WELCOME TO THE 2017, 2018 WEDNESDAY AFTERNOON LECTURE SERIES HERE AT THE NIH. FOR THOSE WHO DON'T KNOW ME, MY NAME IS -- I'M IN THE CELL BIOLOGY NEUROBIOLOGY BRANCH AT NICHD AND I'M PRESENTING ON BEHALF OF MEMBRANE PROTEIN INTEREST GROUP. TODAY WE HAVE ONE OF FEW NAMED LECTURES, THE NIH DIRECTORS LECTURE, WHICH MEANS THAT THE SPEAKER WAS VOTED FOR -- BY DIRECTORS OF THE NIH INSTITUTES. THE REASON I PAUSED OFF MY WRITTEN SCRIPT HERE, BECAUSE IT IS IMPOSSIBLE NOT TO FEEL OVERWHELMED WHEN ONE EVEN ATTEMPTS TO INTRODUCE TODAY'S SPEAKER. AND IT'S NOT REALLY ONLY BECAUSE AFTER PERFORMING TRAIL BLAZING EXPERIMENTS IN THE ION CHANNEL FIELD AS A YOUNG FACULTY AT HARVARD MEDICAL SCHOOL, HE DECIDED THAT HE WAS NOT SATISFIED WITH THE RESOLUTION OF THE EXPERIMENTS THAT THE THEN STUDY WERE PROVIDING HIM. AND DECIDED TO CRYSTALLIZE POTASSIUM CHANNELS WHICH WAS AT THAT TIME CONSIDERED IMPOSSIBLE FEAT BY ESTABLISHED CHRISALOGRAPHERS. NOT ONLY BECAUSE AFTER RECEIVING THE NOBEL PRIZE AT A VERY YOUNG AGE HE CONTINUED TO PUSH THE FRONTIERS ON THE ION CHANNEL AND MEMBRANE PROTEIN RESEARCH FIELD BY SOLVING THE FIRST STRUCTURE OF EUKARYOTIC POTASSIUM CHANNEL IN 2005, INCIDENTALLY THE FIRST EUKARYOTIC MEMBRANE PROTEIN STRUCTURE TO BE SOLVED THROUGH HETEROLOGOUS APPLICATION. ALSO BECAUSE WHEN OTHERS WOULD HAVE CHOSEN TO LEAD FROM THE BACK, THIS IS WHEN I WAS A POST-DOC WITH HIM, HE WOULD REGULARLY COME TO TRIPS STAY UP UNTIL PAST MIDNIGHT, PROCESS CRYSTAL GRAPHIC DATA AND SHOW UP IN THE MORNING AT SEVEN O'CLOCK AND CONTINUE WORK ON IT. NOT ONLY BECAUSE AS LATE AS 2012 WHEN THE STRUCTURE OF THE FIRST TWO FOLD POTASSIUM CHANNEL CAME OUT ONE WAS ENTIRELY FROM EXPERIMENTS DONE BY ROD HIMSELF. AND BECAUSE LIKE REALLY THE PROVERBIAL VISUALLY IMPAIRED MAN TRYING TO GAUGE AN ELEPHANT, THESE ARE REALLY SNIPPETS OF BROAD SCIENTIFIC PERSONNEL. THE HALLMARK OF WHICH IS REALLY HIS DEEPLY PASSIONATE UNTIRING SINGLE MINDED AND SINGULARLY INCITEFUL PURSUIT OF THE PROBLEM. THAT IS ALLOT OF HIS LIFE HOW ION CHANNELS WORK THAT IS EXEMPLARY AND INSPIRATIONAL. I WON'T ATTEMPT TO GO THROUGH HIS AWARDS BECAUSE THEY ARE NOT ERALY GOOD DESCRIPTORS. I'LL SHARE TWO EXPERIENCES. RIGHT AFTER I CAME TO THE NIH, POST-DOC FROM A LAB EXPLAINED TO ME AFTER COMING BACK FROM A CRYO EM WORKSHOP, I MET ROD THERE, HE WAS SURPRISED AND SHOCKED AND I WAS NOT. IN HIS OWN WORDS THAT WAS PUBLISHED IN INTERVIEW IN NATURE HE LIKES TO BE AT THE STEEPEST SLOPE OF THE LEARNING CURVE WHICH WAS REFERRING TO THE LATEST BREAK THROUGHS IN CRYO-EM THAT REVOLUTIONIZED STRUCTURAL BIOLOGY AND THE ION CHANNEL FIELD, THE LATTER MUCH THROUGH ROD'S OWN WORK. WHEN I CAME TO ROD'S LAB AS POST-DOC I WAS SURPRISED HOW SMALL THEIR SEMINAR ROOM WAS FOR LAB OF THAT STATURE. I QUICKLY REALIZED THAT WHAT THAT DID WAS IT FOCUSED EVERYONE'S ATTENTION ON TO THE SCREEN AND ALMOST EVERY GROUP MEETING WE WOULD HAVE AGITATED DISCUSSIONS THAT OCCASIONALLY ENDED WITH ROD'S VOICE SLOWLY RISING OVER THE REST OF US AND AS EVERYONE ELSE QUIETED DOWN, ROD WOULD SHARE ALMOST AS A SO LITTLE QUESTION HIS INCITES ON THE ISSUE. PERSONALLY THESE ARE SOME OF THE DEEPEST MOMENTS OF LEARNING OF MY LIFE THAT I OFTEN YIELDER TO GET BACK TO. WE'RE LUCKY HERE AT THE NIH, ROD TURNS DOWN WAY MORE INVITATIONS THAN HE ACCEPTS. WITHOUT TAKING ANY MORE TIME FROM HIM, THANK YOU FORMENT COOING TO THE NIH, ROB. [APPLAUSE] >> THANK YOU, VERY MUCH FOR THAT MORE THAN KIND INTRODUCTION. AND THANK YOU FOR THOSE WHO INVITED ME HERE. IT'S AN HONOR TO BE HERE. AND TALK ABOUT SOME OF THE WORK FROM MY LAB. IN TERMS OF ADVANCES THAT MY LAB HAS BEEN ABLE MAKE, IT'S A GOOD EXAMPLE OF HOW THE WHOLE ENTERPRISE OF SCIENCE IS AMAZING BECAUSE IN WAY MY CAREER HAS BEEN BASICALLY A PARASITE OF A GREAT TECHNOLOGICAL ADVANCES THAT OTHERS HAVE MADE BOTH IN THE DEVELOPMENT OF ELECTROPHYSIOLOGICAL METHODS, THE DEVELOPMENT OF CRYSTAL GRAPHIC METHODS AND MORE RECENTLY, THOSE WHO DEVELOP THE HARDWARE AND SOFTWARE BEHIND THE NEW REVOLUTION IN STRUCTURAL BIOLOGY THAT ENABLED CRYOELECTRON MICROSCOPY TO BE THE TECHNIQUE OF CHOICE FOR TONIC STRUCTURE NOW. SO IT'S JUST REALLY GREAT TO BE IN A SOCIETY AND IN A WORLD WHERE WE CAN PURSUE SCIENCE WITH SO MUCH AT OUR HANDS. I'M GOING TO TALK ABOUT THE SUBJECT I LOVE, BIOPHYSICS AND BIOLOGY OF POTASSIUM CHANNELS, TWO EXAMPLES OF PROJECTS WE WORKED ON IN THE LAB OVER THE LAST FEW YEARS. FIRST I'LL GIVE A BRIEF INTRODUCTION TO A PARTICULAR ION CHANNEL, POTASSIUM CHANNELS, AND THIS SLIDE IS A CARTOON PICTURE OF A CELL AT CELL MEMBRANE AND NUCLEUS, IN THE MEMBRANE I SHOW SODIUM POTASSIUM ATPASE, THE IMPORTANT PUMP THAT CONCENTRATES INSIDE THE CELL AND SODIUM OUTSIDE THE CELL AND I SHOW A VARIETY OF ION CHANNELS THAT ARE PASSIVE CONDUITS FOR IONS ACROSS THE MEMBRANE. ON THE TOP I SHOW POTASSIUM CHANNELS AND SAY THEY'RE HYPERPOLARIZING. THAT MEANS WHEN POTASSIUM CHANNELS ARE OPEN AS SOME ARE IN AT REST IN MOST CELL MEMBRANES, POTASSIUM RUNS DOWN ITS CONCENTRATION GRADIANT UNTIL IT CHARGES -- LEAVES A NEGATIVE CHARGE BEHIND, WE SAY CHARGES THE CAPACITY OF THIS CELL MEMBRANE SO VOLTAGE ON THE INSIDE, VOLTAGE ACROSS THE MEMBRANE BALANCES THE CHEMICAL DRIVING FORCE SO THAT SAYS TO BE THE POTENTIAL FOR POTASSIUM AND ORIGIN OF RESTING MEMBRANE POTENTIAL IN MOST CELLS. THEN YOU CAN HAVE ANOTHER CHANNEL LIKE A SODIUM CHANNEL, WHEN IT OPENS SODIUM RUSHES DOWN ITS ELECTROCHEMICAL GRADIANT CAUSING IT DEPOLARIZATION OR SWITCH IN THE CHARGE ACROSS THE COMPASSTANT OF THE MEMBRANE. THAT PROCESS IS ALWAYS STOPPED THEN BY SOMETHING LIKE A SODIUM ACTIVATED POTASSIUM CHANNEL, THE SODIUM ENTERING WILL OPEN ADDITIONAL POTASSIUM CHANNELS AND RESTORE THE MEMBRANE POTENTIAL. SO POTASSIUM CHANNELS SET THE RESTING POTENTIAL AND THEY TEND TO STOP EXCITATORY PROCESSES. THEY BASICALLY PUT A GATE ON IT AND SO STOP EXITATION. THE POTASSIUM CHANNEL FAMILY IS VERY LARGE. SO THEY'RE APPROXIMATELY 80 MEMBERS OF POTASSIUM CHANNELS IN US. THIS IS JUST TO GIVE AN IDEA OF BIOLOGICAL ROLES, NOT TO SAY IN DETAIL BUT THE THERE'S CHASES HERE CALLED RECTIFIER AND THEY DO MANY THINGS, REGULATE THE