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.
01:03:42.819,00:00:00.000
[APPLAUSE]
