>>WELCOME TO TODAY'S
NIH NEUROSCIENCE SEMINAR SERIES.
IT'S A REAL PLEASURE--FOR THOSE
WHO DON'T KNOW ME I AM
[INDISCERNIBLE] I'M IN THE
INTRAMURAL OF NICHD AND IT'S A
REAL PLEASURE TO WELCOME AND
INTRODUCE TODAY'S SPEAKER
VASANTHI, JAYARAMAN.
VASANTHI, GREW UP IN INDIA
SPECIFICALLY IN [INDISCERNIBLE].
HE WENT TO HIS NATIVE
[INDISCERNIBLE] FOR
UNDERGRADUATE DEGREE AND THEN
DID A MASTERS AT THE INDIAN
INSTITUTE OF TECHNOLOGY IN
[INDISCERNIBLE] WHERE I BELIEVE
SHE WORKED WITH [INDISCERNIBLE]
WHO'S A VERY PREEMINENT PHYSICAL
CHEMIST BACK THERE IN INDIA AND
THAT GOT HER EXCITED ABOUT
CHEMISTRY AND THEN SHE WAS A
GRADUATE STUDENT WITH TOM SPIRO
AT THE CHEMISTRY DEPARTMENT AT
PRINCETON UNIVERSITY.
THERE VASANTHI, DID
[INDISCERNIBLE] SPECROSCOPY TO
UNDERSTAND HEMOGLOBIN AND SHE
FOLLOWED THIS UP WITH A POST
DOCTORAL FELLOWSHIP WHEN SHE WAS
A POST DOCTORAL FELLOW IN GEORGE
HESS'S LAB AT CORNELL UNIVERSITY
WHERE SHE WAS--THAT WAS SORT OF
HER INTRODUCTION TO THE WORLD OF
PHYSIOLOGY AND ION CHANNELS.
SO WHEN SHE STARTED, I THINK
BACK IN 1997, IN THE CHEMISTRY
DEPARTMENT AT [INDISCERNIBLE]
UNIVERSITY SHE BROUGHT THE 2
EXPERTISE TOGETHER COMBINING
SPECROSCOPY TO STUDY ION CHANEMS
AND THAT IS WHAT HER LAB HAS
FOCUSED ON SINCE THEN.
SHE HAS MOVED FROM WISCONSIN TO
TD DEPARTMENT OF INTEGRATIVE
BIOLOGY AT THE UNIVERSITY OF
TEXAS IN 2002 AND HAS STAYED IN
TEXAS SINCE THEN.
AND NOW SHE'S A PROFESSOR IN THE
DEPARTMENT OF CHEMISTRY AND
MOLECULAR BIOLOGY AT THE
UNIVERSITY OF TEXAS HEALTH
CENTER, SHE'S GOTTEN NUMEROUS
AWARDS AND SHE'S REALLY A
SUCCESSFUL--HAS MOST RECENTLY
BEEN APPOINTED AS CHIEF OF I
BIOPHYSICAL JOURNAL SO WITH
THAT, THANK YOU VERY MUCH FOR
ACCEPTING OUR INVITATION AND I
LOOK FORWARD TO YOUR TALK.
WELCOME TO THE NIH.
>> THANK YOU FOR THIS KIND
INTRODUCTION, YOU CAN HEAR ME
RIGHT?
I ALSO WANTED TO JUST SAY THIS,
WHEN HE SORT OF BROUGHT ALL THIS
TOGETHER AND SAID, I BROUGHT IN
THE SPECROSCOPY AND THE
ELECTROPHYSIOLOGY TOGETHER, I DO
WANT TO GIVE A SHOUT OUT AND SAY
MARK MAYER IS THE 1 WHO TOLD ME,
I DON'T THINK YOU CAN JUST DO
JUST PHYSIOLOGY, THERE'S LOTS OF
PEOPLE OUT THERE WHO ARE DOING
THAT, BRING IN YOUR EXPERTISE IF
YOU WANT TO DO SOMETHING USEFUL
IN THIS FIELD.
AND SO HERE I AM DOING COMBINING
THE TWO
RECEPTORS AND
WORK WE'VE DONE ON SINGLE
MOLECULE AND MORE RECENTLY GONE
ON TO COMPLEXES IN THE SYNAPSES.
--MOST OF THE WORK DONE BY DREW
AND RYAN AND NITTY AND MABINA
DID THE MD SIMULATIONS AND SHE'S
ALSO DONE WORK ON RECEPTORS.
SO THEY BIND GLUTAMATE AND THEY
OPEN CHANNELS WHICH ARE CAT ION
SPVENG AND THAT TRIGGERS THE
ELECTRICAL SIGNALING AND WE USE
A WIDE RANGE OF TECHNIQUES,
EXPECTATIONS LECT ROUGH ATOM
PHYSIOLOGY FUNCTIONAL STUDIES
EXPW DOING FLOURESCENCE WE USE
MOLECULAR BIOLOGY TO USE THE
OTHER FORCE AND SO ON.
WE DO A BIT OF CHEMISTRY SO
WE'VE DONE DAIJ SYNTHESIS SO I
WON'T TALK ABOUT IT BUT PUT IT
OUT THERE IN CASE ANYBODY'S
INTERESTED IN THAT.
MOST OF OUR WORK, SO WE HAVE
DONE ALL THE WAY FROM
VIBRATIONAL FLUORESCENCE AND
MAINLY SINGLE MOLECULE
FLUORESCENCE AND LIFETIME
MEASUREMENTS.
AND WE COMPLEMENT THESE WITH MD
SIMULATIONS WITH ALEX'S LAB AT
UT.
SO MOST OF WHAT I WILL BE
TALKING ABOUT TODAY WILL FOCUS
ON NMDA RECEPTORS AND THEN GO
INTO DELTA OR MOST RECENT WORK
ON DELTA RECEPTORS.
SO A LITTLE BIT ABOUT MDA
RECEPTORS, THE STRUCTURES ARE
WELL KNOWN AND SO YOU HAVE--IT'S
LIKE ALL THE GLUTAMATE
RECEPTORS, IT IS A DIMER OF
DIMERS, EXCEPT THE INTERESTING
PART ABOUT NMDA RECEPTORS, HAVE
YOU BOTH THE GLYSEEN BINDING
DOMAIN AND YOU NEED BOTH GLYSEEN
AND GLUTAMATE HAVE NEGATIVE
COPRAATIVE.
SO IN OTHER WORDS THEN IT IS
LOW.
IF GLUTAMATE IS BOND THEN
GLIESINE IS LOW, SO WE WANT TO
TEST THE UNDERLYING MECHANISM OF
THAT ASPECT OF NMDA RECEPTORS.
GLYSEEN IS FASTER VERSUS THE
ABSENCE, AND THIS IN CASE WE ARE
MEASURING DISASSOCIATION AND
THEN IT'S SEEN HOW MUCH CURRENT
AND LEFT BY ADDING GLUTAMATE.
AND SAME AS THE CASE THE OTHER
WAY AROUND WHERE GLUTAMATE
ASSOCIATION IS FASTER IN THE
PRESENCE OF GLYSEEN VERSUS IN
THE ABSENCE OF GLYSEEN.
