>>THANK YOU FOR COMING.
TODAY EITHER IN PERSON OR
VIRTUALLY.
WE HAVE A NICE TURN OUT TODAY.
I'M KATIE KENT, SENIOR
INVESTIGATOR AT THE NIDCD AND
IT'S MY PLEASURE TO INTRODUCE
TODAY'S NEUROSCIENCE SEMINAR
SPEAKER RYAN HIBBS, WORLD CLASS
STRUCTURAL BIOLOGIST.
IVY KNOWN RYAN SINCE WE WERE
GRADUATE STUDENTS AT UCSD, AT
THE UNIVERSITY OF SAN DIEGO
WHICH DOES SEEM LIKE A LIFETIME
AGO.
VERY MEMORABLE, RYAN HELPED ME
PICK OUT MY FIRST SURF BOARD AND
WE HAD A LOT OF FUN TRIPS ON THE
BAJA PENINSULA.
WHEN HE WASN'T SURFING HE DID
RESEARCH IN STRUCTURAL BIOLOGY
AND HE STUDIED LIGAND
SPECIFICITY AND CONFIRMATIONAL
CHANGES IN ION CHANNEL BINDING
DOMAIN.
I'M NOT A STRUCTURAL BIOLOGIST,
I FIND IF FASCINATING, I THINK I
GOT THAT RIGHT AND AFTER A
SUCCESSFUL Ph.D. WE STARTEDIE
POST DOC AT OSU AND PORTLAND IS
A WONDERFUL PLACE TO DO YOUR
POST DOC AND I DID MINE AS WELL,
SO I GET TO HANG OUT WITH RYAN
SOMEWHERE, BUT IN THE LAB, RYAN
STARTED LEARNING THE NUANCES OF
CRYSTALLOGRAPHY AND SOLVEDLET
STRUCTURALLY UPPER OF AN LIGAND
ION CHAN AND HE WILL THIS
STRUCTURE ALONG WITH SUBSEQUENT
STRUCTURES THAT HIS LAB SOLVED
ARE TRULY LANDMARK
ACCOMPLISHMENTS BECAUSE THESE
SYNAPTIC PROTEINS RESIDE IN THE
MEMBRANE, AND HAD BEEN
PREVIOUSLY EXTREMELY DIFFICULT
TO CRYSTALLIZE TO GET THE
STRUCTURALLY UPPER OF SO IT'S
VERY AMAZING AND HEARD LOTS OF
REALLY AMAZING PAPERS THAT WOULD
MAKE ANY STRUCTURAL BIOLOGIST,A
MAZING, SO IT'S BEEN FUN TO
FOLLOW HIS CAREER, NONETHELESS,
SO HE'S IN HIS LAB, HE'S NOT
ONLY DO CRYSTALLOGRAPHY BUT ALSO
CRYOEM APPROACHES TO OBTAIN NICE
HIGH RESOLUTION 3D STRUCTURES OF
THESE SYNAPTIC PROTEINS WITH
AGAIN A FOCUS ON LIGAND
RECEPTORS SOME OF WHICH WE WILL
LEARN ABOUT TODAY MULTIPLE
CONFIGURURATIONS WHICH I'VE
LEARNED IS IMPORTANT IF YOU WANT
TO KNOW NOT ONLY HOW THE NATURAL
LIGAND BINDS BUT HOW THEY MIGHT
INTERACT WITH THE RECEPTOR OR
MAKE DIFFERENT THERAPEUTIC DRUG
TARGETS AS WELL.
AND AFTER HIS CONTINUED SUCCESS
ADDED UT SOUTHWESTERN, HE WILL
MOVE HIS LAB BACK TO UCSD IN THE
SPRING WHERE HE BEGAN HIS WORK
IN STRUCTURAL BIOLOGY DOING MORE
GREAT WORK AND GETTING TO GO DO
MORE SURFING AS WELL, AGAIN, AND
SO IT'S REALLY MY PLEASURE TO
HAVE YOU HERE TODAY, RYAN SO
THANK YOU FOR COMING.
BUT BEFORE WE HEAR ABOUT YOUR
MOST RECENT WORK I WANT TO
REMIND EVERYBODY TO USE A
MICROPHONE SO PEOPLE CAN HEAR
YOU, AND I WILL BE CHECKING MY
E-MAIL AND WILL BE ABLE TO ASK
QUESTIONS YOU SEND IN THROUGH
THE LIVE CHAT.
SO THANK YOU.
GO AHEAD, ALL YOURS, RYAN.
[ APPLAUSE ]
>>THANK YOU KATIE AND THANK YOU
ALL FOR COMING.
I APPRECIATE YOUR PATIENCE, I'VE
RESCHEDULED AND CANCELED THIS
SEMINAR, KENT INVITED ME AND
THEN THE PANDEMIC HAPPENED AND I
DIDN'T WANT TO DO IT VIRTUALLY
AND SO I'M JUST WONDERFUL POETIC
BE HERE, WONDERFUL FRIENDS AND
COLLEAGUES, PEOPLE LIKE KATIE.
SO I'M GLAD TO BE HERE.
I NL EXCITED TO TELL YOU ABOUT A
PROJECT WE'VE BEEN LOOKING ON
FOR A LONG TIME TRYING TO GET A
STRUCTURE OF THIS PROTOTYPICAL
ION CHANNEL, THE RECEPTOR, THE
STAR OF THE SHOW TODAY, IT'S IN
THE MIDDLE OF THE SCREEN HERE,
THE CRYOEM RECEPTOR AND A THEME
SHALL BE PRESENT THROUGHOUT THE
TALK IS HOW NATURAL PRODUCTS AND
TALKS HAVE BEEN USED TO PROBE
THE STRUCTURE AND FUNCTION AND
PHYSIOLOGY OF THIS FAMOUS
CHANNEL, SO ON THE LEFT WE HAVE
AN INDIAN FROM SOUTH AMERICA WHO
HAS CODED THE TIPS OF THEIR
ARROWS WITH THE EXTRACT FROM THE
PLANT THAT CONTAINS THE MOLECULE
[INDISCERNIBLE] AND MORE
SPECIFICALLY, THAT BINDS AS A
COMPETITIVE ANTAGONIST AT THE
MUSCLE NICTINNIC, THE JUNCTION
CAUSING FLACCID PARALYSIS OF THE
MUSCLES SO IF PARALYZE IT IS THE
PREY AND THEY CAN CAPTURE THEIR
PREY.
SNAKES HAVE--DOES DIFFERENT
THINGS IN TERMS OF STRUCTURE
FUNCTION RELATIONSHIPS AND BOTH
OF THESE HAVE BEEN USED TO
CHARACTERIZE THE RECEPTOR AND I
WILL MAKE AN ARGUMENT TODAY THAT
THERE'S STILL MORE TO LEARN FROM
THESE INTERESTING NATURAL
PRODUCTS.
