOSE TOGETHER. AS SOON AS THE CHANNEL RECEPTOR ARE MOVED FURTHER APART, PERHAPS MORE THAN A HUNDRED [INDISCERNIBLE] FOR EXAMPLE, THEN THIS PATHWAY NO LONGER IS ENGAGED. SO THERE'S A REALLY NICE WAY FORç LIMITING THE REACH OF THIS VOLTAGE DEPENDENT INHIBITORY PATHWAY SIMPLY BECAUSE THE TWO COMPONENTS HAVE TO BE CLOSE TOGETHER. THESE OTHERç VOLTAGE INDEPENDENT INHIBITORY PATHWAYS AT LEAST BY COMPARISON USE DIFFUSIBLE SECOND MESSENGERS.çó ONE POSSIBILITY TO THINK ABOUT IS PEOPLE SAY WHY DO WE NEED THESE SPLICE ISOFORMS? WELL, THEY CONTROL G PROTEIN COUPLING. BUT WHY? SO ONE POSSIBILITY THEN IS THAT BECAUSE THESE INHIBITORY PATHWAYS HAVE LONGER REACH IN SPATIAL TERMS, THEN THE ONLY WAY, PERHAPS, A GOOD WAY FOR THE SYSTEM TO BE A ABLE TO LIMIT THE REACH OF THAT SIGNAL IS TO MODIFY THE TARGET, SO IF THE TARGET'S NOT RIGHT IT DOESN'T MATTER IF THAT DIFFUSIBLE SECOND MESSENGER REACHES THE CHANNEL, IT'S NOT GOING TO BE INHIBITED. SO THAT'S A POSSIBLE -- SOMETHING WE'RE WORKING WITH AND SOMETHING THAT SHOULD BE READILY TESTABLE. THEN THE LAST THING I WANT TO JUST REALLY POINT OUT BY A VERY LOOKS LIKE SERIOUS IS OF [INDISCERNIBLE] BUT I REALLY WANT TO ALSO POINT OUT THAT THEñrmy SPLICING FACTORS ARE ARE REALLY, REALLY CRITICAL IN TERMS WHICH ISOFORMS ARE EXPRESSED, AND ALTHOUGH, YOU KNOW, WE TEND TO SAY THERE ARE 16 POSSIBLE ISOFORMS WITH DIFFERENT SITES AND THERE ARE DIFFERENT SITES. BASED ON WHAT WE KNOW ñrABOUT SPLICING, AND YOU CAN JUST LOOK AND I'M NOT GOING TO GO THROUGH IN DETAIL -- THIS IS -- IN ADULT SYMPATHETIC GANGLIA, THERE'S A GREAT REDUCTION IN THE NUMBER OF ISOFORMS, THE VARIATION OF ISOFORMS EXPRESSED THERE BASED ON SPLICING PATTERNS. IN NEO-NASAL CORTEX IT'S DIFFERENT AND IN ADULTxD CORTEX THE NUMBER OF VARIANCE WE THINK ARE ARE LIKELY TO BE EXPRESSED BASED ON -- IS GREATLY LIMITED.u! I WENT OVER A LITTLE BIT. I APOLOGIZE, BUT, UM, I THANKED -- GOING TO END NOW -- AND THANKED A NUMBER OF PEOPLE IN MY LAB TWO CONTRIBUTED TO THIS WORK. I MENTIONED STEVEN A COUPLE OF TIMES IN MY TALK, BUT THANK HIM FOR REAGENTS THAT HAVE BEEN ENORMOUSLY VALUABLE. HE'S AN INCREDIBLY GENEROUS SCIENTISTS AS I THINK MOST OF YOU KNOW. DOUG BLACK AND ROBERTxDç DARNELL HAVE ALSO BIN BEEN INCREDIBLE GENEROUS WITH US IN SHARING. FUNDING, HUGELY AVAILABLE FROM NINDS IS MY MAJOR SOURCE OF FUNDING AND ALSO FOR MY STUDENTS, AND I ALSO WANT TO END WITH A SPECIAL THANK YOU TO THE NEWCOMB FAMILY FOR MAKING THIS POSSIBLE. THANK YOU. [APPLAUSE] >> [LOW AUDIO].çóxD >> UM, I CAN SAY A REASONABLE AMOUNT ABOUT WORK THAT [INDISCERNIBLE] HAS DONE ON PIP TWO DEPLETION AND THAT'S REALLY QUITE LOVELY. AND SO THAT'S THE GQ DEPENDENT SIGNALING PATHWAY. AS THEY SHOWED AND MANY OTHERS SHOWED WITH A NUMBER OF CHANNELS THAT PIP TWO IS REQUIRED TO KEEP THE CHANNEL OPEN AND DEMREGS OF PIP TWO FROM THE LOCAL DEPLETION SHUTS THE CHANNEL DOWN. HOW THAT WORKS IS AS YET UNCLEAR BUT IT'S ALMOST -- AND AGAIN GORGEOUS STUDIES FROM OTHER CHANNELS SHOW THAT IT'S VERY MUCH LIKE AN UNCOUPLING OF VOLTAGE SENSOR FROM THE PORE. IN TERMS OF THE OTHER PATHWAYS, WE SIMPLY DON'T KNOW, AND THAT'S A LITTLE