SOPHISTICATED WHAT UTILIZES THIS
SO FIRST THIS IS THE LIGAND
BINDING DOMAIN AND IT'S SIMILAR
HERE, THE GLUTAMATE BINDING
DOMAIN, AND IF YOU CAN ZOOM IN,
CAN YOU SEE THIS IS SORT OF THE
CLEFT THAT IT BINDS AND IT'S
KNOWN THAT WHENLET BI LAND
BIEBDS THE CLEFT CLOSES SO IN
ORDER TO MEASURE THE
CONFIRMATIONAL CHANGE OF THE
CLOSURE, THE USED FRONT AND WE
INTRODUCED 2 CYSTINS IN
[INDISCERNIBLE], SO WE REMOVE
ALL THE CYSTINES IN THE EXTRA
CELLULAR SIDE OF THE PROTEIN
THAT ALLOWS US TO ABLE THE CELLS
THAT CYSTINE REACTIVE FLORA FORS
AND THE INTRODUCE THE CYSTINES
IN THE SITES.
Y SO YOU CAN MEASURE THE
DISTANCE BETWEEN THESE 2-POINTS
BY USING FRET, BECAUSE WHEN FRET
WHEN YOU MEASURE THE AMOUNT OF
LIGHT BEING DONATED FROM THE
DONOR TO THE ACCEPTOR, YOU CAN
SEE THE EFFICIENCIES DEPENDENTOT
DISTANCE BETWEEN THE DONOR AND
THE ACCEPTOR.
ALL OF OUR MEASUREMENTS ARE DONE
ON THE FULL LENGTH PROTEIN, SO
YOU KNOW EVEN THOUGH I ZOOMED IN
ON THE ISOLATED DOMAIN AND THE
WAY WE DO IT IS WE EXPRESS THE
PROTEIN?
HEK CELLS, THE MODIFICATION IS
IT'S A PROTEIN AND YOU HAVE
THOSE IN THE SITE WHERE THE
FLORA FLOR WAS INTRODUCED AND
THE OTHER IS THIS WE HAVE A
CANNED WHAT TAG AT C-TERMINUS AT
THE END OF THE PROTEIN, AND
EXPRESS THE PROTEIN AND WE DO AN
NC2 PULL DOWN SO YOU CAN SEE SO
THAT YOU CAN SEE THAT THE VENUE
INTRODUCED THE CELL LYSAIT, YOU
GET INSITU PULL DOWN OFF YOUR
PROTEIN ON THE SLIDE AND THE
MOLECULES ARE FAR APART BECAUSE
WE ARE DILUTED THE BIOTIN ON THE
SLIDE, AND SO YOU CAN SEE THAT
YOU CAN ACTUALLY SEE INDIVIDUAL
MOLECULES IN THE MICROSCOPE AND
THEN YOU POINT TO 1 OF THESE
MOLECULES AND YOU LOOK AT THE
INTENSITY OF THAT MOLECULE IN
BOTH THE DONOR AND ACCEPTOR
FREQUENCIES OR WAVE LENGTHS SO
THIS IS BASICALLY DONOR
EXCITATION, BUT WHAT YOU ARE
SEEING HERE IS THE ACCEPTAR
EMISSION AND AND BELOW THAT IS
THE DONOR EMISSION, AND AS CAN
YOU AS YOU'RE INSIGHTING THE
DONOR, THEY ARE GIVING THAT
ENERGY TO THE ACCEPTOR AND THIS
IS THE FRETTING REGION AND WE
MEASURE 1 MOLECULE AT A TIME SO
WE CAN ACTUALLY MEASURE THESE
SPECIFIC DYNAMICS AND YOU CAN
SEE THAT AT SOME POINT, THE
ACCEPTOR BLEACHES AND YOU CAN
SEE BECAUSE THE DONOR CAN NO
LONGER GIVE ITS ENERGY TO THE
ACCEPTOR, THEN THE DONOR COMES
UP AND THIS CLEARLY SHOWINGS
IT'S A SINGLE FLOR O BLEACHING
STEP SO IT'S A SINGLE ACCEPTOR
AND EVENTUALLY THE DONOR
BLEACHES, SO THAT'S A SINGLE
DONOR SO THE PROTEIN HAS A
SINGLE DONOR AND ALSO YOU KNOW
THAT THE ANTICORRELATION MEANS
THAT YOU'RE FRETTING MOLECULES.
OKAY?
YEAH?
>> [INDISCERNIBLE]
>> THIS IS IN DETERGENT BUT WE
ALSO DO SMA.
>> AS FAR AS CONCERNS WE DON'T
SEE ANY BIG DIFFERENCE WHETHER
YOU DO THE SMA ACID POLYMER THAT
YOU CAN USE TO MAKE NANO DISKS
OFF THE PROTEIN AND AND WE FIND
IT PUNCHES HOLES IN SIMPLISTIC
TERMS OFFCELLS AND WE FIND THERE
ARE SIMILAR IN THE EXTRA
CELLULAR DOMAIN ABE AND WE SEE
DIFFERENCES IN THE C-TERMINUS.
EVERYTHING IS THERE, LIKE I SAID
THE ONLY MODIFICATION WE DID WAS
EXTRA CELLULAR THINGS REMOVED
AND THAT'S A VERY CRITICAL
POINT.
WHAT YOU ARE SEEING HERE IS THIS
REGION AND THIS PARTICULAR
MOLECULE IS DO NOT DOING MUCH
IT'S STAYING IN 1 STATE WHICH IS
FINE AND THE GREEN IS THE
OBSERVED AND THE BLACK IS THE
D-NOISE DATA.
SO REALLY ALL OF WHAT I WILL
SHOW YOU IS BASED ON CHANGES
THAT WE SEE IN THE GREEN BUT THE
BLACK ALLOWS US TO GET BETTER,
YOU KNOW IN TERMS OF FINDING OUT
THE STATES, ET CETERA, SO THAT'S
JUST THE DENOISING THAT WE DO.
SO THIS IS THE HISTOGRAM FOR THE
NERVE AND THIS IS THE HISTOGRAM,
AND YOU CAN SEE IN THESE CASES
IT'S REALLY ONLY 1 STATEMENT THE
PROTEIN IS OCCUPYING.
AND FROM THAT YOU CAN SEE WHAT
WE CAN GET, BECAUSE WE HAVE ALL
THE INTENSITIES, WE HAVE THE
PRESENCE OF INTENSITY IN THE
DONOR, IN IPT GREATER TENSITY IN
THE ABSENCE OF THE DONOR AND SO
SO ON SO WE CAN COUNT FROM THE
THREAD.
THE EXAMPLES I WILL SHOW YOU IS
JUST THIS REGION, THE FRETTING
REGION, NOT BEYOND THAT SO YOU
CAN SEE THAT IN THE FRETTING
REGION THIS PARTICULAR MOLECULE
IS ACTUALLY SWITCHING BETWEEN 2
DIFFERENT STATES, RIGHT?
SO THIS IS THE ACCEPTOR, THIS IS
THE DONOR AND THEN AT SOME
POINT, THE THREAT BECOMES LESS
SO THE DONOR GOES UP AND THE
ACCEPTOR GOES DOWN AND 2 THREAT
EFFICIENCIES AND YOU CAN SEE
IT'S STAYED IF ARE A WHILE
BEFORE IT SWITCHES INTO THE
OTHER STATE.