RHYTHM OF THE HEART, THEY'RE IMPORTANT IN NEURONAL COMPUTATION, IMPORTANT IN SECRETION OF HORMONES SUCH AS INSULIN, THEN THERE ARE CHANNELS CALLED SLOW CHANNELS, POTASSIUM ACTIVATED CALCIUM ACTIVATED OR SODIUM ACTIVATED POTASSIUM CHANNELS IMPORTANT IN A NUMBER OF FUNCTIONS INCLUDING MUSCLE CONTRACTION, AUDITORY SIGNAL PROCESSING, NEURAL EXCITABILITY, TWO LARGE CLASSES OF VOLTAGE DEPENDENT LARGE BRANCHES OF THE FAMILY CALLED VOLTAGE DEPENDENT ION CHANNELS THAT ARE IMPORTANT IN VARIOUS ELECTRICAL SIGNALLING IN NERVOUS SYSTEM AND OUTSIDE THE NERVOUS SYSTEM. AND THEN A FAIRLY MYSTERIOUS CLASS CALLED K 2P CHANNELS THAT WE STILL DON'T KNOW VERY MUCH ABOUT. THESE CHANNELS ALL -- WHAT MAKES THEM A COMMON FAMILY, THEY HAVE A STRUCTURAL ELEMENT CALLED THE SELECTIVITY FILTER THAT LOOKS LIKE THIS, I WON'T TALK ABOUT IT OTHER THAN TO SAY IT IS WHAT MAKES THEM ALL MEMBERS OF THE SAME FAMILY. IT ALLOWS POTASSIUM TO CONDUCT ACROSS THE MEMBRANE AND SODIUM NOT TO CONDUCT. IF YOU LOOK AT STRUCTURES OF THIS FAMILY, YOU LOOK AT THEM FROM FAR AWAY AND YOU REALIZE THAT OUTSIDE OF THAT LITTLE ELEMENT THAT MAKES THE SELECTIVITY FILTER YOU CAN SEE THAT THERE ARE MORPHOLOGICALLY VERY DIVERSE FAMILY. RANGING FROM VERY LITTLE SMALL ONES HERE THAT LOOK LIKE THIS, TO REALLY BIG ONES UP HERE, JUST A VERY VARIED FAMILY. SO WHAT'S GOING ON HERE AND WHAT'S OUR CURRENT UNDERSTANDING OF WHY THIS IS SUCH A BROAD FAMILY? IT'S MY WAY AS A BIOPHYSICISTS OF LOOKING AT THIS, IS WHAT'S HAPPENED IS I DESCRIBE POTASSIUM CHANNELS AS SETTING THE MEMBRANE POTENTIAL AND REGULATING EXCITATORY PROCESSES. THERE ARE MANY WAYS THAT A CELL WANTS TO REGULATE AN EXCITATORY PROCESS. AND THIS CHANNEL FAMILY HAS EVOLVED TO HAVE MANY DIFFERENT MEMBERS THAT RESPOND TO DIFFERENT ENVIRONMENTAL STILLLY. THERE ARE ONES I CALL CHEMICAL FORCES CAUSE THEM TO OPEN, A LIGAND GATED CHANNEL, A LIGAND IN RED BINDS TO THE CLOSED FORM AND CAUSES IT TO OPEN. THAT MIGHT BE A SEED YUM ION -- SODIUM ION OR CALCIUM ION THAT COMES IN DURING EXITATION AND THEN THE EXITATION IS SHUT OFF BY THESE IONS BINDING TO THE CHANNEL AND CAUSING IT TO OPEN. THERE ARE MECHANICAL FORCES OPEN CERTAIN POTASSIUM CHANNELS SO THERE ARE -- THAT MEANS YOU PUSH ON THE MEMBRANE AND THEY OPEN. AND WE'RE STILL TRYING TO UNDERSTAND WHY THAT OCCURS AND HOW THAT OCCURS. THEN THERE ARE ION CHANNELS WHERE SAY ELECTRICAL FORCES CAUSE THEM TO OPEN AND THOSE ARE THE VOLTAGE DEPENDENT ION CHANNELS. IN THAT FIRST SLIDE OF THE CELL, I SAID THAT ION CHANNELS OPEN AND THE IONS MOVE DOWN THEIR CHEMICAL GRADIANT AND SET THE MEMBRANE VOLTAGE BUT THERE ARE THEN CHANNELS THAT ACTUALLY THE VALUE OF THE MEMBRANE VOLTAGE DETERMINES WHETHER THEY'RE OPEN SO THEY'RE RECURSIVE ON THEMSELVES. THOSE ARE THE KIND OF CHANNELS THAT MAKE ACTION POTENTIALS THAT ARE IMPORTANT IN NEURONS FOR PROPAGATING SIGNALS LONG DISTANCES. SO THE IDEA IS THIS FAMILY IS VERY BROAD AND DIVERSE BECAUSE THERE ARE MEMBERS THAT RESPOND TO DIFFERENT ENVIRONMENTAL STIMULI AND IN A SENSE YOU CAN SAY THERE'S A BASIC PORE AND SELECTIVITY FILTER BUT THEN THAT WAS EMBELLISHED WITH OTHER STRUCTURAL ELEMENTS THAT ALLOWED THAT PORE TO RESPOND TO THE DIFFERENT STIMULI. SO I'LL TALK ABOUT TWO MEMBER HEARSAY OF THIS, FIRST ONE IS HERE. TWO MEMBERS OF THIS FAMILY AS EXAMPLES, AND FIRST IS CALLED KIR 3 OR G PROTEIN GATED INWARD RECTIFIER POTASSIUM CHANNEL OR GERC FOR SHORT. THIS KIND OF CHANNELS ROLE IN BIOLOGY IS EXHIBITED HERE. SO THERE ARE TWO EXAMPLES. ON THE TOP OF THIS SLIDE I SHOW A CARDIAC PACEMAKER CELL, ISOLATED FROM THE ATRIUM, THE ESSAY NODE REGION FROM A MOUSE PUT IN A DISH AND THIS IS VOLTAGE AND TIME. THESE LITTLE BLACK SPIKES ARE ACTUALLY DEPO HAIRIZATION, ACTION POTENTIALS AND THIS CELL SPIKES ON A VERY REGULAR BASIS. IN FACT THAT'S THE KIND OF CELL THAT SETS THE PACE IN THE HEART. BUT OF COURSE OUR HEART CAN SPEED UP, OUR HEART CAN SLOW DOWN. AND THE SLOWING DOWN OF OUR HEART IS MEDIATED BY THE STIMULATION OF A CERTAIN KIND OF RECEPTOR. SO IT'S SHOWN HERE ON THIS CELL IF YOU ADD ACETYL CHOLINE, THE NEUROTRANSMITTER TRANSMITED FROM THE VAGUS NERVE THAT SLOWS THE HEART, NOT ONLY IS THE CELL SLOW BUT THIS EXITATION SLOWS IT ACTUALLY STOPS HERE, THAT'S BECAUSE THIS CELL IS ISOLATED IN A DISH AND IT'S NOT UNDER BOTH THE PARASYMPATHETIC AND SYMPATHETIC SIDES OF THE AUTONOMIC NERVOUS SYSTEM SO PURE ACETYLCHOLINE ON HERE CAUSES A HYPERPOLARIZATION AND ONLY AFTER IT'S REMOVED AFTER A WHILE, THE PERIODIC EXITATIONS RETURN. HERE IS A VERY SIMILAR PHENOMENON IN A MOUSE HIPPOCAMPAL NEURON, AND IN THIS CASE IT'S NOT ACETYLCHOLINE IT'S BACLAFIN, AN AGONIST FOR THE GABBA RECEPTOR, G PROTEIN COUPLED RECEPTOR, HERE WHAT'S HAPPENING IS THIS NEURON IS DEPOLARIZED SO THAT THRESHOLD IS REACHED MEANING POTENTIAL AT WHICH THIS NEURON WILL SPONTANEOUSLY SPIKE. SO IT STARTS TO SPIKE THEN YOU COME WITH BACLAFIN AND YOU ACTIVATE THIS RECEPTOR G PROTEIN COUPLED RECEPTOR, IT HYPERPOLARIZES, THE SPIKING STOPS AND INSTEAD OF TAKING THE BACLAFIN AWAY YOU ADD INHIBIT TO OF A PARTICULAR POTASSIUM CHANNEL THE KIR 3 OR GIRC POTASSIUM CHANNEL AND THE SPIKING RETURNS. THAT SHOWS US THAT ACTUALLY WHAT THE AGONIST IS DOING BY STILL HATING THE G COUPLED PROTEIN RECPTOR, HYPERPOLARIZING THE MEMBRANE BELOW THE THRESHOLD SO IT NO LONGER SPIKES. SO WE KNOW ABOUT THIS PATHWAY. IN GENERAL. BOTH THE HEART AND THE NERVOUS SYSTEM A G PROTEIN COUPLED RECEPTOR IS STIMULATED BY NEUROTRANSMITTER AND WHEN THAT'S -- WHEN THE GPCR IS ACTIVATED, IT CATALYZES THE EXCHANGE OF GDP TO GTP ON THE ALPHA SUBUNIT, THE G PROTEIN SUBUNITS THEN RELEASED AND THEY COME AWAY FROM THE GPCR AND ACTIVATE THEIR TARGETS. IF THIS IS GI COUPLED RECEPTOR, THE TARGET, THE SECOND MESSENGER IS THE BETA GAMMA SUBUNIT AND TARGET IS GIRC POTASSIUM CHANNEL. SO THIS CARTOON SHOWS A SCHEMATIC WITH STRUCTURES OF ALL THE COMPONENTS. WHAT I WANT TO ADDRESS HERE IS WHAT HAPPENS TO THIS POTASSIUM CHANNEL WHEN IT MEETS THE BETA GAMMA SUBUNIT OF THE G PROTEIN. THEN WHAT HAPPENS WHEN THIS COMES OVER HERE, THIS ACTIVATES THE POTASSIUM CHANNEL UNTIL THE ALPHA SUBUNIT HAS HYDROIZED