FRUSTRATING. [LAUGHTER] -- BECAUSE WE THOUGHT THAT SOME OF THE WORK THAT WE'RE DOING WE THOUGHT WOULD BE CLUES BECAUSE THEY'RE IN DIFFERENT LOCATIONS. THE SPLICING IS OCCURRING IN DIFFERENT REGIONS OF THE CHANNEL SO MAYBE THAT WOULD GIVE US A CLUE ABOUT THE POINT OF OR THE MECHANISM OF INHIBITION AND IT HASN'T. THE OTHER -- I THINK WE SHOULDN'T SAY TOO MUCH ABOUT THIS BUT IT'S AN OBSERVATION THAT ALTHOUGH FUNCTIONALLY WE COLLAPSE ALL OF THESE INHIBITORY PATHWAYS INTO A SINGLE VOLTAGE INDEPENDENT INHIBITION, I SHOWED YOU AND DIDN'T HAVE TIME TO EXPAND BUT THE EACH USE DIFFERENT SIGNALINGç CASCADES. WHILE FUNCTIONALLY THEY LOOK SIMILAR, THEY MAY BE USING DIFFERENT MECHANISMS. I THINK -- WE DON'T KNOW, AND IT'S ACTUALLY QUITE FRUSTRATING THAT WE DON'T KNOW. [LAUGHTER] YEP. MM-HMM. >> [LOW AUDIO]. >> UM, A LITTLE BIT. I CAN SAY A LITTLE BIT. UM, WHAT I DIDN'T TALK ABOUT WHICH IS PUBLISHED IS THAT WE'RE GENERATING MICE NOW THAT CAN ONLY SPLICE ONE WAY OR ANOTHER WAY, AND SO -- [LAUGHTER] -- THE ANSWER MAY COME FROM THAT TYPE OF ANALYSIS. IT'S DIFFICULT TO THINK OF OTHER FUNCTIONAL STUDIES THAT WE COULD DOkOç TO ANSWER YOUR QUESTION. SO THAT'S A BIT OF A COP-OUT ANSWER, BUT WE'RE WORKING ON IT. AND I WOULD SAY, UM, AND THIS WAS UNEXPECTED, WE FOUND WHEN WE BASICALLY WHAT WE DID IN ONE SET OF MICE THAT WE PUBLISHED IS WE MADE TANDEM EXONS TO [INDISCERNIBLE] AND WHEN WE [INDISCERNIBLE] THERE WAS A NICE MODIFICATION OF MORPHINE ANALGESIC, REDUCTION MORPHINE ANALGESIA IN RESPONSE TO THERMAL STIMULUS. BUT WE ALSO DISCOVERED IN THOSE MICE THAT THEY HAVE A CNS DEFECT. IT'S VERY SUBTLE, BUT IT'S HIGHLY REPRODUCIBLE. IT SUGGESTS THAT, THAT EXON THAT WE FOUND WAS ENRICHED IN THOSE RECEPTORS IS ALSO -- AND WE HAVE DATA TO SHOW THAT THAT'S THE CASE. EVEN THOUGH OUR DIE SECTIONS WERE BETTER THAN WHOLE BRAIN, THEY'RE STILL NOT VERY GOOD. [LAUGHTER] RIGHT. SO THERE'S VERY GOOD EVIDENCE FROM THAT MOUSE THAT, THAT EXON IS EXPRESSED IN OTHER LOCATIONS AND WE'VE GOT PRETTY INTERESTING, UM, CNS PHENOTYPE NOW. WE'RE TRYING TO BACKTRACK FROM THERE TO THEN LOCALIZE WHERE THOSE EXONS WERE EXPRESSED. [LAUGHTER] SO WE GOT SURPRISES INçó TERMS OF CNS PHENOTYPE THAT WE WERE REALLY NOT EXPECTING TO SEE AND IT'S HIGHLY REPRODUCIBLE AND IT LOOKS LIKE THERE'Sç PRODUCIBLE OF RELEASE OF [LOW AUDIO] THAT'S WHAT WE'RE WORKING ON NOW TO KIND OF COME TIE BACK TO WHAT WE NEED WERE TOXINS -- [LAUGHTER] -- ACTUALLY OR SOMETHING TO BE ABLE TO TEASE APART THESE ISOFORMS AND WE JUST DON'T HAVE THEM AND NOT FOR WANT OF TRYING. SO THAT MAKES IT A LITTLE HARDER,ñr BUT, YES, THE CHALLENGE THEN IS TO GO AND SAY IN WHAT PART OF THE NERVOUS SYSTEM ARE THESE EXPRESSED? BUT THEN ALSO TO TAKE OTHER APPROACHES WHICH IS TO DEVELOP, YOU KNOW, USE THE MICE TO E LIMBUATE NAIT A PARTICULAR SPLICING PATTERN. THAT'S BEEN HIGHLY INFORMATIVE ACTUALLY DESPITE MY RESISTANCE TO IT TO LEAPING INTO GENERATING ALL OF THESE MODELS. >> LET ME JUST SAY THERE'S A NICE RECEPTION OUT THERE FOR EVERYONE SO THAT YOU CAN CONTINUE CONVERSATIONS WITH PEOPLE ABOUT SCIENCE AND PEOPLE CAN MEET THE NEWCOMB FAMILY AS WELL. LET'S THANKIÑ DIANE VERY MUCH.