ALL RIGHT, SO, GOING FORWARD
LET'S SEE HERE, NO.
CAN I GO?
OH, I SEE, AKRO BAT, IT'S TRYING
TO UPDATE, OKAY, GOOD.
SO ALMOST ALL THE WORK I'M GOING
TO TALK ABOUT TODAY WAS DONE BOO
BY A TALENTED POST DOC IN THE
LAB [INDISCERNIBLE].
SO NICK O 10IC RECEPTORS THEY
ARE PART OF A FAMILY OF
RECEPTORS, THIS CHANNEL FAMILY
CAN BE DIVIDED INTO 2 BROAD
CATEGORIES THOSE WHOSE INTRINSIC
CHANNELS ARE SELECTED FOR CAT
IONS AND 5 H3 T SEROTONIN
RECEPTORS.
THE OTHER SIDE OF THE FAMILY IS
A SELECTIVE BRANCH INCLUDING
GABBA A RECEPTORS AND GLUE
MARIOUS SEEN RECEPTORS, THE CAT
ION SELECTED BRANCH ARE
GENERALLY NONSELECTIVE AMONG CAT
IONS AND SOME LET CALCIUM AND
SYNAPTIC MISSION, AND THE AN ION
CHANNELS ARE PERMEABLE TO
CHLORIDE, AND BECAUSE THE
MEMBRANE POTENTIAL AT WHICH
CHLORIDE IS AN EQUILIBRIUM FOR
THE NEURON WHEN YOU OPEN ANN ION
CHANNEL LIKE THE GABBA A
RECEPTOR THEY TYPICALLY, POSE
EXCITABILITY AND PLAY INHIBITORY
ROLES IN SYNAPTIC TRANSMISSION.
NOW REGARDLESS OF THE
NEUROTRANSMITTER PHARMACOLOGY
AND THE CHANNEL PROPERTIES THEY
SHARE A CONSERVED ARCH TEJTURE
SO THEY'RE ION CHANNELS, THEY
CAN BE HOMOPENTINE REGIMEN MERS
OR HETEROGENEOUSERY PENTAMERS,
PHYSIOLOGICALLY IN HUMANS,
THEY'RE NEARLY ALL HETEROPENTINE
REGIMEN MERS AS AN EXAMPLE,
THERE ARE 16 SUBUNITS, THAT CAN
ASSEMBLE IN A LARGE BUT DEFINED
NUMBER OF WAYS TO MAKE
FUNCTIONAL PHYSIOLOGICALLY
RELEVANT CHANNELS, ONLY 1 OF
THOSE 16 SUBUNITS, THE ALPHA 7
ASSEMBLES INTO FUNCTIONAL
PHYSIOLOGICALLY HOMOPENTINE
REGIMEN MERS, THE OTHER ASSEMBLE
INTO OBLIGATE HETEROMERS WITH
THE SUBUNIT COMPOSITIONS, SO
REGARDLESS OF THE STOIKIOTETRY,
EACH SUBUNIT CHAIRS A COMMON
TOPOLOGY.
SO FROM'S A LARGE EXTRA CELLULAR
AMINO TERMINAL DOMAIN SO THIS IS
WHERE NEUROTRANSMITTERS BIND,
WITH THE SUBUNITS IN THE PENTINE
REGIMEN MER, FOR TRANSMEMBRANE
SPANNING ALPHA HELIXESS FOR THE
SECOND 1 CALLED M2, THE SECOND 1
OFF THE HELIX IS THE 1S THAT
LINES THE ION CHANNEL, SO IT
DETERMINES MOST OF THE
PERMEATION PROPERTIES AND CAT
ION AND SELECTIVITY.
SO THIS IS THE ONLY CHANNEL
FAMILY I KNOW OF THAT HAS
EVOLVED, THEY USE A COMMON
SCAFFOLD TO OPPOSING ACTIVITIES
SO THE TETRAMERIC, THE THEY'RE
ALL CAT ION CHANNELS SO IT'S
INTERESTING TO TRY TO LEARN FROM
THESE STUDIES ABOUT HOW THIS
COMMON SCAFFOLD CAN GIVE RISE TO
CHANNELS OPPOSING SELECTIVITYS.
OKAY, SO THESE ARE THE
PENTAMERRIC CHANNELS AND WE WILL
FOCUS NOW IN ON NICOTINIC
RECEPTORS FOR THE REST OF THE
TALK BUT I WANT TO MENTION A
DRUG CLASS WE WILL GET INTO AT
THE VERY END AND THAT IS GENERAL
AN THESAURUSSICS AND THIS WILL
COME UP AT THE END BUT WE'VE
BEEN WORKING ON GABBA A
RECEPTORS AND NICOTINIC RECEPTS
IT IN MY LAB AND THESE ARE THE
FAMOUS TARGET OF MANY
ANESTHETICS AND THE WAY
GENERALLAN ESTIMATE THADTHETICS
WORK BROADLY, THEY DON'T ALL
ACTOT SAME CHANNELS, THEY ACT
THROUGH POLYPHARMACOLOGY
MECHANISMS SO THEY DO DIFFERENT
THINGS TO DIFFERENT KINDS OF
CHANNELS, AND BROADLY THE WAY
GENERALLAN ESTIMATE THADTHETICS
DO AND WHAT THEY DO IS THEY
POTENTIAL INHIBITORY CHANNELS
AND INHIBIT EXCITATORY CHANNELS
SO THEY'LL--THE SAME MOLECULE
LIKE PROPOFOL WILL POTENTIAL
GABBA A RECEPTORS AND INHIBIT
NICOTINIC RECEPTORS SO WE WILL
LOOK AT HOW THEY WORKT ON THE
NICOTINIC RECEPTORS AT THE VERY
END.
THIS DETERMINES WHERE THEY'RE
FOUND IN THE BODY, WHAT THEY
DID, THE PHARMACOLOGY,
BIOPHYSICS, WE WORKED ON A FEW
OF THEM, I WILL TAKE YOU THROUGH
THIS TO GIVE YOU A SENSE IN THE
DIVERSITY IN THE FAMILY.
THESE LITTLE COLORED CIRCLES
HERE ARE WHERE NEUROTRANSMITTERS
AND OTHER AGONIST BIND THE
INTERFACES OF ALPHA AND NONALPHA
SUBUNITS SO THIS IS THE MOST
ABUNDANT ASSEMBLY IN THE BRAIN
AND IF YOU KNOCK OUT EITHER
ALPHA 4 BATTA 2 SUBUNITS THEY
WILL NO LONGER ADMINISTER
NICOTINE SO THIS RECEPTOR FORNLS
THE HIGH AFFINITY BINDING SITE,
AND EFFICACIOUS AND SMOKING
CESSATION.