THE GPP BACK TO GDP WHICH POINT THE ALPHA SUBUNIT GETS HIGH AFFINITY FOR THE BETA GAMMA, AND BY MASS ACTION REMOVES IT FROM THE POTASSIUM CHANNEL, IT DIFFUSES AROUND AS THEN ENGAGES GPCR AND AGAIN READY TO BE ACTIVATED. SO WHAT I WANT TO FOCUS ON IN THE FIRST PART OF THE TALK IS THE PROCESS HOW THE BETA GAMMA ACTIVATES THE POTASSIUM CHANNEL. THERE ARE THREE LIGANDS SHOWN HERE. PIC 2, CALLED SIGNALING LIPID, AND ALMOST ALL MEMBERS OF THIS POTASSIUM CHANNEL FAMILY ARE DEPENDENT ON -- IN OBLIGATORY WAY OPT PRESENCE OF THAT. I SHOW BETA GAMMA, THE G PROTEIN SUBUNIT, SUBUNITS THAT ARE COME ALWAYS TOGETHER ADS A SINGLE COMPLEX AND SODIUM IONS. THESE ARE THE TWO LIGANDS THAT I'M GOING TO TALK ABOUT AND HOW THEY SEEM TO ACT ON THE POTASSIUM CHANNEL. SO HERE IS A PICTURE OF THE GIRC POTASSIUM CHANNEL, A CRYSTAL STRUCTRE. THIS PART GOES THROUGH THE MEMBRANE, THIS WOULD BE OUTSIDE THE CELL, AND THIS WOULD BE INSIDE THE CELL. THIS PROJECTS INTO THE CELL. THIS HAS PIP 2 PRESENT BUT NOT BETA GAMMA AND IT IS CLOSED. AND THIS RED LINING IS SHOWING THE LINING OF THE PORE. IT IS A CRYSTAL STRUCTURE IN ABSENCE OF BETA GAMMA AND IT'S CLOSED. HERE IS THE CRYSTAL STRUCTURE OF THE POTASSIUM CHANNEL IN THE PRESENCE OF BETA GAMMA SUBUNITS. WHAT YOU SEE AGAIN IS THE SAME POTASSIUM CHANNEL, THIS IS THE PART THROUGH THE MEMBRANE, HERE IS PIP 2 IN MAGENTA SODIUM IONS AND HERE ARE THE BETA GAMMA SUBUNITS. THE POTASSIUM CHANNEL IS A TETRAMER BE FOUR SUBUNITS AND YOU SEE THAT EACH SUBUNIT ARE ACTUALLY AT THE INTERFACE BETWEEN EACH SUBUNIT, THERE'S A BETA GAMMA G PROTEIN UNIT BOUND TO IT SO A FOUR TO ONE STOICHIOMETRY POTASSIUM CHANNEL. A VERY BEAUTIFUL STRUCTURE AND IN FACT QUITE REMARKABLE THAT IT COULD HAVE BEEN SOLVED IN CRYSTAL FOR DETAILS THAT I WON'T GET INTO NOW. IT WAS A MANY YEAR EFFORT AND FINALLY ACCOMPLISHED BY MATT WARTON, A POST-DOC IN THE LAB AT THE TIME AND NOW RUNS HIS OWN LAB AT THE INSTITUTE IN PORTLAND OREGON. SO THE G PROTEINS BIND, WHAT I WANT TO ADDRESS IS FUNCTIONALLY HOW DOES THIS OPEN THE CHANNEL, HOW MANY DO YOU NEED? IS ONE ENOUGH? DO YOU NEED FOUR? WHAT'S THE AFFINITY? WHAT IS SODIUM DOING? THESE MIGHT -- THESE ARE SIMPLE QUESTIONS, YOU MIGHT WONDER WHY WE DON'T KNOW THE ANSWER UNTIL NOW. BUT IT WILL BECOME APPARENT. WHY THIS WAS NOT AN EASY THING TO ADDRESS. IN A SENSE WE HAVE A PROTEIN WITH ACTIVITY AND WE HAVE LIGANDS THAT ACTIVATE WHY CAN'T YOU TITRATE IT AND WHY DON'T WE KNOW THE ANSWER TO THIS. IT'S A TRICKY THING. BETA GAMMA SUBUNITS ARE BOUND SO TOP ARE UP AGAINST THE MEMBRANE AND IT TURNS OUT THE GAMMA SUBUNIT IS LIP DATED SO REALLY THESE ARE MEMBRANE PROTEINS AND HARD TO CONTROL OR KNOW CONCENTRATIONS. THAT'S ONE OF THE PROBLEMS WITH NOT KNOWING THE AFFINITY OF THESE AND HOW DO YOU GET AT THAT. WHAT HAPPENS WHEN BETA GAMMA BINDS IS THIS. HERE IS SHOWING A ROTATION OF THE CYTOPLASMIC COMPONENT THAT UNWRAPS THE LILIESES HERE TO MAKE THE GATE SO YOU CAN MAKE ROTATION OF THE PART BETWEEN BOUND, THIS WOULD BE BETA GAMMA BOUND, NOT BOUND. YOU WILL SEE WHEN BETA GAMMA BINDS IT CAUSES A ROTATION THAT EXPANDS THE HELICES TOWARDS THE OPEN CONFIRMATION. SO WE CAN SEE MECHANICALLY THAT THE CHANNEL OPENS BUT HOW MANY BETA DOES IT TAKE IS ONE ENOUGH? DO YOU NEED FOUR, DO THEY BIND INDEPENDENTLY WITH WHAT AFFINITY HOW DO YOU GET AT THAT? THE SYSTEM IS A SYNTHETIC LIPID MEMBRANE SYSTEM, THE SYSTEM THAT SCIENTISTS USED TO STUDY THIS PROCESS FOR SEVERAL DECADES ARE CELLS WHOLES CELLS ELECTRICALLY OR EXCISING MEMBRANE PATCHES. THE GOOD THING ABOUT WHOLE CELLS IS EVERYTHING YOU NEED FOR THE PROPER RESPONSE IS THERE. SO IF YOU STUDY A CARDIAC PACEMAKER CELL IT HAS EVERYTHING IT NEEDS. YOU DON'T KNOW WHAT'S IN THERE AND YOU DON'T KNOW HOW MUCH OF THE COMPONENTS ARE IN THERE. SO IN A SYNTHETIC LIPID SYSTEM MEMBRANE SYSTEM YOU HAVE TWO CHAMBERS AND A LITTLE PARTITION. ON THAT PARTITION YOU PAINT A LIPID MEMBRANE THEN RECONSTITUTE COMPONENTS IN THERE SO YOU KNOW WHAT LIPIDS ARE THERE. THEY'RE SYNTHETIC OR ISOLATED, YOU KNOW EVERY COMPONENT, YOU ADD THE MEMBRANE PROTEINS YOU ADD THE G PROTEINS YOU ADD EVERY O -- THE PIP 2 AND YOU KNOW HOW MUCH OF WHAT YOU PUT IN THERE. HERE IS AN EXAMPLE OF EXPERIMENT YOU CAN DONA SYNTHETIC SYSTEM YOU CAN'T DO IN A CELL, YOU MAKE MEMBRANES WITH DIFFERENT MOLE FRACTIONS OF PIP 2 IN THEM AND YOU CAN LOOK HOW PIP TWO WILL ACTIVATE THE CHANNEL IN THE PRESENCE OF G PROTEINS AND OTHER NECESSARY COMPONENTS, YOU CAN LOOK AT THE PIP 2 DEPENDENTS. IF YOU HAVE HAMS IN THE MEMBRANE -- CHANNELS IN THE MEMBRANE AND YOU HAVE A ALPHA SUBUNIT BOUND TO GDP AFTER STIRRING ARTIFACT YOU GET NO ACTIVATION OF POTASSIUM CURRENT BUT IF YOU ADD THE BETA GAMMA SUBUNITS AFTER STIRRING ARTIFACT YOU SEE THE POTASSIUM CHANNELS TURN ON. SO THIS IS VERY NICE CONTROLLED SYSTEM. THEY TURN ON UNTIL YOU APPLY ALPHA GDP THEN SHUT OFF LIKE HAPPENS IN A CELL BECAUSE THE ALPHA NOW HAS HIGH AFFINITY FOR THE BETA GAMMA SUBUNIT AND COMPETES AWAY FROM THE CHANNEL. SO THAT'S HOW YOU DO THIS BUT IN FACT, WITH THIS KIND OF SYSTEM YOU CAN LOOK AT THE SODIUM DEPENDENCE OF ACTIVATION AND LOK AT PIP 2 DEPENDENCE IN THIS WAY BUT ACTUALLY, THE BETA GAMMA DEPENDENTS TAKES AN EXTRA TRIP, THAT'S WHAT I WANT TO SHOW YOU. THE PROBLEM IS YOU CAN MAKE A MEMBRANE WITH KNOWN MODEL FRACTION OF PIP 2 BECAUSE YOU MIX TOGETHER BUT YOU CAN'T DO THE SAME WITH THE BETA GAMMA, THE BETA GAMMA IS A PROTEIN AND WHEN YOU MAKE THE MEMBRANE YOU HAVE LIPID IN DECANE AND YOU PAINT IT ON TO ORGANIC MIXTURE WHICH YOU CAN'T ADD A PROTEIN TO. SO THE PROBLEM THEN WITH THE BETA GAMMA SUBUNIT IS TO WORK, IT HAS TO BE LIPIDATED AND BOUND TO MEMBRANE, IT'S ITSELF INSOLUBLE SO YOU CAN'T CONTROLLABLY PUT A KNOWN AMOUNT OF BETA GAMMA IN THE MEMBRANE. HERE IS THE WAY YOU GET AROUND THIS. INSTEAD YOU MAKE A LIPID MEMBRANE WITH NICKEL NTA KNOWN ORGANIC MIXTURE MAKE THE MEMBRANE, NOW YOU TAKE YOUR BETA GAMMA SUBUNIT, REPLACE THE LIPID GROUP WITH A HIS TAG AND THAT WILL THEN BIND TO THE NICKEL NTA HEAD GROUP, IN OTHER WORDS STICK TO THE MEMBRANE, AND SO YOU CAN THEN CONTROL THE CONCENTRATION OF BETA GAMMA IN