THE DIFFERENCE BETWEEN THIS
MOLECULE AND THIS MOLECULE IS
THAT EVEN THOUGH THIS IS SORT OF
SIMILAR 2 STATES, YOU SEE THAT
HERE THE MOLECULE IS ACTUALLY
FLUCTUATING A LOT AND EVERY
THRUCTUATION YOU GET, THERE IS
ANTICORRELATION SAYING IT IS
REALLY THREAT DIFFERENCES AND
THIS MOLECULE IS FLUCTUATING
BETWEEN THE 2 STATES.
SO WHAT YOU GET FROM SINGLE
MOLECULE THREAT IS NOT JUST THE
THREAT DISTANCES BUT ALSO THE
DYNAMICS OFF THE PROTEIN IN HOW
IT SWITCHES BETWEEN THE STAKES,
BUT THE DYNAMIC SYSTEM LIMITED
IN THE MILLI SECOND TIME SCALE,
IT'S NOT FASTER OR VERY LONG OR
SLOWER DYNAMICS ARE ALSO GOING
TO BE MISSED BECAUSE HAVE YOU
PHOTO BLEACHING.
SO IT'S SORT OF IN THE SWEET
SPOT OF 5 MILLI SECONDS, 200
MILLI SECONDS.
OKAY?
SO THIS IS WHAT RYAN FOUND WHEN
HE DID THE ISOLATED LIGAND
BINDING DOMAIN AND THE GRAY IS
THE OBSERVED DATA AND THE BLUE
IS JUST THE DENOISING TO SEE
WHAT STATES ARE THERE, BUT
PRETTY MUCH ALL OF OUR
MEASUREMENTS, CAN YOU JUST
OVERLOAD THE GRAY AND SEE THE
DIFFERENCES, OKAY, EVEN THOUGH
YOU'RE SEEING THE BLUE, BEING
SHOWN HERE, IT'S EVEN WITH THE
OBSERVED DATA YOU CAN SEE WHAT'S
GOING ON.
BUT BOTTOM LINE IS THIS THE APO
STATE WHEN THERE IS NO LIGAND IN
BOTH SIDES AND WHEN MOTIONING
THE GLUTAMATE, THE GLUE MARIOUS
SEEN BINDING SITED, YOU SEE IT'S
AT A CERTAIN DISTANCE, AGAIN THE
ABSOLUTE DISTANCE DOESN'T MEAN
MUCH BECAUSE YOU'RE MEASURING
BETWEEN FLORA FLORS BUT ALSO I
WOULD LIKE YOU TO SEE THE
TRANSITIONS I SHOWED YOU IN THE
PREVIOUS SLIDE AND THIS SORT OF
GIVES YOU THE NUMBER OF
TRANSITIONS THAT ARE HAPPENING
BETWEEN THE DIFFERENT STATES AND
YOU CAN SEE THAT THERE'S A
CERTAIN NUMBER OF TRANSITIONS
THAT IS HAPPENING IN THEA, PO
STATE.
NOW WHEN YOU GO TO THE GLUE
MARIOUS SEEN STATE, YES, THE
CLEFT CLOSES AND THIS IS
CLEARLY, IT'S KNOWN IN THE
STRUCTURE SO IT'S NOT LIKE WE
ARE SHOWING THAT THE CLEFT
CLOSED, THIS HAS BEEN KNOWN FOR
A WHILE, BUT WHAT IS INTERESTING
IS THAT THE TRANSITIONS ARE VERY
LESS AND THE PROTEIN IS VERY
STABLE.
THAT'S WHAT YOU EXPECT, RIGHT?
YOU HAVE A GLUE MARIOUS SEEN
BOUND AND THEN CLEARLY THE
INTERACTION WILL STABILIZE THE
CLEFT AND SO THEREFORE THE
TRANSITION WILL BE REALIZED.
IT'S NOT SURPRISING BUT AGAIN IT
CONFIRMED WHAT WE WOULD THINK
SHOULD HAPPEN.
BUT WHAT IS INTERESTING IS WHEN
YOU ADD GLUTAMATE TO THE OTHER
SITE, WHAT YOU SEE IS GLYSEEN
BINDING SITE, SLIGHTLY SHIFTS
BETWEEN THESE STATES WHERE IT'S
GOING MORE INTO THE OPEN STATE
BUT IT'S A SLIGHT SHIFT BUT
AGAIN YOU CAN START SEEING THAT
THE TRANSITIONS ARE GETTING
MORE.
SO THEN THE DPLIEWT MATE IS
BOUND, THE GLUE MARIOUS SEEN
BINDING CLEFT IS STARTING TO
OPEN UP AND THE TRANSITIONS,
IT'S A BIT MORE UNSTABLE THERE,
IT'S STARTING TO FORM, IT'S
STARTING TO HAVE MORE OF THESE
TRANSITIONS, BUT EVEN THAT IS
PROBABLY NOT AS DRAMATIC AS WHAT
YOU SEE HERE.
YOU DON'T HAVE GLYSEEN IN THE
GLYSEEN BINDING SITE, YOU JUST
HAVE GLUTAMATE AND YOU SLEEP
APNEA AND OBESITYY THAT REALLY
THE LIGAND BINDING DOMAIN IS
LOOKING AT A LOT OF DIFFERENT
CONFIRMATIONAL STATES AND
THERE'S A LOT OF DYNAMICS, SO IN
OTHER WORDS, THIS GLUTAMATE
BINDING HERE IS AFFECTING THE
GLUE MARIOUS SEEN BINDING SITE
ALOESTERICALLY AND THAT IS SORT
OF GOING TO AFFECT THE RATE AT
WHICH GLYSEEN BINDS TO THE
PROTEIN.
SO THE SAME THING IS TRUE IN THE
GLUTAMATE BINDING SITE AND I
WILL NOT BORE YOU CAN THE DETAIL
EXCEPT TO SAY THAT AGAIN, THE
APO STATE IS MORE OPEN, MORE
DYNAMIC, GLUTAMATE ALONE IS
FAVORING MORE CLOSE, LESS
DYNAMIC BUT THEN YOU HAVE
GLYSEEN IN THE SITE.
YOU SEE THAT THE DYNAMICS IS
REALLY GETTING VERY LARGE, OKAY?
SO TO MAKE SURE THAT THIS IS
WHAT WE ARE MEASURING IS A
POSSIBILITY, WE NEEDED MB
SIMULATION BUT WE ALSO WANTED TO
SEE WHAT INTERFACE IS
CONTRIBUTING TO THIS NEGATIVE
COOPERATIVE DEAL.
AND YOU WILL SEE THAT IN THE
NMDA RECEPTORS THERE'S 2
DIFFERENT INTERFACES THIS, IS 1
INTERFACE WHICH IS BASICALLY
THIS IS THE GLUE MARIOUS SEEN
BINDING SITE AND THAT'S
GLUTAMATE BINDING SITE AND THIS
IS THE OTHER INTERFACE WHEN YOU
TURN AT 90-DEGREES AND THAT'S
AGAIN THE GLUE MARIOUS SEEN AND
GLUTAMATE, AND GLUE MARIOUS SEEN
AND GLUTAMATE.
SO WHAT THEY DID WAS TOOK JUST
THE INTERFACES AND SAID LET'S DO
AN MD SIMULATION AND SEE HOW
GLUE MARIOUS SEEN ALONE OR
GLUTAMATE ALONE OR GLUTAMATE AND
LYSINE AFFECTS THE CLEFT BETWEEN
THESE AND IF IT CAN BE SORT OF
CORRELATED TO THE SINGLE
MOLECULE THREAT WE GOT ON THE
FULL LENGTH RECEPTOR, OKAY?