ALPHA 3 BETA 4 THIS RECEPTOR
SUBTYPE IS FAMOUSLY CALLED THE
GANGLIONIC RECEPTOR BECAUSE IT'S
FOUND IN THE AUTONOAMIC GANGLIA
THIS IS PART OF THE PERIPHERAL
NERVOUS SYSTEM, THEY'RE'S
SYMPATHETIC AND PARASYMPATHETIC
BRANCHES AND, NEURONS COME OUT
OF THE SPINAL CORD, FORM A
SYNAPSE BEFORE THEY GET TO THE
TARGET ORGANS AND AT THAT
SYNAPSE THE NEUROTRANSMITTER IS
THE CHOLINE AND THE POST
SINATTIC RECEPTOR IS ALWAYS THE
ALPHABETTA TINNIC 3 AND 4
RECEPTOR SO WHY AM I SHOWING YOU
A HEROIN NEEDLE HERE?
WELL THEY'RE NOT JUST FOUND ON
THE PERIPHERY, THEY'RE ON THE
BRAIN AND REASON FOR REWARD AND
ADDICTION, SPECIFICALLY
[INDISCERNIBLE] AND SELECTIVE
ANTAGONIST FOR ALPHA 3 BETA 4
ARE REMARKABLY EFFICACIOUS IN
BROAD FORMS OF ADDICTION, SO
OPIOIDS AND AMPHETAMINES, KRO
CAIN, ALCOHOL, NICOTINE, EVEN
SUGAR.
SO THERE'S EFFORTS UNDER WAY TO
DEVELOP SELECTIVE COMPOUNDS AND
DON'T BROTHER THE RECEPTORS IN
THE PERIPHERY SOMEHOW IN
ADDICTION.
ALPHA 7 IS THE BIZARRE MEMBER OF
THE FAMILY, AND IT'S FOUND NOT
ONLY IN THE BRAIN, VERY
ABUNDANTLY BUT ALSO IN THE
PERIPHERY AND NONNEURONAL CELLS,
NONEXCITABLE CELLS, FOUND IN
MACROPHAGES AND EPITHELIAL CELLS
AND IT'S THOUGHT TO BE VERY
IMPORTANT IN INFLAMMATION, SO
AGONIST IN THE PERIPHERY OF THE
RECEPTOR, CHOLINE RELEASED BY
THE VAGUELE NURVE HAVE POTENT
ANTIINFLAMMATORY EFFECTS.
AND THEN THE FOCUS TODAY, THE
MUSCLENIQUE
MUSCLE NICOTINIC RECEPTOR, AND
THEN IN ADULTS THE GAMMA IS
REPLACED.
SO A GOOD PLACE TO START IS WITH
CLAUDE AND COLLEAGUES, HE GOT
AHOLD OF THIS STRICLNOS PLANTS
THAT THE POISON THEY HAD.
AND THEN JOHN NEW PORT LANGLY
FAME USUALLY COINED THE SERM
RECEPTIVE SUBSTANCE, CONCEPT
ULTIMATELYIZING THE NOTION OF A
CELL SURFACE RECEPTOR AND HE WAS
CONTRASTING THE ACTIONS OF
NICKEE TEEN SO THIS WAS TOBACCO
PLANT AND LEAVES AND FLOWERS,
NICKEE TEEN IS AN AGONIST FOR
THESE RECEPTORS CONTRASTING THAT
WITH THE ANTAGONIST AND THE
RECEPTORS AND FIRST KIND OF
MODEL CITIZEN LEAKULAR
PHARMACOLOGY EXPERIMENTS.
SO WHY DO WE KNOW SO MUCH ABOUT
THE MUSCLE NICOTINIC RECEPTOR,
WE HAVE THESE ALSO IMPORTANTLY,
DISCOVERED THAT ELECTRIC FISH
AND EELS HAVE THEIR ELECTRIC
ORGANS ARE PACKED WITH
CRYSTALLINE DENSITY OF ION
CHANNELS THAT HOMOLOGOUS TO THE
CHO LINE RECEPTORS AND THESE
PROVIDE AN ABUNDANT NATURAL
RESOURCE FOR CHEMISTS AND
BIOLOGIST TO STUDY.
SO HOW DO YOU ISOLATE THIS?
YOU NEED AN AFFINITY AGENT AND
THAT CAME IN THE FORM OF SNAKE
VENOM TOXINS, 3 FINGERED VENOM
TOXINS THAT BIND IRREVERSIBLY TO
NICOTINIC RECEPTORS.
AND THEN THEY PUT THESE
TOGETHER, THE ABUNDANT NATURAL
SOURCE, AFFINITY REAGENT, AND
PERFORMED THE FIRST PURIFICATION
OF AN ION CHANNEL AND THESE ARE
THE FIRST ELECTRON MICROGAFFS IN
1 OF THESE AND YOU CAN START TO
SEE IF YOU CAN SEE THAT FAR, I
CAN'T SEE IT FROM HERE BUT THERE
IS LITTLE ROSETTES WITH DOTS IN
THE MIDDLE AND THAT'S LOOKING
DOWN THE LONG AXIS OF THE ION
CHANNEL.
SO REALLY NEAT HISTORY AND THEN
IN TERMS OF STRUCTURAL BIOLOGY,
THE HERO HAS BEEN NIgE L WHO
MERELY SINGLE HANDEDLY FIGURED
OUT HOW TO GET STRUCTURES OF
THIS RECEPTOR SO HE WAS WORKING
WITH THE TORPEDO RAY AND HE
FOUND IF YOU DID A CRUDE
ENRICHMENT OF THESE MEMBRANES
AND YOU LET THEM SIT FOR A
MONTH, SOMETIMES THEY WOULD ROLL
UP INTO THESE BEAUTIFULLY
HELICAL ORDER TUBES, SO THESE
STRAIGHT LINES HERE, THESE ARE
THESE TUBES AND HE CAN THEN USE
THESE APPROACHES TO ROW
CONSTRUCT STRUCTURES OF
INCREASINGLY HIRE RESOLUTION OF
THESE RECEPTORS AND HERE'S 1,
CARVED OUT OF BALSA WOOD AND I'M
GLAD WE DON'T HAVE TO DO BALSA
WOOD RECONSTRUCTIONS IMORP.
THIS IS AMAZING BUT THIS
SPECIFIC EXAMPLE HERE
ILLUSTRATES THE HAZARDS OF
WORKING WITH AT LOW RESOLUTION
WHICH IS THAT 1 CAN MAKE
MISTAKES IN WHERE YOU PUT THINGS
AND THE SUBUNIT DOESN'T BELONG
HERE AND THIS IS GAMMA UNIT
BETWEEN THE 2 ALPHA SUBUNITS
WHICH WAS WORKED OUT BY ANOTHER
BIOCHEMIST.
THIS IS I THINK THE HIGHEST
RESOLUTION STRUCTURE THAT HE'S
BEEN ABLE TO GET TO, 2012, WE
MIGHT CALL DHS SOMETHING LIKE
6-ANGSTROM RESOLUTION, HERE'S
THE EXTRA CELLULAR DOMAIN WHICH
IS MOSTLY BETA STRAPPEDS AND
THESE ARE NOT RESOLVED FROM EACH
OTHER.