MEMBRANE. HERE IS WHAT THE EXPERIMENT LOOKS LIKE. SO HERE IS CURRENT AND TIME AND THIS EXPERIMENT IN THE BLACK TRACES THE NICKEL NTA LIPID MOLE FRACTION IS ZERO. WHEN YOU ADD THE CURRENT LEVEL IS ZERO, YOU HAVE TWO MICROMOLARS SOLUBLE HIS 10 BETA GAMMA YOU GET ESSENTIALLY NO CURRENT, A LITTLE NOISE BUT SATURATE WITH THE SAME MEMBRANE WITH LIPIDATED BETA GAMMA AND YOU KNOW YOU HAD CHANNELS IN THE MEMBRANE NOT ACTIVATED BY SOLUBLE BETA GAMMA. NOW REPEAT THIS EXPERIMENT WITH A NEW MEMBRANE WITH A CERTAIN MOLE FRACTION OF NICKEL NTA LIPID SO THAT'S ABOUT TWO IN EVERY -- TWO LIPID MOLECULES MOLE FRACTION .0019. TWO IN 10,000 LIPID MOLECULES ARE -- HAVE NICKEL NTA, TWO IN A THOUSAND HAVE NICKEL NTA HEAD GROUP SO YOU COME WITH YOUR TWO MICROMOLAR BETA GAMMA AND YOU GET AMOUNT OF CURRENT, AND NOW YOU SATURATE TO FULLY -- YOU PUT -- YOU NOW PUT LIPIDATED BETA GAMMA IN MEMBRANE TO FULLY ACTIVATE THE CHANNELS AND THEN SAY OKAY, .6 OF THE MAXIMUM ACTIVATION OCCURRED UNDER THAT CONDITION. SO THERE'S -- THAT'S HUH YOU DO THIS EXPERIMENT BUT YOU LOOK AND SAY WAIT A MINUTE, HOW DO YOU KNOW WHETHER YOU GOT THIS LEVEL BECAUSE YOU DIDN'T HAVE ENOUGH NICKEL NTA LIPID IN THE MEMBRANE OR BECAUSE YOU HAD ENOUGH BUT YOU DIDN'T FULLY SATURATE THE NICKEL N TA WITH THE SOLUBLE BETA GAMMA. IN OTHER WORDS TWO EQUILIBRIUM HERE, ONE BETWEEN LIPID AND SOLUBLE BETA GAMMA AND THE OTHER BETWEEN TETHERED BETA GAMMA IN THE CHANNEL. BETA GAMMA IN SOLUTION DOES NOT ACTIVATE THE CHANNEL DIRECTLY. YOU CAN GET AT THAT -- GET THE ANSWER TO THAT QUICKLY BY THE FOLLOWING EXPERIMENTS. SO THIS IS SHOWING THIS NORMALIZED CURRENT AS A FUNCTION OF SOLUBLE BETA GAMMA AT THE KNOWN LIPID MOLE FRACTION. WHAT YOU CAN SEE IS THAT IT SATURATES AT VALUE OF A LITTLE BIT ABOVE .6. IT DOESN'T GO TO ONE. WHY DOESN'T IT GO TO ONE? IT SATURATED SO IT MUST BE THAT YOU FULLY SATURATED THE LIPID IN THE MEMBRANE BUT YOU DIDN'T HAVE ENOUGH OF THEM. IN THE MEMBRANE TO FULLY ACTIVATE THE CHANNEL. YOU CAN THEN TEST THAT BY INSTEAD OF HAVING A FIXED MOLE FRACTION OF LIPID, INCREASING THE SOLUBLE BETA GAMMA, INSTEAD WHAT YOU DO IS YOU HAVE A FIXED SOLUBLE CONCENTRATION. FIXED SOLUBLE CONCENTRATION, MAKE DIFFERENT MEMBRANES WITH DIFFERENT MOLE FRACTIONS AND HERE YOU CAN SEE ACTIVATION IS ASYSTEM TOTTIC TO ONE. JUST AS YOU EXPECT IF YOU GET ENOUGH SATURATED LIPID IN THE MEMBRANE, YOU ACTIVATE THE CHANNEL. IN FACT YOU CAN NOW TAKE THESE TWO GRAPHS AND MAKE ANOTHER GRAPH. YOU CAN SAY WELL, I HAVE THIS RELATIONSHIP AND THIS RELATIONSHIP. LET ME PLOT THIS AXIS, THIS X AXIS AGAINST THIS AXIS AND WHAT I'M GOING THE LOOK AT IS SATURATION OF THE LIPID IN THE MEMBRANE. THIS IS ACTUALLY BASICALLY A ISOTHERM SHOWING THE SATURATION OF LIPID IN THE MEMBRANE WITH BETA GAMMA. WHAT IT TELLS YOU IS IF YOU HAVE THIS HIS 140 TAGS TWO MICROMOLAR CONCENTRATION OF SOLUBLE BETA GAMMA YOU WILL SATURATE THE LIPIDS IN THE MEMBRANE. SO THERE'S A LITTLE FUNNY RESULT SO I WILL SHOW YOU THAT ACTUALLY HAS A QUANTITATIVE INTERPRETATION THAT IS IMPORTANT WHAT HAPPENS IF I DO THE SAME EXPERIMENT WITH A HIS 4 TAG INSTEAD OF HIS 10 TAG? YOU GET A VERY FUNNY RESULT AT FIRST. WITH HIS 4 BETA GAMMA YOU SEE IN FACT YOU'RE ASIMILAR TOTTIC TO A HIGHER VALUE BUT AFFINITY IS LOWER. YOU CAN SEE WHAT'S GOING ON IF YOU DO THE SAME ANALYSIS HERE. INSTEAD WE NOW MAKE THE GRAPH THE EQUIVALENT TITRATION GRAPH WITH HIS 4. WHAT YOU CAN SEE IS WHEN YOU USE HIS 4 BETA GAMMA, YOU GET LOWER AFFINITY BINDING TO THIS MEMBRANE BUT THE MAXIMUM VALUE WITHIN ERROR IS THREE TIME IS WHAT FOR THE HIS TEN, THREE TIME AS MUCH BETA GAMMA IN THE MEMBRANE. REALIZE WHAT'S HAPPENING BY APPEALING TO CRYSTAL STRUCTURES WITH POLYHISTOTEEN WITH NICKEL NTA GROUPS. THERE'S A RULE THE NICKEL NTA GROUP IS COORDINATED BY TWO HISTIDINES AND THE TWO HISTIDINES HAVE TO BE AT LEAST ONE HISTIDINE APART SO AT LEAST ONE IN THREE POSITION. THEN YOU REALIZE A HA, A HIS 10 CAN BIND UP TO THREE NICKEL NTAs AND IT DOES. WHEREAS A HIS 4 CAN ONLY BIND ONE SO YOU HAVE A FIELD OF NICKEL NTA AND HIS 4 IS LOWER AFFINITY BUT THREE TIMES AS MANY IN THE MEMBRANE. WHEN YOU USE HIS 10, EACH HIS 10 TAG ON THE BETA GAMMA RECRUITS THREE LIPIDS, BINDS WITH HIGH AFFINITY. SO WHAT THIS COMES DOWN TO IS THE FOLLOWING WE NOW HOW MUCH BETA GAMMA WE'RE PUTTING ON THE MEMBRANE. WE KNOW IF WE USE TWO MICROMOLAR HIS 10 FULLY SATURATE THE MEMBRANE, YOU KNOW THE CONCENTRATION OF THE MEMBRANE BECAUSE IT'S CONCENTRATION OF TWO DIMENSIONAL CONCENTRATION OF THE HIS 10 -- TWO DIMENSIONAL CONCENTRATION OF THE NICKEL NTA LIPID DIVIDED BY THREE BECAUSE EACH USES UP TO THREE LIPIDS SO WITH THIS BACKGROUND YOU CAN GET TO THE EXPERIMENT I WANT TO SHOW YOU. THAT IS, HERE IS A GRAPH SHOWING THE NORMALIZED CURRENT SO ACTIVATION OF THE CHANNEL AS A FUNCTION OF NICKEL NTA LIPID AND THE SODIUM CONCENTRATION. I TOLD YOU I WANTED TO GET TWO LIGANDS, YOU WILL SEE WHY. I CAN'T VERY WELL BUT HOPE YOU CAN. THESE SETS OF CURVE VERSUS A SURFACE AN THESE SURFACE CORRESPONDS TO A MODEL THAT I'LL EXPLAIN IN A MINUTE. ANOTHER REPRESENTATION OF THIS SAME SURFACE IS SLICES ALONG FIXED CONCENTRATION OF SODIUM HERE. SO NORMALIZED CONCENTRATION OF LIPID NTA MOLE FRACTION, THE UNIT I'M USING FOR THE BETA GAMMA CONCENTRATION ON THE MEMBRANE. AND WHAT YOU'RE SEEING IS VERY STEEP ACTIVATION CURVES THAT GET SHIFTED AT DIFFERENT SODIUM CONCENTRATIONS. I'LLMENT CAN BACK THE THAT. SO THE MODEL IS THE SIMPLEST IN THIS SYSTEM. WE SEE FOUR BETA GAMMAS BOUND. THE MODEL SAYS LET'S ALLOW ONE, TWO, THREE, FOUR BETA GAMMAS, ONE, TWO, THREE, FOUR SODIUMS TO BIND INDEPENDENTLY IF THEY WANT, WE CAN PHUT IN COOPERATIVETY TERMS TO SEW IF COOPERATIVE. YOU ASK HOW MANY DO YOU NEED? HOW MANY DO YOU NEED TO OPEN THIS POTASSIUM CHANNEL? HOW MANY ARE REQUIRED? WHETHER ARE AFFINITIES? THE ANSWER IS YOU NEED FOUR BETA GAMMAS TO OPEN IT AND STATISTICALLY -- THE -- YOU CAN'T GET THEM TO WORK WELL EVEN IF YOU SAY LET THREE OPEN, AT LEAST THREE. SO YOU NEED FOUR. MOREOVER THEY BIND IN A HIGHLY COOPERATIVE MANNER. SO THE KD FOR EACH SEQUENTIAL BETA GAMMA IS .3 TIME IT IS KD, KD OF THE THIRD