AND SHE SAW THAT MEASURING THE
SAME SITES AS THE SINGLE
MOLECULE FRET, SHE FOUND THAT
BASICALLY THE TRENDS THAT WE ARE
SEEING THEN YOU HAVE GLUTAMATE
AND GLYSEEN AND BINDING DOMAIN
IS STARTING TO OPEN THE APO
STATE AND THE MOST UNSTABLE
BEING GLUTAMATE WHEN YOU ARE
MEASURING AT THE GLSEEN AND
GLYCINE FOR THE GLUTAMATE.
THESE MATCH VERY WELL WHAT WE
SAW IN THE SINGLE MOLECULE FRET,
WHEN SHE DID THE BACK-TO-BACK,
THE TRANSFER NOT UNTILING THE
SAME AS WHAT WE SAW IN A SINGLE
MOLECULE AND IN FACT IN SOME
CASES, IT WOULD SORT OF GO TO
POSITIVE COOPERATIVITY BECAUSE
OF THE TRENDS.
SO, WE THINK THAT WILD THIS
INTERFACE MAY BE, I'M NOT SAYING
IT'S NOT IMPORTANT, ALL I'M
SAYING IS IT'S NOT COOPERATED
HF-IT'S NOT CONTRIBUTING AS MUCH
TO THE NEGATIVE COOPERATIVE.
SO HAVING ESTABLISHED--SORRY, I
WILL GO BACK.
HAVING ESTABLISHED THAT THIS
INTERFACE MIGHT BE CRITICAL FOR
THE TEST, WE DECIDED TO CROSS
LINK THIS USING A NATURAL AMINO
ACID AND SEE IF INDEED THIS IS
CROSS AND SHOULD BE AFFECTING
NEGATIVE COOPERATIVELY.
SO WE INTRODUCE A NATURAL AMINO
ACID AND BE CROSS LINKED AND AS
YOU CAN SEE THE RISK, SOME
FRACTION OF THE PROTEIN THAT'S
CROSS LINKED, WE CAN SEE AN
EFFECT IF THAT IS AN EFFECT AND
AS EXPECTED, THE NEGATIVE
COOPERATIVITY IS SIGNIFICANTLY
REDUCED, SO IF YOU SEE THIS IS
THE WILD-TYPE, THIS IS THE--THE
GLUTAMATE SO THE RATIO TELLS YOU
IF THERE IS NEGATIVE
COOPERATIVITY.
SO THIS HAS NEGATIVE
COOPERATIVITY.
AND IT'S VERY SITE SPECIFIC, SO
THEN YOU HAVE THIS UNNATURAL
AMINO ACID BEING INTRODUCED AT
720.
YOU SEE THAT IT DOESN'T HAVE AN
EFFECT BECAUSE IT'S NOT RIGHT AT
THE INTERFACE BUT WHEN YOU
INTRODUCE IT AT 723, YOU SEE IT
HAS AN EFFECT AND AGAIN, AT 712
IT DOESN'T.
SO THIS INTERFACE WHICH IS RIGHT
AT THE INTERFACE WHEN IT'S CROSS
LINKED ACROSS, IT DOES HAVE AN
EFFECT ON NEGATIVE
COOPERATIVITY, FURTHER SHOWING
THAT THIS INTERFACE IS CRITICAL
FOR NEGATIVE COOPERATIVE.
BUT AGAIN, I DON'T WANT YOU TO
LEAVE THINKING THAT IS THE ONLY
INTERFACE THAT'S IMPORTANT FOR
THE ALOESTERRIC CROSS TALK
BETWEEN GLYCINE AND GLUTAMATE,
WE DID ANALYSIS WHERE HE CAME TO
US AND SDZ, HEY, LISTEN WE HAVE
THIS MUTATION IN THIS PATIENT
AND OUR ELECTROPHYSIOLOGICAL
STUDIES IS SHOWING THAT EVEN
THOUGH THE MUTATION IS IN THE
GLYCINE BINDING DOMAIN, IT'S NOT
AFFECTING THE GLYCINE IN TERMS
OF FUNCTION BUT IT IS AFFECTING
GLUTAMATE BINDING IN TERMS OF
FUNCTION AND THAT GLUTAMATE
BINDING IS MUCH LESS EFFICIENT.
SO AGAIN [INDISCERNIBLE] DID MD
SIMULATIONINGS TO SEE HOW IT'S
AFFECTING THIS MUTATION IS
AFFECTING THE PROTEIN AND IT'S
ACTUALLY AT THIS BACK-TO-BACK
INTERFACE.
IT'S A PROLEAN THAT'S BEEN
MUTATED TO HISTIDINE, IT
ACTUALLY FORMS A HYDROGEN 1
ACROSS, THIS IS THE GLUE MARIOUS
SEEN BINDING SITE AND THIS IS
THE DPLIEWT MATE BINDING SITE.
IT FORMS THIS HYDROGEN BINDING
ACROSS AND KIND OF PROVES THE
LOWER PART OF THE GLUTAMATE
BINDING DOMAIN, SUCH THAT IT NOW
SEEMS TO POP OPEN MORE THEREFORE
GLUTAMATE IS NOT ABLE TO BIND AS
EFFICIENTLY.
SO IT'S IN THOSE TO BACK
INTERFACE AND IT'S PULLING THIS
THING MORE OPEN SO THAT NOT NOW
IT'S NOT BINDING ASENTIOUS
FICIENTLY.
SO OF COURSE THIS IS MD
SIMULATIONS, WE WANT TO VERIFY
IT WITH SINGLE MOLECULE THREAT
AND I WILL JUST SHOW YOU
THE--AGAIN THE SAME LOOKING AT
GLUTAMATE ACROSS AND SEEING HOW
THE MOTIONS ARE, SO I WILL JUST
SHOW YOU THE CATONNATED FRET
STATES, YOU CAN SEE ALL THE FRET
STATES THE PROTEIN IS EXLORING
AND THIS IS THE WILD-TYPE AND
THIS IS WHAT I SHOWED YOU
BEFORE, THERE'S NOT MUCH MOTION
YOU SEE IN THE WILD-TYPE BUT IN
THE MUTANT YOU CAN SEE THAT IT'S
FLUCTUATING A LOT BETWEEN THE
STATES.
AND IT'S GOING FROM A HIGH FRET
STATE TO A MUCH LOWER FRET STATE
THEREFORE THE CLEFT IS OPENING
MORE.
SO THIS SORT OF SHOWS YOU WHERE
YOU GO FROM MUTATION FUNCTION TO
MOLECULAR DYNAMIC SIMULATION TO
SINGLE MOLECULE SHOW HOW THE
DYNAMICS IS INVOLVED IN
DICTATING FUNCTION.
OKAY?
SO JUST TO SHOW YOU A LITTLE BIT
MORE ABOUT NMDA BEFORE I JUMP
INTO THE DELTA RECEPTORS, ALL OF
THIS WHAT I SHOWED YOU WAS IN
THE EXTRA CELLULAR PART BUT OF
COURSE WHAT MOST OF THIS IS
INTERESTED IN IS THE CHANNEL
PART, RIGHT BUT WHEN YOU'RE
DOING FRET, YOU DON'T WANT FLORA
FLORS INSIDE THE CHANNEL SO DREW
STARTED DOING THIS WORK DECIDED
TO PUT THE FLORA FLORS JUST
OUTSIDE OF THE TRANSMEMBRANE
PART TO SEE THE MOTIONS OF THE
TRANSMEMBRANE PART, THE WITH
RESPECT TO WHAT WE ARE SEEING AT
THE LIGAND BINDING DOMAIN
MOTIONS.