THE TRANSMEMBRANE DOMAIN,
TUBEULAR DENSITIES, CLEARLY
ALPHA HELICAL AND YOU CAN SAY, I
LET ABOUT, SOME ABOUT WHERE THE
SUBUNITS ARE, YOU CAN TALK ABOUT
SECONDARY STRUCTURE BUT WHAT'S
MISSING HERE IS RESOLUTION
SUFFICIENT POSITION SIDE CHAINS
WITH CONFIDENCE AND IF YOU WANT
TO TALK ABOUT LIGAND
INTERACTIONS IF YOU WANT TO TALK
ABOUT WHAT'S LINING THE
PERMEATION PATHWAY TO UNDERSTAND
ION SELECTIVITY, YOU NEED TO
KNOW WHERE THE SIDE CHAINS ARE
AND SO THIS IS THE YEAR I
STARTED MY LAB WAS 2012 AND WE
STARTED CHIPPING AWAY AT HOW DO
YOU MAKE THEE RECEPTORS RECOMBIN
ANTLY AND THAT'S WHERE WE WORKED
ON NEURONAL SUBTYPES AND WE
TRIED TO GET RECEPTORS USING
APPROACHES FOR THE MUSCLE
RECEPTOR AND FAILED OVER AND
OVER AGAIN.
WE COULDN'T GET THIS
PENTAMERCOMPRISING 4 DIFFERENT
TYPES OF SUBUNITS TO EXPRESS AT
PRECCABLE LEVELS.
SO WE RECENTLY WENT BACK TO THE
TORPEDE'S RAY AND WORKED WITH A
CHEMIST TO FIGURE OUT HOW TO
ISOLATE THIS IN QUANTITIES AND
QUALITIES SUFFICIENT FOR HIGH
RESOLUTION CRYOEM STUDIES.
SO WE GET SOME OF THE ELECTRIC
ORGAN, WE FRACTIONATE MEMBRANES,
THERE'S, I DON'T KNOW 50 YEARS
OF BEAUTIFUL BIOCHEMISTRY ON
WHAT KIND OF SALT, WHAT Ph,
HOW DO YOU GET RID OF THE SAV
OLDING PROTEINS THAT KEEP IT IN
THE RICE PLACENTA?
DETERMINANTS PRESERVE FUNCTION
AND GOOD BIOCHEMISTRY?
THAT WAS FUN TO READ AND WE
TALKED TO ARTHUR CONLYNN ABOUT
THIS A LOT AND [INDISCERNIBLE]
AND THEN WE EXTRACTED AND HOW DO
WE AFFINITY PURIFY IT SO A
FRIEND AND COLLEAGUE AT UC
BOULDER SIPGHTICIZED A NEW
REAGENT FOR US, SO THIS HAS THIS
AMMONIUM ON THE END OF IT AND WE
TETHER THAT THE PRIMARY MEANS SO
WE CAN ATTACH AND IT WORKS
BEAUTIFULLY.
IN THE ELECTRIC ORGAN 25% OF THE
PROTEIN IS THE NICOTINIC
RECEPTOR SO YOU DON'T HAVE TO DO
MUCH.
AND NICOTINIC WE ALLUDE OFF THE
COLONY IN 2 WAYS.
ORGANIZATIONSIGEINALLY WE WERE
ALLUDING WITH CARBOCOL, AND THAT
ALLOWED US TO GET STRUCTURES BUT
WE WANTED APO STRUCTURES WITH
ALL KINDS OF MOLECULES BOUND AND
THEN WE SWITCHED TO CHOLINE
WHICH HAS THE SAMEOT END BUT
MUCH LOWER AFFINITY SO WE CAN
ALLUDE OFF THE COLUMN AND RUN
THE FILTRATION AND GET RID OF
CHOLINE AND GET WHATEVER WE WANT
FOUND.
BEAUTIFULLY PURE, AFTER THIS
STEP, AND THEN WHEN WE RUN
ANALYTICAL GEL FILTRATION, WE
SEE A MIXED POPULATION, WE HAVE
A MONITOR MER, DIMER POPULATION,
HERE MONITOR MERMEANS 1
PENTAMERAND DIMER MEANS 2
PENTINE REGIMEN A MERS STUCK
TOGETHER BECAUSE THERE'S A CROSS
LINK IN THE RECEPTOR AT THE
C-TERMINAL RESIDUE OF TELTA DUB
UNITS.
WHY?
DON'T KNOW, MAYBE IMPORTANT FOR
PACKING AT HIGH DENSITY IN THAT
MEMBRANE BUT WE KNOW WE CAN
REDUCE THOSE AND FUNCTION ISN'T
AFFECTED SO WHAT DO WE DO NEXT,
WE HAVE A PURIFICATION APPROACH
BUT WE WANT TO BE SMART ABOUT
HOW WE'RE GOING TO MAKE OUR EM
SAMPLE TO HOPEFULLY PRESERVE
FUNCTIONAL PROTEINS WHEN WE LOOK
AT CONFIRMATIONAL CHANGES, WE
CAN HAVE CONFIDENCE THAT THEY'RE
RELEVANT.
SO WE WANT TO TEST LIPIDS IN
CONSTITUTION TO SEE WHICH
SUPPORT FUNCTION, SO WE TAKEAUR
RECEPTOR, WE ADD LIPIDS AND WE
TRY DIFFERENT COMPOSITIONS OF
LIPIDS.
WE REMOVE DETERGENT WITH THE
BIOBEADS THAT ABSORB THE
DETERGENT.
AND WE DO THIS WITH DIFFERENT
KINDS OF LIPIDS.
WE FOUND SOY [INDISCERNIBLE]
EXTRACT WORKS BEAUTIFULLY AND WE
CAN SEE CHARACTERISTIC OPENING
AT BOTH POSITIVE AND NEGATIVE
VOLTAGES AND WE CAN BLOCK THIS
TOXIN.
SO WE'RE HAPPY TO ABOUT THAT SO
THEN WE TAKE OUR PURIFIED
RECEPTOR AND DETERNLG AND THEN
THE SIEWTION THE SAME
COMPENSATION AND WE ADD A NANO
DISK AND IN THIS FIRST CASE, WE
ADDED THE SNAKE VENOM TOXIN I
MENTIONED.