BETA GAMMA IS .3 TIME IT IS KD OF THE SECOND, IS .3 TIME IT IS BETA GAMMA OF THE FIRST WHICH MEAN IT IS FOURTH BINDS WITH 37 -- MEANS THE FOURTH BINDS 37 TIMES HIGHER AFFINITY THAN THE FIRST SO HIGHLY COOPERATIVE WITH ETCH OOH OTHER. THE FACT THAT YOU NEED FOUR AND THE FACT THAT THEY'RE COOPERATIVE IS THE REASON WHY YOU GET THESE EXTREMELY STEEP ACTIVATION CURVES SO YOU MIGHT LOOK AND SAY THEY ACTIVATE BUT REALIZE THIS IS ALMOST SQUARE SHAPED. AND IT'S BECAUSE OF THE HIGH STOICHIOMETRY, AND EVEN IF THERE WERE NOT COOPERATIVETY, THIS WOULD BE SIGMOIDAL BECAUSE IF IT REQUIRES -- IT'S A POWER 4 RELATION BUT ON TOP OF THAT, YOU HAVE THE COOPERATIVETY WHICH MAKES IT ALMOST A SQUARE STEP. AND THE SIGNIFICANCE OF THAT WILL COME WHEN WE LOOK AT CONSIDER MANY MINUTE THE EFFECT OF SODIUM AND HOW IT SEEMS TO SHIFT THESE CURVES TO THE LEFT. AND MAKE THEM STEEPER. BUT FIRST, LET ME JUST GIVE YOU AN INTUITIVE PICTURE OF THIS COOPERATIVETY. RECALL THAT I SHOWED YOU THE STRUCTURE OF GIRC IN ABSENCE OF BETA GAMMA AND IN THE PRESENCE OF THE BETA GAMMA WITH THIS MORPH THAT SHOWED THE CONFIRMATIONAL CHANGE THAT TAKES PLACE BY ENTERPOE LATING BETWEEN THE CLOSED AND THE OPEN STRUCTURE. SO HERE IS A CARTOON VERSION OF THAT. HERE IS A CLOSED GIRC CHANNEL IN AN OPEN ONE AND IN THIS PICTURE IS -- SUGGESTIVE BY SHOWING BETA GAMMA FITTING WELL THE OPENERS BUT NOT THE CLOSED. IMAGINE IF THAT IS THE CASE AND THE FACT THAT THIS HAPPENS AS REGION BODY ROTATION IN THE CRYSTAL STRUCTURE, HIT MAKES SENSE THIS IS ALL OR NONE. ONCE IT ROTATES ALL FOUR BINDING SITES ARE RECEPTIVE ARE A GOOD FIT WHEN IT ROTATES BACK, THEY'RE NOT. SO THIS SIMPLE PICTURE GIVES RISE TO THIS VERY INTUITIVE FEEL FOR THE ORIGIN OF THE HIGH COOPERATIVETY. IN THIS SYSTEM. INCREASED SODIUM, THE MAXIMUMS INCREASE AND ALSO THE CURVES IN THE STEEP PART SHIFT TO LOWER BETA GAMMA CONCENTRATIONS. WHAT'S HAPPENING IN THE MODEL SODIUM BINDING -- THE PRESENCE OF SODIUM CAUSES BETA GAMMA TO BIND WITH HIGHER AFFINITY. SUCH THAT A CHANNEL WITH FOUR SODIUMS BOUND, BETA GAMMA WILL BIND SEVEN TIMES HIGHER AFFINITY. SO IT'S THIS CROSS COOPERATIVETY BETWEEN SODIUM AND BETA GAMMA. AND THAT CAUSES THIS SHIFT. NOW, THE SIGNIFICANCE OF SODIUM IN THIS SYSTEM, IS THE FOLLOWING. THIS IS AN INHIBITORY SIGNAL. SO WHEN FOR EXAMPLE IN THE NERVOUS SYSTEM, GAB BAY IS RELEASED ON TO THE BABB BAY G RECEPTOR -- GABA B RECEPTOR, IT'S ACTIVATED AND LEADS TO POTASSIUM CHANNEL WHICH HYPERPOLARIZES THE NEURON AN SILENCES IT. WHEN DOES SODIUM CHANGE IN A NEURON? A HIGHLY EXCITED NEURON HAS SODIUM ENTERING AS BASIS OF EXITATION. AND NEURONS WITH HIGH EXCITABILITY GET HIGHER SODIUM UNDER THE MEMBRANE, PARTICULARLY IN THE DENDRITES, AND IN LONG EXTENSIONS BUT EVEN DOCUMENTED WELL IN THE CELL BODY SO A HIGHLY EXCITED NEURON HAS A HIGH CONCENTRATION OF SODIUM. WHICH WOULD MEAN THAT THE INHIBITORY INPUT DUE TO STIMULATION OF GABA B RECEPTOR IS STRONGER BECAUSE OF SHIFT IN THE CURVE. THAT'S THE IDEA, THAT'S WHAT WE THINK IS THE BIOLOGICAL SIGNIFICANCE OF THE SODIUM EFFECT. IT SAYS THAT A MORE EXCITED NEURON WILL GET MORE INHIBITION. SO IF YOU LOOK AT THIS YOU SAY WELL HOW MUCH MORE? THAT WOULD DEPEND ON WHERE YOU ARE ON THIS X AXIS, INSIDE A CELL WHEN YOU STIMULATE THE G PROTEIN COUPLED RECEPTOR. HOW MUCH BETA GAMMA IS RELEASED ON TO THE MEMBRANE BY THE GPCR? IF YOU ARE OUT HERE, SUPPOSE AT A PHYSIOLOGICAL INTERNAL CONCENTRATION OF FOUR AND YOU GO UP TO 16, YOU WOULD SAY IT INCREASES BUTNA'S NOT VERY SUBSTANTIAL. YOU MIGHT HAVE HAD A 20 OR 30% INCREASE IN YOUR POTASSIUM CONDUCTANCE. MAYBE THAT WILL HAVE AN EFFECT, MAYBE NOT. BUT IF YOU'RE DOWN HERE IN THIS STEEP PART YOU CAN HAVE A VERY, VERY LARGE EFFECT. SO IT REALLY DEPENDS ON WHERE YOU ARE ON THIS ACTIVATION CURVE. IN OTHER WORDS, THIS IS GENERATED IN THE LAB, AND A BILAYER SYSTEM BUT WHERE ARE YOU IN A CELL? THEN THINKING ABOUT THIS, MADE US REALIZE THIS IS INTERESTING, WE CAN WORK BACKWARDS USING THESE DATA TO FIGURE OUT HOW MUCH DATA IS GENERATED INSIDE A CELL. IN FACT, THIS EXPERIMENT KIND OF CAME UP WHILE I WAS PADDLING WITH MY FRIEND AND PLEAING BRUCE BEAN TELLING HIM ABOUT THESE RESULTS AND HE SAYS THAT REMINDS ME OF SOMETHING WE HAVE SEEN. SO HERE IS THE EXPERIMENT THAT WAS CARRIED OUT IN BRUCE BEAN'S LAB. THAT WHEN YOU THEY LOOKED AT A BUNCH OF NEURONS FROM THE SUBSTANTIA NIGRA DOPAMINE NEURONS THEY ADD BACLAFIN. YOU CAN SEE THIS IS CURRENT, YOU CAN SEE HYPERPOLARIZATION. HERE IS A CASE WITH ZERO MILLIMOLAR SODIUM IN THE PIPETTE SO WHOLESALE RECORDING YOU PUT THE PIPETTE ON THE CELL IN THE SOLUTION EXCHANGES INSIDE THE CELL. SO IN THIS CASE IT HAS NO SODIUM IN THE PIPETTE OR INSIDE THE CELL. HERE IS A CASE WITH 27 MILLIMOLAR SODIUM CHLORIDE. YOU CAN SEE THE CURRENT NOW TO THE SAME AMOUNT OF BACLAFIN IS LARGER, IT'S DIFFERENT CELL. SO THEY DO A BUNCH OF CELLS IN ZERO SODIUM AND A BUNCH WITH 27 MILLIMOLAR. ON AVERAGE YOU GET A MUCH LARGER RESPONSE IN 27 MILLIMOLAR. SO THEN YOU TAKE THE CURVES FROM THE BILAYER EXPERIMENTS THAT 32 AND 0 AND 32 IS SUPPOSED TO BE 27 MILLIMOLAR BUT THAT'S THE MISTAKE THAT HAPPENS WHEN YOU COMMUNICATE BETWEEN NEW YORK AND BOSTON BIT'S CLOSE ENOUGH. SO YOU NOW GENERATE A CURVE THAT IS BASICALLY WE CALL AN AMPLIFICATION CURVE WE DIVIDE BY THIS CURVE AND GET THIS CURVE AND THEN YOU ASK WELL, WHAT'S THE FOLD EFFECT AND WHERE DO YOU FALL HERE? SO YOU FALL RIGHT HERE. IT'S AN EIGHT FOLD INCREASE ON AVERAGE, A BIG DISTRIBUTION, NOT ALL NEURONS ARE ALIGNING. AVERAGE EIGHT FOLD THEN YOU SAY HOW MUCH NTA LIPID MOLE FRACTION DOES THAT CORRESPOND TO AND CONVERT IT INTO A G BETA GAMMA CONCENTRATION INSIDE THE CELL. I WON'T GO INTO WHAT THAT ABSOLUTE CONCENTRATION IS RIGHT NOW. I THINK THE SIGNIFICANCE IS, IT'S VERY BEAUTIFUL THAT NOTICE THAT THAT IS FALLING RIGHT IN A VERY STEEP PART OF THESE CURVES. IT'S NEAR THE BOTTOM OF THIS ONE AND NEAR THE TOP OF THIS ONE. SO AS IF THE SYSTEM HAS EVOLVED TO PLACE THE STEEP PART OF THE G PROTEIN BETA GAMMA ACTIVATION CURVES, SUCH THAT CHANGES IN SODIUM WILL AMPLIFY THE RESPONSE WHICH THEN MEANS A NEURON UNDERGOING A LOT