SO THIS IS THE APO STATE, AGAIN
IT'S PRIMARILY CLOSED.
I SAY SHORTER, AND WOULD SUGGEST
THAT THE TRANSMEMBRANE PART IS
MORE PACKED AND THE--IN THE
PRESENCE OF GLUTAMATE IS
GLYCINE, IS IT TARTS TO PROBE
SOME MORE DISTANT STATES AND
SUGGESTING THAT MAYBE THAT IS
THE OPEN CHANNEL FORUM AND IN
FACT THE FRACTION THAT IS IN
THESE STATES MATCHES WELL THE
B-FRACTION OF THE CHANNEL THAT'S
OPEN WHEN YOU HAVE BOTH GLYCINE
AND GLUTAMATE.
WE THINK THIS PROBABLY
REPRESENTS THE DESENSITIZED
STATES.
BUT WHAT IS INTERESTING IS THE
FACT THAT YOU SEE LESS
TRANSITIONS BETWEEN THE STATES
IN THE RESTING APO STATE BUT AS
YOU SEE MORE TRANSITIONS IN THE
GLUTAMATE GLYCINE STATES BETWEEN
THE DIFFERENT STATES.
SO IF YOU ARE--IF YOU REMEMBER
IN THE AGONIST BINDING DOMAIN,
THE APO WAS MORE DYNAMIC BUT AS
THE GLUTAMATE AND GLYCINE IS
LESS DYNAMIC AND JUST THE
OPPOSITE HAPPENS THEN IT'S IN
THE TRANSMEMORY RESPONSE BRINE
PART WHERE THE APO STATE OR
CLOSED STATE IS LESS DYNAMIC, IT
LIKES TO BE CLOSED, BUT THE
GLUTAMATE GLYCINE IS MORE
DYNAMIC ALLOWING THE CHANNEL TO
OPEN IT.
SO I THINK ABOUT IT AS A BUNCH
OF ROSES, IF YOU HAVE ROSES AND
YOU HAVE THEM BY THE STEM THEN
THE TOP WILL FLOP OPEN RIGHT?
SO THAT ALLOWS THE STEMS TO BE
CLOSED, THE TRANSMEMBRANE
SEGMENTS TO BE CLOSED WHILE THE
TOP IS FLOPPING,A ROUND ASK JUST
LIKE THE APO STATE.
WHEREAS IN THE OPEN STATE, IF
YOU IMAGINE HOLDING THE BUNCH OF
ROSES WITH JUST THE TOP, THEN
THE BOTTOMS--THE STEMS START TO
MOVE AND THEREFORE THE OTHER
CHANNEL OPENS, OKAY?
SO THIS IS JUST--IT'S NOT JUST
TRUE IN THE RESTING STATE BUT
IT'S TRUE IN THE DESENSITIZED
STATES BUT THE TOP IN THE
CHANNEL IS ABLE TO BE IN A
CLOSED STATE.
SO, WITH THIS BACKGROUND WE THEN
DECIDED TO LOOK AT THIS DATA
RECEPTORS, DELTA RECEPTORS ARE
ORPHAN RECEPTORS AND THAT'S WHAT
THEY'RE CLASSIFIED AS BECAUSE NO
LIGATED ACTIVITY WAS SEEN FOR
THESE DELTA RECEPTORS AND SO
IF--JUST TO SHOW YOU THIS IS HOW
AMPORECEPTORS LOOK BASICALLY YOU
HAVE ACTIVATION AND
DESENSITIZATION AND THEN YOU
APPLY LIGAND, I'M SORRY, AMPO
RECEPTORS FAST AND NMDA
RECEPTORS ARE SLOWER AND CAN YOU
SEE THAT THEY'RE NICELY COUPLED
AT THE TOP AND IF YOU LOOK AT
THE DELTA RECEPTORS WHICH
[INDISCERNIBLE] WHO WAS HERE
THEN WENT ON TO HAVE THE
INDEPENDENT LAB IN INDIA, HE IS
THE 1 WHO DID THE DELTA RECEPTOR
STRUCTURES, AND HE'S--HE SAW
THAT THE TOP IS VERY DECOUPLED,
COMPARED TO THESE 2, RIGHT?
SO LIKE I TOLD YOU THIS SORT OF
REMINDS YOU OF A FLOWERED THING,
IT'S A BUNCH OF ROSES, IT'S
DECOUPLED AND THE WE THINK THE
REASON THERE IS NO ACTIVITY IS
BECAUSE THE CHANNEL IS PROBABLY
DESENSITIZED OR YOU COULD THINK
OF IT AS GLYCINE BINDING DOES
NOT DO ANYTHING.
AND NOW I SAID GLYCINE, AND
YOU'RE LIKE WAIT. THIS IS
GLUTAMATE.
THAT'S THE THING, THE DELTA
RECEPTOR BYPASSING THEY'VE BONE
MD SIMULATIONS AS WELL AS
STRUCTURES AND THEY FOUND IT WAS
NOT APPROPRIATE FOR GLUTAMATE
BINDING, IT WAS PROBABLY GLYCINE
THAT WAS THE LIGAND THAT BOUND
TO THESE RECEPTORS SO EVEN
THOUGH IT'S UNDER GLUTAMATE
RECEPTORS BECAUSE AS YOU CAN SEE
IT HAS VERY SIMILAR STRUCTURE,
RIGHT?
IT HAS--VERY SIMILAR TO THESE
AMPORECEPTORS EXCEPT DECOUPLED
AT THE TOP, SO IT WAS CLASSIFIED
BUT PEOPLE KNOW THAT THESE WERE
BINDING.
BUT NO ACTIVITY WAS SEEN WITH
THE RECEPTORS.
SO WHAT DO THEY DO?
WHY ARE THEY THERE?
BEFORE WE GO ON, I WANTED TO
SHOW YOU SINCE THIS IS A CRYOEM
STRUCTURE 1 STARTS TO WONDER
THAT IT'S JUST IN THE CRYOEM AND
THEN YOU CAN SEE ALL THE STATES
SO WE JUST DID THAT FIRST TO
CONFIRM THAT INDEED IT IS
DECOUPLED AND OUR DATA IS VERY
CLOSE TO WHAT [INDISCERNIBLE]
STRUCTURES SHOWS SO IT IS
SUGGESTING THAT BY ITSELF, THEN
YOU STUDY IT, IT IS REALLY
DECOUPLED LIKE THE STRUCTURE, SO
IT'S NOT THAT.
SO LIKE I SAID, WHAT DO THESE
DO, YOU KNOW WHAT ARE THE
CONTEXT OF THESE DELTA RECEPTORS
IN TERMS OF PHYSIOLOGY MPLET A
LOT OF LABS INCLUDING
[INDISCERNIBLE] LAB HAS TAKEN
THESE DELTA RECEPTORS AND THEY
HAVE SHOWN THAT THEY ACTUALLY
FORM THESE TRANSSYNAPTIC LIKE
CONNECTIONS, SO YOU HAVE NRX,
AND THEN CBLN, AND THEN THE GLUE
AND THEN THEY ALL COME TOGETHER
TO STABILIZE, TO GIVE THE
SYNAPSE A STRUCTURAL STABILITY,
OKAY?