WE THOUGHT AT THE BEGINNING WE
WERE GOING TO NEED SOME KIND OF
BIG ASYMMETRIC MOLECULE STICKING
OUT OF THE SIDE OF THE RECEPTOR
THAT WOULD ALLOW US TO ALIGN
PARTICLES IN 3 DIMENSIONS, SO IN
CRYOEM WE'RE AVERAGING HUNDREDS
OF THOUSANDS OF MILLIONS OF
PARTICLES FROM DIFFERENT
ORIENTATIONS TO GENERATE A 3D
RECONSTRUCTION, THESE RECEPTORS
CEMET RICK OR PSEUDOSYMMETRIC
AROUND THE 5 FOLD ACCESS AND,A
LIENING PARTICLES STARTS USING
ONLY LOW RESOLUTION INFORMATION
SO YOU'RE LOOKING FOR BIG
FEATURES AND AT LOW RESOLUTION,
YOU CAN'T TELL, WE WOULD BT
THINK AN ALPHA FROM A BETA FROM
A GATE KEEPER AMIA FROM A DELTA
SUBUNIT AND IF YOU SUPER IMPOSE
THOSE ON TOP OF EACH OTHER AND
FIND A HIGH RESOLUTION, YOU WILL
BLUR OUT BECAUSE YOU WILL SUPER
IMPOSE ALPHAS AND SO WE ADD BUNG
AROTOXIN SO WE THOUSAND DOLLAR
IT WOULD GIVE IT WINGS TO,A
LIEN.
THEN WE COLLECTED CRYOEM DATA
AND WE SEE THESE DIMERS AND
PENTAMERS AND HERE'S A SIDE
VIEW, WHERE THEY'RE PARALLEL TO
EACH OTHER MAYBE IN 1 LARGE NANO
DISK AND MAYBE YOU CAN SEE
BETWEEN THESE EXTRA CELLULAR
DOMAINS, THERE ARE THESE WINGS
STICKING OUT, THAT'S THE
NEUROTOXIN, THIS IS THE FIRST
DATA SET WE COLLECTED ON THESE
AND WE COULD SEE THESE FEATURES,
VERY EXCITING BUT WE HAVE THESE
DIMERS AND PENTINE REGIMEN MERS
AND THEY ADOPT DIFFERENT
ORIENTATIONS RELATIVE TO EACH
OTHER AND WHICH WOULD BE A
PROBLEM ARE FOR RECONSTRUCTING A
3D STRUCTURE.
SO WE TRIED SOME REDUCING
REAGENTS AND PEOPLE SHOWED 30
YEARS AGO, YOU COULD ADD THE
ETHAN OLDER PEOPLE AND REDUCE
THESE AND FUNCTION WAS
IDENTICAL, YOU COULD RECONSITUTE
THEM AND WE FOUND THE SAME
THING.
SO WE ADD BME AND WE RUN THE
FILTRATION, TO CLEAN IT UP AND
WE SHIP THE DIMER OVER TO THE
MONITOR MERAND THAT'S WHAT WE US
THEN FOR STRUCTURAL BIOLOGY.
>>ALL RIGHT, SO HERE'S AN
EXAMPLE OF THE PATHWAY FOR
PROCESSING 1 OF THESE DATA SETS
SO WE COLLECT THOUSANDS OF
ELECTRON MICROGAFFS, I SECOND
QUARTERLED A COUPLE OF THE
RECEPTORS UP HERE THAT ARE
SINGLE PENTINE REGIMEN MERS, NOT
DIMERS OR PENTINE REGIMEN MERS,
2 D CLASSIFICATION TO GET A DATA
SET AND GET A SENSE OF DATA
QUALITY.
IN THE UPPER LEFT HAND KARNER WE
HAVE THE EXTRA CELLULAR DOMAIN,
TRANSMEMBRANE DOMAIN, WRAPPED IN
THE SCAFFOLD AND NOTHING
STICKING OUT OF THE BOTTOM.
CONTRAST THAT WITH THIS 1 HERE
AND YOU CAN SEE NICE DENSITY FOR
THE INTRACELLULAR DOMAIN AND
WHEN WE MOVE ON TO 3D
CLASSIFICATION, WE SEE SOMETHING
SIMILAR WHERE HALF THE PARTICLES
ARE LACKING THE IMPOSSIBLE TO
BUILD RACELLULAR DOMAIN,
PROBABLY DAMAGED FROM
INTERACTING WITH THE AIR WATER
INTERFACE, SOMETHING LIKE THAT,
THAT'S A GUESS, SO WE THROW OUT
THOSE PARTICLES THAT ARE IN THAT
CLASS BECAUSE WE ONLY WANT
INTACT RECEPTORS AND THEN WE
REFINED HIGH RUZ LIEWGZ AND WE
SEE THAT THE RESOLUTION'S FAIRLY
ISOTROPIC THROUGH THE WHOLE
RECEPTOR.
REALLY IMPRESSIVELY BEAUTIFUL
STRUCTURES COMPARED TO OTHER
PROTEINS WE'RE WORKING WITH.
ALL RIGHT.
SO THIS IS THE APO STRUCTURE IN
THE ABSENCE AND PRESENCE OF
DOPED IN CHOLESTEROL.
SO THERE'S A LOT OF INFORMATION
IN THIS AREA, ON LIPID FUNCTION
AND ESPECIALLY CHOLESTEROL AND
IN THE TORPEDO ELECTRIC RAY,
IT'S LIKE 40% CHOLESTEROL, IT'S
SUPER ABUNDANT IN OUR BRAINS,
AND WE WANT TO LOOK AT
CHOLESTEROL BINDING SEAT SITES
SO WE COLLECTED AND WE CAN SEE
THAT SOME OF THESE RED PEEKS
GET--APPEAR WHEN YOU ADD
CHOLESTEROL AND SOME SIMPLY GET
STRONGER SOPHISTICATEDY WE'RE
ABLE TO IDENTIFY OR ASSIGN LOWER
AND HIGHER AFFINITY CHOLESTEROL
SITES.
SO THIS IS THE EXTRA CELLULAR
DOMAIN, THESE THERAPIST IS
GLYCANS THAT COME OFF AND IN THE
INTRACELLULAR DOMAIN AND THEN
THIS IS THE TRANSMEMBRANE DOMAIN
IN THE SCAFFOLD,ER SO HERE'S THE
ARK RANGEMENT OF SUBUNITS
ALREADY WELL DELINEATED BY OTHER
PEOPLE AND THEY HAVE THE
LEFT-HAND SIDE OF THE
NEUROTRANSMITTER BINDING POCKET
AND THEN GAMMA AND DELTA
SUBUNITS MAKE WHAT WE CALL THE
COMPLIMENTARY SIDES OF THESE
NEUROTRANSMITTER BINDING POCKETS
SO THAT'S WHERE
NEUROTRANSPOLITTERS BIND.
I WILL SHOW YOU WHAT AN ALPHA
AND NONALPHA SUBUNIT LOOK LIKE
BECAUSE IT TURNS OUT WE DIDN'T
NEED ALPHA BUNKINGAR O TOXIN TO
PROPERLY REGISTER THE ALIGNMENT
OF THESE PARTICLES.