OF EXITATION WHEN IT GETS INHIBITORY INPUT, THAT INPUT WILL BE STRONGER DUE TO THE ACTIVATION -- TO THE EFFECT THAT SODIUM IS HAVING ON THE AFFINITY OF BETA GAMMA FOR THE CHANNEL. SO THE CONCLUSION FROM THIS PART OF THE WORK IS THAT THE GIRC CHANNEL HAS FOUR BETA GAMMAS BIND IN HIGHLY COOPERATIVE, THIS GIVES RISE TO THIS STEEP DEPENDENCE OF CHANNEL ACTIVATION ON G BETA GAMMA. INTRACELLULAR SODIUM CONCENTRATIONEN CREASES AFFINITY OF SODIUM IN THE CELL, GPCR STIMULATION STIMULATES G BETA GAMMA CONCENTRATIONS ON STEEP DEPENDENCE SO WELL MATCHED. AND THE ABOVE PROPERTY GIVES RISE TO THIS SODIUM AMPLIFICATION SUCH THAT NEURONAL ACTIVITY WILL MAKE INHIBITOR RESPONSE MUCH STRONGER. THEN THIS I DIDN'T REALLY GET INTO BUT BETA GAMMA IF YOU TURN INTO AFFINITY, IT'S QUITE LOW AFFINITY. IT'S A HIGH CONCENTRATION GENERATED. THIS IS CONSISTENT WITH THE IDEA THAT SOON AS THE GPCR STIMULUS IS TURNED OFF, AND WITHIN THE FEW SECONDS THAT IT TAKES FOR THE ALPHA SUBUNIT TO HYDROLYZE GTP TO GDP, THE STIMULUS IS SHUT OFF ON THE POTASSIUM CHANNEL AND IT'S CLOSED. A QUICK TURN OFF ONCE THE STIMULUS IS REMOVED. SECOND THING I WANT TO TELL YOU, IT'S A DIFFERENT CHANNEL, IT'S A DIFFERENT EXPERIMENT. BUT IT'S KIND OF YOU WILL SEE IT HAS A CONCLUSION. THAT IS VERY CONSISTENT WITH G PROTEIN GATED CHANNEL. THE G PROTEIN GATED CHANNEL IS A SWIPE THAT TURNS ON COOP RA TVLY AND LOOK WHAT HAPPENS -- COOPERATIVELY AND LOOK WHAT HAPPENS IN THIS CASE. THIS IS A NEW A POST-DOC WHO CARRIED THE FUNCTIONAL EXPERIMENTS I JUST TALKED ABOUT. THIS EXPERIMENT I WILL TELL YOU WAS CARRIED BY RICH HEIGHT OWN LAB AT MEMORIAL SLOAN-KETTERING. YOU WILL SEE THE QUESTION WE'RE AFTER, IT INVOLVES A -- THE EXPERIMENT BUZZ CARRIED OUT WITH A CHANNEL CALLED SLOW 2, THE EXPERIMENT WE WERE INTERESTED IN IS THIS. SO WHAT I WANT TO SHOW YOU HERE, IS A SET OF PICTURES. WHEN ELECTROFIZZ IDEAL GIST RECORDS FROM A NEURON, OR CELL WITH CHANNELS LIKE THIS ONE IN IT HERE IS WHAT YOU SEE. YOU SEE IN THIS RECORDING THIS IS CURRENT ON THE Y AXIS TIME ON THE X AXIS, THIS IS A SODIUM ACTIVATED POTASSIUM CHANNEL, IT'S IN THE ABSENCE OF SODIUM ABSENCE OF SODIUM YOU SEE NO CHANNEL ACTIVITY AS YOU RAISE THE SODIUM CONCENTRATION YOU START TO SEE WITH SOME PROBABILITY THE CHANNEL OPEN AND AS YOU RAISE IT FURTHER YOU SEE MORE OPENING AND IN FACT YOU REALIZE THAT THERE ARE AT LEAST TWO CHANNELS IN THAT MEMBRANE. WHAT THE PHYSIOLOGIST SEES IS FLUCTUATIONS, THE RANDOM HOPPING OF THE INK SINGLE MOLECULE ION CHANNELS AND HOW YOU U SEE THE STOCHASTIC NATURE OPENING AND CLOSING AND WHEN WE TALK ABOUT A CHANNEL BEING OPEN, WE'RE NOT -- WE HAVE IN MIND AN OPEN STATE BUT ACTUALLY THE PHYSIOLOGIST KNOWS THAT MEAN THERE'S SOME PROBABILITY IT WILL BE OPENING AND CONDUCTING. SO WE TALK ABOUT OPEN PROBABILITY. IT LOOKS LIKE THIS, YOU CAN CALCULATE THE OPEN PROBABILITY AND MAKE A GRAPH SODIUM SON CONCENTRATION, OPEN PROBABILITY AND IT LOOKS LIKE THAT. THE QUESTION IS WHAT IS GOING ON STRUCTURALLY? WHAT DO YOU SEE? LET'S DEVELOP THIS, AND I WILL JUST SAY THAT ELECTRON MICROSCOPY ENABLES THIS EXPERIMENT BECAUSE IN CRYSTALLOGRAPHY YOU COULDN'T DO IT, CRYSTALLOGRAPHY YOU CAN DO WHAT WE SHOWED YOU, SOLVE THE STRUCTURE OF CHANNEL IN ABSENCE OF LIGAND, SOLVE IN PRESENCE OF LIGAND, AND YOU CAN GET THE TWO CONFIRMATIONS AN SHOW THE MOVIE I SHOWED YOU OF A MORPH BETWEEN THE OPEN AND CLOSED BUT WHAT'S REALLY -- WHAT DOES THAT MEAN? IN TERMS OF THE FLUCTUATIONS AND WHAT DOES IT MEAN IN NUMBER OF CONFIRMATIONS THAT HAPPEN BETWEEN THE CLOSED AND OPEN STATE. THAT'S WHAT WE'RE AFTER HERE. IN CRYSTALLOGRAPHY YOU COULDN'T DO IT BECAUSE A GOOD CRYSTAL REQUIRES IDENTITY OF WHAT'S IN THE UNIT CELL WHICH GENERALLY IMPLIES AN IDENTITY OF STRUCTURE UNDER A GIVEN CONDITION. WHEREAS IN A CRYO-EM EXPERIMENT, YOU ARE TAKING YOUR PROTEINS SPREAD OUT IN SOLUTION NOT TOUCHING EACH OTHER, AND IN A SENSE YOU SHOULD BE ABLE TO GET THE STRUCTURE, ALL STRUCTURES THAT ARE PRESENT IN THE POPULATION OF PROTEINS. IN PRINCIPLE YOU SHOULD DO A TITRATION OF THIS STRUCTURE, THAT'S WHAT WE'RE AFTER HERE. THE GENERAL STRATEGY IS TO COLLECT DATA UNDER DIFFERENT CONCENTRATIONS OF SODIUM AND SOLVE THE STRUCTURES AND USE SOMETHING CALLED CLASSIFICATION TO ASK WHAT PROBABILITY ARE OPEN, WHAT PROBABILITY ARE CLOSED AND WHAT WE WERE PARTICULARLY INTERESTED IN IS HOW MANY STRUCTURES DO WE SEE AND DO WE SEE INTERMEDIATE STEPS ON THE WAY BETWEEN CLOSED AND OPEN. THAT'S WHAT WE'RE AFTER. THE WAY WE DID IT IS COMBINED OUR DATA SETS, FOUR OR FIVE, WE COULDN'T DO ALL FIVE. BECAUSE ONE OF THEM THE FIFTH ONE WAS COLLECTED OWN A DIFFERENT MICROSCOPE AND COULDN'T COMBINE THE DATA BUT WE LIKED THE IDEA OF COLLECTING THE DATA SETS AND THROWING THEM TOGETHER AND PROCESSING THEM TOGETHER THAT WAY WE PRODUCE ANY BIAS WHEN PROCESSING. CLASSIFY THEN ASK WHAT DATA SET DO YOU COME OUT OF, WE ASK THAT LATER. FIRST LET ME SHOW YOU AN OPEN AND CLOSED CONFIRMATION OF THE CHANNEL SO HERE IS THE OPEN VERSION, SHOW AS BLUE AND HERE IS CLOSED VERSION. THIS SHOWS THE LINING OF THE PORE YOU CAN SEE THERE'S A BIG CONFIRMATIONAL CHANGE RIGHT HERE, IN THIS MOVIE. SO THIS IS AGAIN A MORPH BETWEEN THE CLOSED CONFIRMATION AND THE ABSENCE OF SODIUM, AND OPEN CONFIRMATION IN PRESENCE OF HIGH SODIUM. THIS MOVIE DIDN'T COME FROM INTERMEDIATES. I'M SHOWING YOU EXTREMES RIGHT NOW SO YOU HAVE A SENSE OF WHAT WE ARE LOOKING FOR. SO YOU SEE IT GOING BACK AND FORTH BETWEEN THE CLOSED AND THE OPEN STRUCTURE. YOU CAN SEE MECHANICAL EXPANSION IN THIS PART IN BLUE THAT OPENS UP THE HELICES THAT FORMS THE GATE OF THIS CHANNEL INSIDE THE MEMBRANE. THOSE ARE THE END POINTS. BACK TO THIS TITRATION. WHAT YOU DO WHEN YOU CLASSIFY, FIRST IN THIS CASE LITTLE COMMENT ABOUT THIS, IN MOST CRYO-EM STRUCTURES YOU SEE, PEOPLE ARE OFTEN YOU WILL SEE THEM START WITH A VERY LARGE NUMBER OF PROTEIN PROTEIN PARTICLES CALLED AND YOU MIGHT NOTICE THAT THE FINAL STRUCTURE PRESENTED REPRESENTS A SMALL FRACTION OF THE TOTAL PARTICLES. THAT'S BECAUSE THAT SMALL FRACTION WAS CLASSIFIED AND GAVE RIDES TO A BEST STRUCTURE. THAT'S OKAY TO DO. BUT IN THIS