SO IN FACT, IF YOU HAVE SAY IF
ANY OF THESE HAVE MUTATIONS OR
ARE NOT ABLE TO FORM BIG
STRUCTURAL COMPONENTS, THEN YOU
HAVE ISSUES LIKE GATE BECAUSE
GLUD2 IS IN THE CEREBELLUM SO
IT'S INVOLVED IN GAIT ISSUES, OR
IF IT'S IN GLUD1, THEN IT'S
MEMORY SO THIS SORT OF
STRUCTURAL STABILITY IS
EXTREMELY CRITICAL BUT THEN IT
MADE US THINK YOU KNOW IS IT
POSSIBLE THAT HAVING THESE--I
SHOULD ALSO ADD THAT
[INDISCERNIBLE] LAB HAS SHOWN
THAT IT AFFECTS THESE CLUSTERS
AND THERE ARE AT LEAST NRX
MOLECULES THAT ARE IN ANY GIVEN
CLUSTER.
SO IT MADE US THINK THAT THERE'S
A POSSIBLE LED-BASED CLUSTERS
BRINGING THESE DOMAINS TOGETHER
AND IF THAT'S THE CASE COULD
THIS PROTEIN BE A LIGATED
CHANNEL IN THIS PARTICULAR
SITUATION AND AGAIN THIS IS JUST
A CARTOON, NO STRUCTURES ARE
KNOWN OF THE FULL LENGTH OF THIS
COMPLEX, IT'S THE GLUE D,
INDIVIDUAL STRUCTURE IN THE
COMPLEX WITH THE AMINO TERMINAL
DOMAIN, SO THE FULL LENGTH IS
KNOWN, SOME COMPONENTS ARE KNOWN
AND THIS IS A CARTOON JUST SO
THAT YOU KNOW THAT.
SO WHAT WE DECIDED TO DO WAS DO
FULL MEASUREMENTS TO SEE THE
DISTANCE ACROSS THE AMINO
TERMINAL DOMAIN IN THE PRESENCE
AND ABSENCE OF THESE INTERACTING
PROTEINS.
AND OF COURSE INTENSITY BASED
MEASUREMENTS SUGGIEST THAT MAYBE
THE FRET IS MORE IN THE PRESENCE
OF THIS AND NOT IN THE PRESENCE
OF THAT BUT ANYBODY WHO DOES
FLUORESCENCE KNOWS THAT
INTENSITY IS NOT THE WAY TO GO
BECAUSE YOU COULD HAVE OTHER
REASONS FOR THIS SO WE DID
LIFETIME BASE MEASUREMENTS AND
SO THE FILM MEASURES ALSO SHOWED
THAT INDEED THESE AMINO TERMINAL
DOMAIN OFF THE DELTA RECEPTORS
CAME TOGETHER, THEN YOU
HAD--THEN YOU HAD THIS
INTERACTING PROTEINS
NEUREXPECTATIONSIN AND
CEREBELLUM.
OKAY?
SO HAVING THIS INFORMATION THAT
INDEED THESE 2 DO COME TOGETHER,
NOW THE QUESTION IS, CAN IT BE
GATED BY GLYCINE.
SO [INDISCERNIBLE] WHO IS A POST
DOC AND RESEARCH ASSIST APT
PROFESSOR IN MY LAB, DECIDED TO
LACK AT THIS--AT CLUSTERS LIKE
THIS, I KNOW IT'S SORT OF
ANYBODY DOING ELECTROPHYSIOLOGY
KNOWS THIS IS A TERRIBLE WAY OF
DOING EXPERIMENTS, RIGHT?
BECAUSE TIME IS VERY LOW AND IN
FACT THAT IS AT THE BOTTOM OF
THE DISH SO EVEN THOUGH IT LOOKS
LIKE IT'S NOT THIS, IS AT THE
BOTTOM OF THE DISH, SHE JUST
PATCHES A CELL THAT'S SURROUNDED
BY ALL THESE CELLS AND NOW WE
HAVE A GLUE D2 NRX AND
CEREBELLIN EXPRESSED IN THE
CELLS AND WE WANTED TO SEE WHAT
HAPPENS WHEN WE ADD GLYCINE AND
WE GOT THIS REALLY NICE CURRENTS
WHERE GLYCINE ACTIVATES THE
CHANNEL, THIS ACTIVATES THE
CHANNEL, GLUTAMATE DOESN'T DO
MUCH, MAYBE A LITTLE BIT.
WE TRIED A LOT OF OTHER AMINO
ACIDS AND WE DON'T SEE CURRENTS.
BUT I DO HAVE TO TELL YOU,
WARNING, THIS IS VERY DIRTY PREP
AND WE'VE DONE A LOT OF FOLLOW
UP AFTER WE DID THESE
EXPERIMENTS IN THE LAST 9 MONTHS
IT'S BEEN PRETTY MUCH ALL FOLLOW
UPS.
SO WHAT DO I MEAN BY SAYING IT'S
A DIRTY PREP.
FIRST OF ALL AS YOU KNOW, HEK
CELLS ARE TERRIBLE, THEY HAVE A
LOT OF ENDOGENOUS PROTEINS.
WE REALIZE THAT OUR SPECIFIC
BATCH OF HEK CELLS DOESN'T HAVE
ANY GLUE D2, AND IT HAD LITTLE
ENDOGENOUS GLUE D1 AND 2.
WHICH IS FINE.
IF IT WAS THERE IT DOESN'T
AFFECT OUR EXPERIMENTS BECAUSE
YOU'RE NOT DOING MUTATIONAL IS
IT THE STUDIES IN THAT
PARTICULAR SET OF EXPERIMENTS,
BUT THEN, IF YOU WANTED TO STUDY
MUTATIONS IN GLUE D, THEN YOU
NEED A KNOCK OUT OF THE GLUE D 1
AND 2 IN THESE HEK CELLS, AND
DAVID IN HIS LAB HAS MADE THE
KNOCK OUT WHICH IS GREAT SO THAT
EXISTS, SO NOW THIS HAS BECOME
LIKE A--THIS IS THE BEST WAY TO
DO SCIENCE, RIGHT?
BECAUSE A LOT OF LABS ARE ALL
INVOLVED IN DIFFERENT DIRECTIONS
TO MAKE THIS WORK.
BECAUSE WE WANT TO MAKE THIS
WORK.
THEN WHAT WE REALIZED IS THE HEK
CELLS IN THE LAB WAS VERY OLD SO
WE SAID LET'S BUY SOME HEK CELLS
FROM, YOU KNOW ATCC, AND THEN
WHEN CANNED WHAT DID THE CONTROL
EXPERIMENTS SHE STARTED SEEING
CURRENTS AND THEN WE REALIZED
THAT THAT IT TOOK US 9 MONTHS SO
WE WERE TRYING TO FIGURE OUT
WHERE IS THIS COMING FROM AND WE
LOOKED AT TRANSPORTERS BECAUSE 1
OF THE--I WAS TALKING TO A LOT
OF DIFFERENT PEOPLE AND THEY
SUGGESTED MAYBE IT'S A
TRANSPORTER AND WE STARTED DOING
WESTERN BLOTS FOR TRANSPORTERS
AND WE FOUND THAT IN FACT SNAT
2, SODIUM COUPLED NEUTRAL AMINO
ACID TRANSPORTER, NEEDED
CONTROLS.