SO HERE'S AN ALPHA SUBUNIT, 1 OF
THE 2, SO EXTRA CELLULAR
DOMAININNA, LOOP CALLED LOOP C
WHICH IS SHOWN HERE IN THIS SIDE
VIEW, SO IT CAPS THE
NEUROTRANSMITTER BINDING POCKET,
AND FORMS A LOT OF INTERACTIONS
WITH AGONISTS, LIKE CHOLINE, THE
LOOP THAT GIVES THE FAMILY ITS
NAME CONNECTS THE
NEUROTRANSBINDING SITE, IT SITS
LIKE A BALL IN A SOCKET SO WHEN
DMIEWR O TRANSMITTERS BIND, THEY
CAN PULLOT CONFIRMATION OF THE
TMD OPEN GATES AND ALLOW IONS TO
GO THROUGH.
THE 4 TRANSMEMBRANE SPANNING
ALPHA HELIXES AND INTRACELLULAR
DOMAIN, SO I WILL SUPER IMPOSE A
NONALPHA SUBHIEWNT ON HERE AND
WE CAN SEE WHAT THE DIFFERENCES
ARE AS WELL AS SIMILARITIES SO
HERE'S ALPHA VERSUS DELTA
SUBUNIT.
SO I HOPE YOU CAN APPRECIATE
THAT THE CORE OF ALL 3 OF THESE
DOMAINS ALIGNS VERY WELL.
WHERE ARE THEY DIFFERENT?
THEY'RE DIFFERENT IN THE C-TERM
NIGH AND IN THE LOOP F AND ALSO
LOOP C IS DIFFERENT BUT IT'S NOT
SHOWN VERY WELL HERE.
SO APPARENTLY THE C-TERM INI AND
THE F, WHICH ARE FOUND IN THE
DAM ADELTA AND BETA SUBUNITS,
ARE ENOUGH IN SYMMETRY, CAN YOU
SEE THEM STICKING OUT TO
PROPERLY,A LIEN PARTICLES SO
THIS FORMS THE BACK SIDE OF THE
BINDING POCKET ON THESE SUBUNITS
AND THE C-TERNIMI EXPEND UP
WHICH IS NOTTINE IN THE SUBUNITS
AND HUNDRED BEEN SEEN IN THE
STRUCTURES BEFORE.
ALL RIGHT SO 1 OF THE BIG
QUESTIONS FROM THE FIELD IS
GAINING MECHANISM, HOW DOES THIS
TRANSMITTER OPEN AND CLOSE HE'S
CHANNELS AND WHAT IS THE GATING
CYCLE, SO WE HAVE CHANNELS IN
ARRESTING ESTATE, YOU APPLY
AGONIST LIKE ASTEOCHOLLINE, THEY
BRIEFLY ACTIVATE AND IN THE
SUSTAINED PRESENCE OF AGONIST,
THEY DESENSITIZE AND NEARLYY ALL
ION CHANNELS DO THIS.
WHEN NEUROTRANSMITTER
CONCENTRATION DROPS THEY CAN DO
THIS AND CONVERT BACK TO A
RESTING STATE.
SO WE HAVE AN APO RESTING ESTATE
STRUCTURE AND AGONIST BOUND
TRUCTURE, WE DO NOT HAVE AN
ACTIVATED STATE STRUCTURE, I
WOULD HAVE LOVED TO SHOW YOU AN
ACTIVATED STRUCTURE TODAY,
THERE'S A LOT OF DEBATE ABOUT,
YOU KNOW WE HAVE SOME ACTIVATED
STATE STRUCTURES OF HOMOPENTINE
REGIMEN MERS OF GLUE MARIOUS
SEEN RECEPTORS OF THE SUBTYPE
FROM OUR LABS, SERIES POINTSA
TONIC RECEPTORS, AND
[INDISCERNIBLE], BUT WE LACK ANY
HETEROPENTINE REGIMEN
MERSTRUCTURES AND WHETHER
THEY'RE FAIRLY SYMMETRIC OR
ASYMMETRIC, PEOPLE ARE ARGUING
ABOUT, SO WE WOULD LIKE TO SEE
THAT BUT WE DON'T HAVE IT, I AND
I HAVED WHYS ABOUT HOW TO GETD
IT AND I WILL TALK ABOUT IT THEA
THE VERY END BUT I CAN COMPARE
AN RESTING STRAIGHT STRUCTURE
ORAL AGONIST BOUND DESENSITIZED
STRUCTURE.
SO THIS IS THE LARGE VESTIBULE,
THESE SPHERES ARE JUST TO GIVE
YOUR EYES A SHAPE--SO WE CAN
ZOOM ON THE CONSTRIBLGHTED PART
HERE THROUGH THE TRANSMEMBRANE
SPANNING PORG OF THE RECEPTOR,OT
LEFT SIDE ARE DIAMETERS FOR SOME
OF THESE KEY POINTS ALONG THE
PERMEATION PATHWAY OR AMINO
ACIDS THAT LINE THE CHANNEL,
SHOWING THE ALPHA SUBUNITS ON
OPPOSITE SIDE HERE 22 TYPE FOR
HYDRATED IONS TO GO THROUGH AND
WE LOOK AT DESENSITIZED STATE IN
COMPARISON WHAT WE SEE IS A
WIDENING OF THE TOP HALF THE
COURSE, THE TIGHT HALF IS NEAR
THE TOP AND IT'S 2 AND HALF
ANGSTROMS IN DIAMETER AND THAT
OPENS UP TO 8-ANGSTROMS IN
DIAMETER.
SO CHANGES IN THE EXTRA CELLULAR
DOMAIN PULL OPEN THE TOPS OF THE
HELIXES DURING ACTIVATION AND WE
THINK THAT THE BOTTOM CLOSES
BECAUSE THE BOTTOM IS ABOUT AS
TIGHTLY CLOSED IN THE
DESENSITIZED AND RESTING STATES.
SO HOW DO WE,A SIGN
CONFIRMATIONAL STATES OR
PHYSIOLOGICAL STATES TO THESE
STATIC STRUCTURES WE'RE LOOKING
AT.
THIS IS AN UNANSWERED QUESTION
IN THE FIELD, HOW TO DO THIS
KIND OF RIGOROUSLY.
WE CAN DO THAT WITH THE
STRUCTURE WE SEE THAT WHILE WE
THINK IT SHOULD BE DESENSITIZED
BECAUSE AGONIST IS BOUND AND
IT'S EQUILIBRIUM, THERE WILL BE
A BIT OF LEAK OF IONS IN MD
SIMULATIONS.
DOES THAT MATCH THE PREDICTED
SINGLE CHANNEL CONDUCT ABTS,
WELL MD IS NOT VERY GOOD AT
MATCHING SINGLES CHANNEL CONDUCT
ANTS FROM ELECTROPHYSIOLOGY
EXPERIMENTS SO WHAT DO YOU SAY,
IS THIS CONDUCTING,
NONCONDUCTING INTERMEDIATE SO
IT'S HELPFUL TO GO BACK TO THE
CLASSIC STUDIES ON HOW BIG ARE
THE IONS THAT CAN GET THROUGH
THE ACTIVATED STATES OF THESE
CHANNELS.