CASE WE COULDN'T DO THAT. WE HAVE TO KEEP TRACK OF EVERYTHING. WE CAN'T THROW THINGS OUT. THE ONLY COULDING THING HAPPENED HERE WERE THINGS IN THE BEGINNING THAT ABSOLUTELY PIECES OF ICE NOT PARTICLE AND EVERYTHING ELSE HAS TO BE KEPT BECAUSE WE HAVE TO NORMALIZE TOTAL NUMBER OF PARTICLES TO GET THESE NUMBERS. ONE THING TO BE CAREFUL IN CRYO-EM I SEAL PEOPLE REFERRING TO A CONFIRMATION AS SIGNIFICANT AS A FUNCTION OF THE CONDITIONS UNDER WHICH IT WAS COLLECTED BUT IF YOU REALIZE IT'S ONLY TEN OR SOME FRACTION OF THE PARTICLES THAT'S NOT FAIR BECAUSE IT'S A SUBPOPULATION OF PARTICLES AND YOU DON'T KNOW REALLY WHAT THE WHOLE POPULATION IS TELLING YOU. SO THAT'S SOMETHING YOU HAVE TO BE CAREFUL ABOUT. SO WITH THIS CLASSIFICATION METHOD YOU SEE IN THE FIRST CONCENTRATIONS AND FOUR DATA SETS HERE, YOU CAN SEE IN FACT IF YOU ASK FOR TEN CLASSES WHAT YOU ESSENTIALLY FIND IS ONE UNIQUE CLASS. THE REST ARE ALMOST THE SAME. IN THE HIGHER CONCENTRATION SEPARATELY YOU FIND TWO MAIN CLASSES YOU GET TEN CLASSES BUT COMPARE AND REALIZE THEY'RE ESSENTIALLY TWO UNIQUE CLASSES IN HERE. IF YOU ASK THE FIRST FOUR DATA SETS AFTER THIS CLASSIFICATION, YOU ASK WHERE DID EACH CLASS COME FROM? SO WE ASKED FOR TEN CLASSES AND I JUST TOLD YOU, THERE ARE ONLY TWO UNIQUE ONE BUS RIGHT NOW, IT -- BUT RIGHT NOW LET'S KEEP IT SEPARATE AND SAY WHAT HAPPENS IS A FUNCTION OF SODIUM CONCENTRATION SO PROCESS THEM TOGETHER NOW YOU MAKE THESE GRAPHS AND YOU SAY OKAY WHICH PARTICLES CAME UNDER -- CAME OUT WHAT IS THE FRACTION OF CLASS 3 AT 20 MILLIMOLAR, CLASS 340 MILLIMOLAR, 80, 160, AND NOTICE THIS CLASS THREE IS THE ONLY ONE CHANGING IN A SIGNIFICANT WAY. IT'S GROWING. THE REST ARE NOT DOING ANYTHING. AS A NET THEY'RE DROPPING A LITTLE BIT BECAUSE THEY'RE GOING INTO CLASS 3. BUT THERE'S NO SYSTEMATIC CHANGE OF THE RED CLASSES SO WHAT YOU SEE IS THIS APPEARANCE OF CLASS 3. IF YOU THEN REPEAT OVER AND OVER AGAIN REALLY STARTING AT THE BEGINNING TO LOOK AT THE REPRODUCIBILITY OR PRECISION OF THIS PROCESS, YOU CAN SEE THAT THIS IS WHAT CLASS 3 DOES, CLASS 3 IS ALWAYS, THESE ARE FIVE INDEPENDENT STRUCTURE DETERMINATIONS. YOU CAN SEE CLASS THREE GROWS AS INCREASE SODIUM CONCENTRATION. WHEREAS AS EXAMPLES, CLASS 1 AND 9, IS TO NOT, WHY DID I PICK ONE AND NINE? CLASS THREE IS UNIQUE AND THE OTHER CLAYSES ARE ALL NOT CLASS 3, AND THEY'RE A LITTLE DIFFERENT FROM EACH OTHER, ONLY IN THE ROTATION OF CYTOPLASMIC DOMAIN WITH RESPECT TO THE PORE. ONE IN NINE ARE MOST EXTREME, WE CAN TELL THE DIFFERENCE BETWEEN THEM AND WE WANTED TO KNOW IF SODIUM AFFECTED THE POPULATION, THE DENSITY OF THEIR POP -- OF THEIR CONTRIBUTIONS. AND THE ANSWER IS NO. THEY DON'T CHANGE AS A FUNCTION OF SODIUM. SO WHAT IS HAPPENING HERE, IS YOU MANGE A GRAPH LIKE -- MAKE A GRAPH LIKE THIS OPEN PROBABILITY AND NOW HERE OPEN PROBABILITY IS ACTUALLY OPEN PROBABILITY OF THE STRUCTURE. I HAVE GONE BACK YOU CAN TAKE OUT IMAGES AND PUT RED CIRCLES ON THE CLOSED ONES AND BLUE CIRCLES ON THE OPEN ONES, IT'S IN THE THAT YOU CAN TELL FROM LOOKING AT THE IMAGE THEY'RE OPEN AND CLOSED. IT'S THE AFTER DETERMINING STRUCTURE AND CLASSIFYING YOU KNOW EVERY PARTICLE THAT WENT INTO EVERY CLASS AND YOU CAN ASK WHAT IMAGE DID THAT COME FROM AND WHERE WERE YOU AND YOU CAN NOW PUT COLOR ON IT. IT CAME FROM THE POST CLASSIFICATION. WHETHER YOU CAN SEE AS SODIUM INCREASED YOU GET A CONVERSION FROM CLOSED TO OPEN. WHAT'S STRANGE IS YOU DON'T SEE INTERMEDIATE STRUCTURES. AT LEAST WITHIN RESOLUTION ABILITY. MID 3 RANGE HERE. YOU CAN SEE A LARGE CONFIRMATIONAL CHANGE, IF SOMETHING SIGNIFICANT WERE HAPPENING IN BETWEEN WE SHOULD SEE, WE CARRIED THIS OUT WITH FOUR FOLD SYMMETRY AND WE ANALYZED WITHOUT IMPOSING SYMMETRY BECAUSE WE ASSUMED IF INTERMEDIATE STATES IT MAYBE ONE SUBUNIT GOES AT A TIME BUT WE SAW NO EVIDENCE OF THAT. SO IT SEEMS THAT IT'S AGAIN A HIGHLY COOPERATIVE PROCESS AND IN FACT, THE HILL CO-EFFICIENT FOR THIS IS ABOUT THREE AND A HALF SOMETHING LIKE THAT. THERE ARE SUBUNITS HERE SO LOOKS LIKE AS YOU INCREASE SODIUM WHAT YOU SEEK IS YOU SEE A POPULATION OF CLOSED CHANNELS AND APPEARANCE OF OPEN CHANNELS AT SOME PROBABILITY THAT GROWS AS SODIUM CONCENTRATION INCREASES AS HARD AS WE LOOK, SO FAR WE CONOT SEE INTERMEDIATE STATES. SO MY -- OUR EXPLANATION FOR THIS WOULD BE THE LOW ENERGY CONFIRMATION ONE THE CLOSED SET THAT ARE VERY CLOSE TO EACH OTHER, JUST SITE ROTATIONS CYTOPLASMIC DOMAIN AND THE OPEN STATE. ALL THE STATES OF COURSE IT'S NOT MAGIC, IT DOESN'T TURN INTO ONE, BUT IT MEANS THE ENERGY OF INTERMEDIATE STATES HAS TO BE HIGHER THAN THE ENERGY OF CLOSED OR THE OPEN. THERE ARE RELATIVE WELLS, HIGHER IN BETWEEN SO IT SPENDS LITTLE TIME IN BETWEEN BECAUSE WHAT WE ARE TRYING TO DO IS LOOK FOR THE DISTRIBUTION OF STRUCTURES AS A FUNCTION OF SODIUM CONCENTRATION. WHAT WE SEE IS ONLY CLOSED AND ONLY OPEN. A HIGHLY CONVERT SETTERED PROCESS. IN FACT, IF YOU COMPARE THE ELECTROPHYSIOLOGY SHOWN HERE IN BLUE WITH THE CRYO-EM ONLY GOES TO 300 MILLIMOLAR, IT'S SURPRISING HOW CLOSE THESE CURVES ARE TO EACH OTHER AND SURPRISING FOR A FEW REASONS, ONE WHEN OWE -- SOMEBODY MILE ASK BUT WHEN YOU FREEZE OR WHEN YOU PREPARE THIS EVAPORATION, DO WE HAVE THE SODIUM CONCENTRATION VERY WELL CONTROLLED. WE DON'T KNOW. BUT THESE ARE THE DATA. THERE'S ONE OBVIOUS DIFFERENCE. THE BLACK CURVE IS CLEARLY GOING TO ONE. IF WE WENT TO 600 MILLIONLY MOLAR SODIUM WOULD BE UP CLOSE TO ONE. HERE WE CLEARLY ARE NOT GOING TO ONE. SO THERE IS A DISCREPANCY AT THE ASIMILAR TOTTIC POINT OF THESE, AND WHAT THAT DISCREPANCY MUST MEAN IS WITHIN THE SET OF CHANNELS THAT WE DEFINE AS OPEN BECAUSE THEY HAVE UNDERGONE LARGE CONFIRMATIONAL CHANGE, THERE ARE SMALL DIFFERENCES WE DON'T SEE THAT CAUSE THEM NOT TO CONDUCT. THIS IS VERY COMMON. FEW CHANNELS FUNCTIONALLY IF YOU DRIVE THE STIMULUS TO OPEN, VERY FEW GO TO OPEN PROBABILITY OF ONE. SOME DO BUT MANY DO NOT. THIS COULD BE AS SUSSING AS BLOCKING ION IN THE PORE THAT WE CAN'T SEE. AT THE CURRENT RESOLUTION OR SMALL KINK SOMEWHERE, SMALL PROMOTIONAL DIFFERENCE, IT IMPLIES THERE ARE SUBTRAITS WITHIN WHAT DEFINE