IF YOU GROW THEM ISOLATED YOU
DON'T GET AS MUCH AS IF YOU GROW
THEM TO FORM THESE CLUSTERS.
SO I'M LEAVING IT OUT THERE FOR
ALL YOUR TRANSPORTER PEOPLE TO
FIGURE OUT WHY, WHAT IS
HAPPENING TO MESS UP OUR GLUE D
DATA?
WHAT IS HAPPENING?
BUT THAT'S ALL I CARE ABOUT.
BUT WE DO HAVE SOME SNAT 1, I
DON'T KNOW IF THIS IS ANYTHING
TO WRITE HOME ABOUT, BUT WE DO
SiRNA, TO GET RID OF SNAT 2.
IN THIS BACKGROUND NOW THAT WE
HAVE LESS CURRENT THIS IS THE
SiRNA TREATED 2 WHERE THE
CURRENTS ARE VERY SMALL SIMILAR
TO WHAT WE'RE SEE NOTHING OUR
PREVIOUS DATA AND THIS IS THE
NEGATIVE TO SHOW THAT YOU KNOW
THIS IS THE SCRAMBLED CONTROL.
IT SHOWS THAT YOU CAN--IT'S NOT
AN SiRNA ISSUE, BUT IT'S
SPECIFIC TO SNAT.
AND THEN ON THAT WE THEN
EXPRESSED GLUE D2 AND CEREBELLUM
AND WERE ABLE TO GET BACK OUR
RESULTS.
SO I THINK IT'S IMPORTANT TO BE
CAREFUL WHEN YOU DO THESE
EXPERIMENTS TO GET A BLANK
BACKGROUND BEFORE YOU START
DOING THESE CONNECTIONS.
FOR SOME REASONS, THE CLUSTERS
DO THINGS TO THESE CELLS AND I
DO NOT KNOW WHAT.
I'M JUST PUTTING IT OUT THERE,
WE ALSO DID THE FIRST 1 AND 2 TO
SHOW THAT YES, IT'S PROBABLY
SNAT 2, THAT'S THE MAIN CULPRIT.
AND YEAH, SO THEN WHILE ALL OF
THIS IS HAPPENING LISA WAS JUST
GETTING VERY FRUSTRATED AND
SAID, YOU KNOW WHAT, I AM DONE
WITH THESE CLUSTER MEASUREMENTS,
WE HAVE TO GET ENOUGH
BACKGROUND, I WILL GO TO A MORE
PURE SYSTEM AND SO SHE ACTUALLY,
YOU NOTICE IT DOESN'T MATTER, AS
LONG AS YOU'RE NOT IN CLUSTERS
YOU DON'T GET CURRENT, IF YOU'RE
NOT IN CLUSTERS IF YOU ARE
MEASURING INDIVIDUAL CELLS,
PEOPLE WHO ARE THE TRANSPORTER
FIELD KNOW THAT IF YOU TAKE
INDIVIDUAL CELLS AND ADD GLYCINE
OR [INDISCERNIBLE], YOU DON'T
GET MUCH CURRENT.
SO SHE SAID I WILL GO TO
INDIVIDUAL CELLS AND SEE HOW I
CAN GET THIS TO WORK?
AND SO THE FIRST THING WE DID
WITH THE INDIVIDUAL CELLS WAS,
OKAY, IF INDEED, FIRST LET'S
VERIFY IF YOU CAN CROSS LINK THE
TOP AND GET CURRENTS SO WE DID
THE CHEMICAL METHOD OF DOING
STUFF.
SO SHE USED, SOPHISTICATED WE
HAVE BASICALLY CYSTINES
INTRODUCED ON BOTH SIDES AND WE
USED BI FUNCTIONAL CYSTINE CROSS
LINKERS OF VARIOUS LENGTHS AND
YOU SEE THAT WHEN YOU USE A SHOT
CROSS LINKER, YOU GET--YOU GET
CURRENTS AND THEN WHEN YOU USE
LONG CROSS LINKER, THIS IS
ROUGHLY THE LINK THAT IS THERE
IN THE CRYO EM STRUCTURES, IT'S
ABOUT 21-ANGSTROMS SO WHEN YOU
GET TO 25, YOU DON'T SEE ANY
CURRENTS SO THIS CRYO EM
STRUCTURE IS PROBABLY THE
DESENSITIZED STATE.
AND OF COURSE, YOU WANT TO, ALL
OF THIS MEANS NOTHING BECAUSE
YOU COULD HAVE DIFFERENT AMOUNTS
OF PROTEIN BEING EXPRESSED SO
SHE DID ELEMENTS TO LOOK AT
PROBABILITY OF OPENING AND YES,
THIS IS MORE OPEN.
BUT THEN THE FINAL SET OF
EXPERIMENTS SHE DID IS SHE SAID,
OKAY, I'M GOING TO EXPRESS GLUD2
IN THESE CELLS AND WE WILL
PURIFY AND SO THAT IT'S SORT OF
AND ON THEN ADD THIS ON TOLET
PIPETTE, THE CEREBELLUM ALONG
WITH GLIESINE AND THEN ACTIVATE
THE CHANNELS AT THE SINGLE
CHANNEL MODE.
SO GLUE D2 ALONE WHEN YOU HAD
GLYCINE IN THE PIPETTE, YOU GET
NO SINGLE CHABLES WHEN YOU ADD
CEREBELLUM WITH GLYCINE, YOU
DON'T SEE MUCH, BUT IF WE RECORD
FOR LONG ENOUGH TIME WE ACTUALLY
SEE 1 OR 2 SMALL LENGTHS, I WANT
TO ADD THAT THERE, BUT THEN YOU
ADD ALL 3, YOU START SEEING
OPENINGS.
OKAY?
AND SINCE THIS TIME, SHE HAS
ACTUALLY PURIFIED, A GLUE D 2
ALSO AND DIP MEASUREMENTS THAT
YOU HAVE GLUE D2 ASK CBLN
ALTOGETHER AND LOOK AT THE
CHANNELS IN BILAYERS AND SHE
SEES SINGLE CHANNELS WHEN SHE
HAS ALL 3 COMPONENTS.
THIS IS AS OF LAST WEEK SO I
DIDN'T INCLUDE THAT DATA YET.
SO SO THE NEXT FINAL PART WAS WE
WANTED TO MAKE SURE THAT WHAT WE
SAW IN THE SINGLE CHANNEL
RECORDING WAS REFLECTED IN OUR
SINGLE MOLECULE THREAT.
SO AND THE TERMINAL DOMAIN
ADDING NEW CEREBELLIN AND
COMPLEX TO SEE WHAT HAPPENS AND
THIS IS WITHOUT NRX AND
CEREBELLUM, THIS IS THE RAW
DATA, THIS IS THE HISTOGRAM
WITHOUT NRX AND CEREBELLIN AND
YOU CAN CLEARLY SEE THAT THE TOP
COMES TOGETHER.
SO THOSE RECEPTORS ARE NOT
ORPHAN RECEPTORS, THEY ARE
ACTUALLY GLUE MARIOUS SEEN
NEGATED CHANNELS.
THE ROLE IN TERMS OF PHYSIOLOGY
IS, WE STILL DON'T KNOW WHAT
THAT GLYCINE AND DC CURRENTS DO,
AND AND BEYOND THAT, BUT
CLEARLY, IT IS BOUND TO THESE,
IT SEEMS THAT YOU CAN GET THESE
CHANNELS.