THIS IS MY FAVORITE FOR THE NICK
O THENNIC RECEPTOR, SO THIS IS
BRITTLE'S GROUP IN 1980 WHERE
MAY PUT THROUGH A SERIES OF CAT
IONS MANY ORGANIC CAT IONS
THROUGH THESE TYPES OF ION
CHANNELS TO SAY HOW BIG SHOULD
THE CHANNEL BE AND MAN, THIS IS
SUCH SUCH BEAUTIFUL WORK, SO
WHATEVER, ALMOST 20 YEARS BEFORE
ROD Mc KINON GO THE STRUCTURE
OF KCSA, POTASSIUM WAS SHOWING
IT WOULD GO THROUGH AS A
DECARBONATED CAT ION.
SO IT CAN BE SELECTIVE THIS IS
TOTALLY DIFFERENT ANIMAL, YOU
COULD DRIVE A TRAIN THROUGH THIS
ION CHANNEL.
IT'S NOT TRYING TO SELECT AMONG
DIFFERENT CAT IONS IT'S TRYING
TO LET THROUGH ANY CAT IONS
MUSCLE CONSTRUCTION AND HAVE IT
WORK EVERY TIME.
IT'S VERY DIFFERENT SO BASED ON
THIS KIND OF STUDY, WE WOULD
CONCLUDE THAT IONS, SODIUM,
POTASSIUM, CALCJUM WHATEVER,
WOULD BE DOING THAT AS A
HYDRATED SPECIES.
SEVEN AND A HALF ANGSTROMS ON
THE DIAGONAL, IN DIAMETER SO
WHAT DID WE SEE, WE SAW
CONSTRUCTION OF 4 AND HALF
ANGSTROMS IN THIS DESENSITIZED
STATE SO BASED ON THAT I WOULD
SAY THIS IS A REASONABLE
ESTIMATION FOR A DESENSITIZED
STATE STRUCTURE BECAUSE IT'S
WEIRD THAT THE CONSTRUCTION IS 4
AND HALF RESTRICTIONS IN
DIAMETER.
SODIUM'S SMALLER THAT THAN AND
THIS IS A POLAR CONSTRUCTION,
THE AN ION HAVE PHOTO RECEPTORS
AT THE BOTTOM AND IT'S EASIER TO
THINK ABOUT HYDROPHOBIC WHERE
THEY CAN STICK TOGETHER, WATER
PONENT BE HAPPY THERE, THESE ARE
POLAR RESIDUES MAKING IT POLAR
GATE.
SO THERE'S STILL ARGUMENT ABOUT
WHAT ARE THESE GOING TO BE
LEAKY, WHAT IS A REAL OPEN STATE
LOOK LIKE ON THE CAT ION
SELECTIVE SIDE OF THE FAMILY.
SO, UNSOLVED PROBLEM.
WE WILL SWITCH FROM CHANNEL
BIOPHYSICS TO PHARMACOLOGY AND
WE WILL CONTRAST THESE 2 NATURAL
PRODUCTS.
SO ALPHA BUNG AROTOXIN, COMPETES
WITH AFTIO ALCOHOLIN AND BINDS
IRREVERSIBLY, THE OFF RATE'S
VERY SLOW.
IT COMES FROM A BLAPT, IT ALSO
COMPETES WITH AFTIO ALCOHOLIN
BUT IT HAS COMPLEX ACTIVITY SO
IN EMBRYONIC NICOTINIC
RECEPTORS, THE 1S THAT HAVE THE
DELTA SUBUNIT,
THEY--[INDISCERNIBLE] IS A
CHANNEL OPENING BEFORE YOU GET
PARALYSIS.
SO, LOOK AT THE STRUCTURE WITH
THE BUNG AROTOXINAR O TOXIN,
THIS IS THE FIRST STRUCTURE WE
GOT ACCIDENT I MENTIONED WE TRY
TO USE IT TO,A LIEN PARTICLES
WHICH DID WORK AND RATHER THAN
SHOWING YOU ALL THESE
CONFIRMATIONS OF THE PERMEATION
PATHWAY, I WILL SUMMARIZE THE
DATA IN GRAPHS LIKE THIS SO WE
HAVE ON THE Y-AXIS, PORE, THE
LONG AX INCREASE IN BODY TO THE
CHANNEL VERSUS PORE DIAMETER SO
IN RESTING STATE WE CAN SEE THAT
APO STRUCTURE I SHOWED YOU
BEFORE, THE TOP OF THE POUR IS
TIGHTLY CLOSED, VERSUS THE
DESENSITIZED STATE THAT OPENING
UP AT THE TOP OF THE PORE,
THAT'S WHAT I'M SHOWING IN THIS
GRAPH, WHAT DOES THIS LOOK LIKE
IN BUNG AROTOXIN BIND OR DO?
I WILL NOT SHOW YOU ALL THE
DETAILS FOR ALL THEE LIGANDS.
I KNOW MOST OF YOU DON'T CARE
ABOUT THIS UNLESS IT'S YOUR
PROTEIN OF INTEREST.
I FEEL THE SAME BUT I THINK THIS
IS AN INTERESTING EXAMPLE.
[LAUGHTER]
IT'S TRUE, RIGHT?
IT'S GOT 3 FINGERS, 1, 2, 3, THE
SECOND FINGER INSERTS DEEPLY
INTO THE SUBUNIT INTERFACE AND
IT BRINGS ALONG HAPPENING THIS
R-GENNINE RESIDUE AND IT THE
AGONIST INCLUDES BASIC NITROGEN,
FORMAL POSITIVE CHARGE,
R-GENERATEDDINE IS A THE SAME
THING IN A SENSE AND IT SITS IN
THE ARROWMATIC RESIDUES HERE
THAT ALSO STABLEALIZE THE
AMMONIUM OF ALCOHOLIN OR
CARBOCOL, IT'S REPLACING THOSE
INTERACTIONS, HIJACKING THAT
SAME SET OF INTERACTIONS AND IT
FORMS A PIE SANDWICH ON THIS AND
IT BRINGS ALONG HALF OF THIS CAT
ION PIE SANDWICH.
YOU KNOW SO IT COMPIETS WITH THE
NEUROTRANSMITTER AND KEEPS LOOP
C FROM CLOSING WHICH IT LIKES TO
DO AND - IT KEEPS THIS WHOLE
INTERFACE FROM BECOMING MORE
COMPACT WHICH IS WHAT HAPPENS
WHEN AGONIST BIND, SO IT
PREVENTS THE CONFIRMATIONAL
CHANGES THAT LEAD TO CHANNEL
OPENING AND MAKES SENSE WE WILL
STABILIZE A RESTING STATE.