AS OPEN SUBTLY DIFFERENT. SOME CONDUCT AND SOME DON'T. THAT ACCOUNT FOR THIS DIFFERENCE. THE SUMMARY FROM THIS PART IS WE SEE AN ENSEMBLE OF CLOSE CONFIRMATIONS, AND SODIUM INDEPENDENT. THEY FLUCTUATE AROUND AND DON'T -- THEIR RELATIVE POPULATIONS OF WHAT WE CALL CLOSED, THE RED CHANNELS, DO NOT CHANGE. THE OPEN CONFIRMATION EMERGES IN A SODIUM DEPENDENT MANNER WITHOUT STABLE INTERMEDIATES, THAT IS THE SODIUM DEPENDENT GATING IS CONCERTED AND AGAIN, AS I MENTIONED AT HIGH RESOLUTION IN PRINCIPLE WE SHOULD BE ABLE TO DISCERN DIFFERENCES. COMING BACK TO THE QUESTION DO I REALLY BELIEVE THERE ARE ABSOLUTELY NO INTERMEDIATES? THERE MUST BE BUT THEY'RE LOW POPULATED. AT LEAST IF ONE IS MORE HIGHLY POPULATED THEY'RE VERY CLOSE TO WHAT WE'RE CALLING CLOSED OR VERY CLOSE TO WHAT WE'RE CALLING OPEN. AND NOT SIGNIFICANTLY DIFFERENT FROM EITHER OF THOSE. SO WE DON'T DISCERN THEM. I WANT TO END WITH ACKNOWLEDGMENTS, THE G PROTEIN GATED POTASSIUM CHANNEL STRUCTURAL WORK WAS CARRIED OUT BY MATT WARTON. WEWE WONING DID THE FUNCTIONAL DATA, COKI TAHA ALREADYA MEASURES STUDIES A SIMILAR SYSTEM IN THE HEART, WEWE STUDIES THEMENT IS IN THE NERVOUS SYSTEM. BUT I TALKED MOSTLY ABOUT WEWE'S DATA AND OUR COLLABORATORS, BRUCE BEAN AND KAKO WEIR FROM HARVARD CARRIED OUT THE EXPERIMENTS ON DOPAMINE NEURONS AND THE SLOW TUBE POTASSIUM CHANNEL TITRATION WAS CARRIED OUT BY RICH HYTE. THANKS VERY MUCH. [APPLAUSE] >> THE SODIUM TITRATIONS ARE BEAUTIFUL AND IT'S AMAZING TO SEE THIS SORT OF STRUCTURAL CORRELATE OF THE CONCERTED OPENING. I'M WONDERING IF YOU WERE TO MAKE A HYPOTHESIS AS TO WHAT AN INTERMEDIATE MIGHT LOOK LIKE? >> AS TO WHETHER? >> HYPOTHESIS WHAT AN INTERMEDIATE LOOK LIKE, IF YOU WERE TO SUGGESTION IT HAD ONE OPEN PLUS THREE CLOSED, THAT -- COULD YOU -- IS IT POSSIBLE TO MAKE A HYPOTHETICAL MODEL AND THEN GO BACK TO THE DATA SET AND SEE IF THAT'S HELPS YOU BRING -- >> YOU COULD DO THAT. SO THE QUESTION IS, YOU KNOW, ACTUALLY TO RESTATE THE QUESTION, OF COURSE INTERMEDIATES HAVE TO OCCUR BECAUSE IT GETS FROM CLOSED TO OPEN. SO THEY DO OCCUR BUT THEY'RE RARE OBVIOUSLY SO WE DON'T SEE THEM. THEY'RE VERY UNDERPOPULATED COMPARED THE WHAT WE DO SEEK. BECAUSE THEY'RE AT A HIGHER ENERGY LEVEL PRESUMABLY. WHAT MUST THEY LOOK LIKE? TURNS OUT IF YOU TRY TO OPEN THIS THING, IF YOU TRY TO OPEN ONE SUBUNIT AT A TIME YOU RUN INTO PROBLEMS BECAUSE IN A SENSE THE WAY THE HELICES WRAP AROUND EACH OTHER, IT'S HARD TO MOVE ONE WITHOUT MOVING ALL OF THEM. SO IT SEEMS MY GUESS IS IT WOULD HAVE TO INCH ITS WAY OUT, NOT ONE SUBUNIT CAN SNAP ALL THE WAY AND SECOND AND THIRD SOME ORDER FASHION, IT'S A DIFFUSIONAL PROCESS WHERE THEY ALL OPEN AT ONCE BECAUSE IN A SENSE THEY BUMP INTO EACH OTHER AS THEY OPEN. >> THANKS. >> GOING BACK TO THE GIRC STRUCTURE YOU HAVE THIS BEAUTIFUL HIGH RESOLUTION DATA. CAN YOU TELL US HOW SODIUM MODULATES BETA GAMMA? AND ALSO MIGHT THE SAME MECHANISM APPLY TO THE SLOW CHANNEL? >> SO THE WAY I THINK SODIUM -- I MEAN SODIUM IS PRETTY SUBTLE. SODIUM IS -- WHAT I WILL SAY IS THIS. SO THE ANSWER IS I DON'T REALLY KNOW. BUT WHAT I DO KNOW IS SODIUM -- RECALL THAT IS CYTOPLASMIC DOMAIN ROTATED AND CAUSED A TWISTING OF THE -- IN THE DIRECTION THAT OPENS THE HELICAL BUNDLE THAT MAKES THE GATE. SODIUM IS BOUND AT THAT INTERFACE BETWEEN THE CYTOPLASMIC DOMAIN AND THOSE HELICES. AND IT MAKES VERY SPECIFIC WHAT LOOK LIKE STRUCTURAL INTERACTIONS. IN MY VIEW SODIUM TIGHTENS UP BASICALLY BETTER TRANSFER IT IS ROTATION OF THE CYTOPLASMIC DOMAIN TO OPENING OF THE -- OPENING OF THE HELICES THAT MAKE THE GATE. SO I THINK THAT'S WHAT'S HAPPENING GIVEN WHERE IT SITS. AND THEN HOW DOES THAT CONNECT TO THE SLOW 2 AND COULD THE SODIUM BE HAVING A SIMILAR EFFECT? I DON'T THINK SO. IT'S BECAUSE -- SO AS HARD AS WE HAVE LOOKED FOR WHERE SODIUM BINDS IN SLOW 2 WE CAN'T SEE IT. WHICH IS A LITTLE BIT SURPRISING FOR THE FOLLOWING REASON. WE HAVE ALSO DETERMINED STRUCTURES THAT QUITE GOOD RESOLUTION OF SLOW 1, IN CLOSE AN OPEN CONFIRMATIONS. THAT IS A COUSIN OF SLOW 2 BUT CALCIUM IS THE ACTIVATED LIGAND INSTEAD OF SODIUM. THERE WE SEE WHERE CALCIUM BINDS, VERY CLEARLY. TWO SITES PER SUBUNIT, EIGHT ALL TOGETHER. IF WE LOOK IN THE CORE RESPONDING SITES, WE ACTUALLY SEE DENSITY THAT WE THOUGHT WAS SODIUM IN ONE OF THEM. WHAT WE CALL THE RCK 2 SITE. BUT WHEN WE MUTATE IT, TO MAKE IT GO AWAY, WE STILL GET SODIUM ACTIVATION. SO WE DON'T KNOW, AND I THINK SODIUM WORKS, NOTICE IT WAS A VERY LOW AFFINITY, IT'S A LOT OF SODIUM, OPENS THAT CHANNEL, TAKES A LOT. SO I THINK IT'S PROBABLY MULTIPLE LOW AFFINITY SITES AND IT'S WHY WE DON'T SEE THEM. IT'S WORKING ON THIS GATING RING STRUCTURE THAT KIND OF HAS THIS VERY SPECIALIZED MOTION THAT IS ALMOST THE SAME IN THE CALCIUM AN SODIUM ACTIVATED ONE BUT VERY DIFFERENT THAN ROTATION THAT HAPPENS IN THE GERC. -- GIRC. YES. >> THANK YOU FOR A WONDERFUL TALK. I WAS WONDERING IF THE GIRC CHANNEL WHEN REFERRING TO PIP 2, ARE YOU TALKING ABOUT PIP 4, 5 TOO OR IN ANY OTHER VERSION? >> I'M TALKING -- SO IT'S A GOOD QUESTION. ALL -- WE HAVE LOOKED AT A LOT OF SUBSTITUTED PIPs. AND THEY HAVE -- MANY OF THEM ACTIVATE. BUT IT'S REALLY INTERESTING BECAUSE MANY OF THEM ACTIVATE WITH NOT ONLY DIFFERENT AFFINITIES BUT TO DIFFERENT MAXIMUM VALUES. IT'S A WHOLE BUNCH OF DATA WE HAVE NOT PUBLISHED BUT THE ONE WE ARE TALKING ABOUT HERE IS PEP 4, 5. >> THANK YOU. >> BUT THE OTHER PIPs HAVE INTERESTING EFFECTS AND IN FACT SOME ARE COMPETITIVE ON THIS ONE. THEY BIND BUT DON'T ACTIVATE YOU CAN ONLY STUDY BY MIXING THEM IN THAT WAY TO SEE IT. IT MUST HAVE TO DO WITH REGULATION IN A WAY NO ONE KNOWS RIGHT NOW. YEAH. >> DOES IT SHARE ANY HOMOLOGY WITH ANY OTHER PIP 2 BINDING PROTEIN? FOR EXAMPLE PH DOMAINS, THINGS LIKE THAT? >> DOES WHAT? >> TYPICALLY PIP 2 BINDS THE PH DOMAINS SO DOES IT HAVE SIMILAR STRUCTURE LIKE THAT? >> THE STRUCTURE IS QUITE DIFFERENT. THE PIP BINDING SITE IS QUITE DIFFERENT IN THE GIRC CHANNELS. SO IN A FEW OF THEM WE HAVE NICE STRUCTURES PIP 2 BOUND LIKE THIS ONE AND IN ONE CALLED KR 2. IT'S A DIFFERENT MODE OF BINDING. >> THANK YOU. [APPLAUSE]