AND I'M GOING TO END WITH THAT
AND JUST THANK ALL THE PEOPLE
WHO WORKED ON THIS, GISTY HELPED
US GET STARTED TO THE SINGLE
MOLECULE 38 AND ALEX IS OUR
COLLABORATOR ON MD SIMULATION,
SO WITH THAT I WILL STOP.
[ APPLAUSE ]
>> SO I THINK FOR THE NRX,
SHOWED US [INDISCERNIBLE] DO YOU
NEED BOTH?
OR IF YOU ADD 1 OR THE OTHER BY
ITSELF [INDISCERNIBLE]?
>> YEAH, THIS IS JUST WITH CBLN,
AND WE'VE ALSO DONE NRX ALONE.
THE TOP 1 IS THE CBLN, NO IT
DOESN'T, YOU NEED BOTH.
I TAKE IT BACK.
SO CBLN ALONE LIKE I SAID, ONCE
IN A WHILE YOU SEE THESE BLIPS
BUT I DON'T KNOW IT COULD JUST
SOME RANDOM BACKGROUND ACTIVITY
BUT WE DON'T SEE THAT AS OFTEN
WITH GLUD ALONE BECAUSE IT HAS
THE ABILITY TO CROSS.
>> DO YOU THINK THAT THEY'RE
MAKING [INDISCERNIBLE]?
>> NRX IS THE 1 THAT'S
MAKING--THE CLUSTERS IS ABLE TO
DO THAT AND WE HAVE NRX DIMER
WHICH WE HAVE MADE FROM
COILED--IT'S NOT A DIMER--I
DON'T THINK IT'S A DIMER.
I DON'T KNOW.
AT LEAST WE DON'T KNOW IT'S A
DIMER IN PHYSIOLOGY, IT'S NRX BY
ITSELF, SO WE MADE A COIL COIL
NRX DIMER WHICH IS WHAT REALLY
MAKES THE ACTIVITY, YEAH.
>> REALLY INTERESTING WORK DO
YOU KNOW ANYTHING ABOUT
[INDISCERNIBLE]?
>> YES, SO IT CAN--AGAIN,
EVERYBODY'S VERY PRELIMINARY,
ALL THIS HAPPENED OVER THE LAST
FEW YEARS.
SO SHE'S DONE--IT'S OF COURSE
SODIUM, POTASSIUM, POTASSIUM CAN
GO THROUGH AND THAT SHE DID THE
POTASSIUM BECAUSE WE WANTED TO
GET RID OF THE SNAT, WHICH WAS
KILLING US AND THEN CALCIUM,
THERE IS SOME, BUT IF YOU ADD
LIKE ZINC FOR INSTANCE IT
INHIBITS THE CHANNEL.
>> [INDISCERNIBLE]
>> YES.
WE WE'VE TRIED THEM.
SO IT'S VERY LITTLE.
NO IT'S NOT--IT'S NOT LARGE, SO
THAT IS DEFINITELY ON THE LIST
OF EXPERIMENTS TO DO.
WE REALLY FEEL LIKE THAT WOULD
REALLY HELP US AND NOW THAT
WE'VE GOTTEN RID OF ALL OTHER
BACKGROUNDS, SO I THINK IT WILL
BE EASIER TO SEE THE CALCIUM.
YEAH.
>> RELATED QUESTION.
ARE THERE ANY OTHER
[INDISCERNIBLE] RECEPTOR?
>> OH YEAH.
ABSOLUTELY.
SORRY, I DONT WANT TO GO INTO IT
BUT WHEN I SAID THE PILLARS,
THAT'S A GOOD POINT BECAUSE MOST
OF THE WORK HAS FOCUSED ON THE
SECONDARY MESSENGERS PATHWAY
BECAUSE THE C-TERMINAL DOMAIN IS
EXTREMELY CRITICAL IN MEDIATING
THAT PATHWAY AND EVEN THERE,
THERE IS A BIT OF A CONTROVERSY
WHERE SOME PEOPLE THINK GLYCINE
IS NECESSARY TO MEDIATE THE
C-TERMINAL IMMEDIATE AND
INTRACELLULAR SIGNALING
PATHWAYS.
OTHERS SAY I DON'T NEED ANY OF
THIS.
ALL YOU NEED IS--THE LAB SAYS
ALL YOU NEED IS THE AMINO
TERMINAL DOMAIN MAKING THIS
ACROSS, AS LONG AS YOU HAVE SOME
PROTEIN CONNECTING THE
C-TERMINUS TO THE AMINO TERMINAL
DOMAIN, IT'S ALL FINE.
LTP AND STILL FINE.
ABOUT YOU OUR OTHER LABS SHOW
THAT IF YOU MUTED THE GLUE
MARIOUS SEEN BINDING SITE FOR
INSTANCE THEN LTP IS DISRUPTIVE
SO THEY'RE LIKE, NO, GLYCINE
BIND SUGGEST IMPORTANT BUT THEY
SAID IT AFFECTS THE C-TERMINAL
DOMAIN, NOTHING TO DO WITH
CHANNEL ACTIVITY SO NOW WE'RE
TRYING TO--BUT THE MUTANT WAS
MADE BUT THE KNOCK OUT WAS KNOCK
OUT AND KNOCK-IN OF THAT MUTANT
CHANNEL MUTANT WAS MADE BUT WE
DID SEE A SMALL CHANGE IN
BEHAVIOR BUT THEY SAID IT'S NOT
THAT SIGNIFICANT AND I AGREE,
THAT'S NOT THE MAIN THING BUT I
THINK THE CHANNEL ACTIVITY IS
THERE.
IT MAY NOTHING LIKE THAT BE THE
MAIN POINT.
IT'S THERE.
YEAH.
>> CAN YOU REPEAT THE QUESTION
FOR THE VIDEOCAST AUDIENCE.
>> WHEN COMPARING THESE IN THE
ALSTERRERY DOMAIN THAT SHE'S THE
SEQUENCE FOR EVOLUTIONARY
SIGNAL
DIFFER?
>> I THINK THAT WOULD BE A GREAT
COLLABORATION WITH LUCY.
>> AND THEN I HAD A QUESTION IF
YOU CAN REPEAT THE QUESTION IN
YOUR MIC, DO I UNDERSTAND THIS
THAT IN THIS CASE, YOU HAVE THE
DELTA RECEPTOR SMA, EXTRACTED.
>> OKAY, THIS WAS DONE ON CELLS,
AND THE SMA PURIFIED NRX
WAS--BECAUSE WHEN WE USE
DETERGENT PURIFIED NRX AND THE
PURIFIED WASN'T STABLE THAT'S
WHY WE WENT WITH PURIFIED NRX
INSTEAD OF CEREBELLIN.
>> [INDISCERNIBLE] WITH BOTH
PROTEINS IN THE EXTRACTED
DEPARTMENT.
>> WE'RE TRYING.
>> THANK YOU VERY MUCH.
>> IT'S THERE.
IT'S BEING DONE AS WE ARE
SPEAKING.
NOT JUST BY OUR LABS, SEVERAL
LABS.
SO A LOT IN THIS FIELD AT THIS
POINT BECAUSE A LOT OF PEOPLE
ARE WORKING ON THIS.
>> WELL, IF THERE ARE NO OTHER
QUESTIONS, THEN LET'S JOIN IN
THANKING OUR SPEAKER AGAIN FOR A
WONDERFUL PRESENTATION.
[ APPLAUSE ]