SO WHAT ELSE IS INTERESTING
ABOUT THIS.
WELL, THERE ARE CRITTERS THAT
HAVE EVOLVED SENSITIVITY TO THE
SNAKE TOXINS AND THEY'RE REALLY
NEAT SO THEY INCLUDE THINGS LIKE
THE MONGOOSE AND COBRA.
SO HOW DO THEY DO THAT?
IN THE CASE OF THEE 2 ANIMALS
THIS TRANSCRIPT O FAN RIGHT HERE
ON LOOP C THAT STICKS OUT FROM
THE CORE OF THE RECEPTOR CREATES
A GLYCOSYLATION SITE.
SO CAN YOU IMAGINE A BIG SUGAR
BRANCH THING COMING OFF LOOP C
WOULD INTERFERE WITH TOXIN
BINDING.
THAT'S PRETTY COOL.
HONEY BADGERS AND PIGS, THE SAME
TRANSCRIPT O FAN RESIDUE AND
REPLACED WITH R-GENINE, IT
DECREASES AFFINITY ENOUGH SO IT
DOESN'T POINCH THE NEUROMSKULAR
JUNCTION IN ANIMALS AND THEN
THERE ARE NEURONAL NICOTINIC
SUBSTITUTIONS, AND IT'S A LARGE
PEPTIDE BUT NONE THE LESS,
TYROSEEN RESIDUES, THIS IS
TYROSEEN RESIDUE RIGHT HERE, PUT
A POSITIVE CHARGEOT END OF IT,
YOU NO LONGER GET HIGH,A FINNITY
BINDING AND INHIBITION BY A
NEUROTOXIN.
ALL RIGHT, SO THAT'S BUNG
AROTOXIN.
SO WHAT ABOUT Q-ARY, AND I
TALKED TO [INDISCERNIBLE] IS
QARY IS THOUGHT TO BE A HIGH
OPPOSED ANTAGONIST RECEPTOR.
SO HE ALLUDED WITH CARBOCOLOFF
THE PROTEIN AND HE GOT THE
STRUCTURE THAT HAD TO OUR
SURPRISE, D2 BOUND IN 1 AGONIST
SITE AND 1 FOUND IN THE OTHER.
SO THEN WE WENT AND DID WHAT WE
SHOULD HAVE DONE WHICH WAS READ
THE LITERATURE, YOU SHOULD
ALWAYS READ THE LITERATURE AND
FOUND THAT OF COURSE, THIS SITE
HAS A 500 FOLD HIGHER AFFINITY
FOR D2 AND THIS SITE AS HAS A 2
FOLD FOR HIGHER, SO IT MAKES
SENSE, AND I'M NOT GOING TO TRAG
IT THROUGH THE DETAILS BUT I DO
HAVE A SLIDE IF YOU WANT TO ASK
ON WHY DOES IT PREFER THESE
SITES AND IT DOES BUT IT MAKES
SENSE.
>>I SAID ALL RIGHT, I WANT TO
GET A STRUCTURE, DON'T YOU OF
THE RECEPTOR BOUND TO SEE WHAT
THE CONFIRMATION OF THE POUR
LOOKS LIKE, I SAID AND WE SAID
YES, LET'S DO THAT, 200
MICROMOLAR FOR THIS ANTAGONIST
AND HE THEN FOUND IT BINDS BOTH
THE ALPHA GAMMAA AND ALPHA DELTA
INTERFACES AND IT BYPASSEDS IN
THE CHANNEL BLOCK SITE AND THIS
WAS THE BIG SURPRISE, IS THAT IT
BINDS IN THIS KIND OF DOWN IN
THE TRANSMEMBRANE DOMAIN BUT ON
THE EXTRA CELLULAR SIDE, JUST
POKING OUT OF THE EXTRA CELLULAR
SIDE OF THE INTERFACE.
AND THE WAY IT FITS IN THERE, IS
THIS M4, THE LAST HELIX FROM
THIS ALPHA SUBUNIT BECOMES
DETACHED, SO THIS INTERACTION
HERE WITH THIS LITTLE SHORT
HELIX HERE THAT MAKES US
INTERACTION WITH WHAT WE CALL
THE COUPLING REGION, THAT
INTERACTION IS SEVERED, THIS
TINNING OPENS UP AND ALLOWS
D-TUBO TO BIND IN THERE.
SO WE THEN SPENT TIME AND I WILL
WALK YOU THROUGH IT OF USING TOO
MUCH PURINE OR IS THIS SHOWING
US SOMETHING INTERESTING ABOUT A
MODULATORS SITE, A NEW DRUGGABLE
SITE OR INSIGHT INTOS HOW
CHANNELS WORK AND I WILL TAKE
YOU THROUGH THAT.
I THINK THERE IS SOMETHING
INTERESTING THERE.
SO BUT A BASIC QUESTION WAS WHAT
CONFIRMATIONAL STATE DOES T-DUBO
STABILIZE, SO I'M SHOWING 3
TRACES I SHOWED YOU BEFORE, THE
RESTING STATE ON THE LEFT WHICH
IS CLOSED TIGHTLY AT THE TOP AND
MIDDLE AND WE SEE THAT BOTH
STRUCTURES WITH D-DUBO BOUND
LOOK LIKE DESENSITIZED CONFIRM
AS OF THE FORE.
WHICH MAKES SENSE GIVEN THAT AS
I TOLD YOU T-TUBO CAN ACT AS A
PARTIAL AGONIST SO WE CAN THINK
OF IT AS A LOW EFFICACY AGONIST
THAT STABILIZES DESENSITIZED
STATE WITHOUT MUCH CHANNEL
OPENING.
OKAY.
SO WHAT ABOUT THIS SITE, THIS
SITE WILL BE THE FOCUS OF THE
REST OF THE TALK.
GREAT.
SO HERE IS--I WILL SHOW YOU 2
STRUCTURAL OVERLIES, HERA 1 ON
THE LEFT WHERE WE'RE SUPER
IMPOSSESSING THE ALPHA SUBUNITS
AND THE RESTING STATE IN GRAY
AND THE DESENSITIZED STATE IN
GREEN AND MOST OF THE CORE OF
THE STRUCTURES OVERLAY QUITE
WELL, THE CONFIRMATION OF THE
PORE IS DIFFERENT.
I'VE SHOWED YOU THE THESE GRAFS,
THE DESENSITIZED STATE IS MORE
OPEN, AND THEN WHAT WE STARTED
NOTICING IS AFTER ONLY REALLY
LOOKING AT THESE D-TUBO SITE,
THERE'S SOMETHING FUNNY GOING ON
WITH THE CONFIRMATION OF THE
C-TERM NIGH OF THE M4 HELIX, SO
THE VERY END OF THE RECEPTOR
POKING OUT INTO THE EXTRA
CELLULAR SPACE FROM JUST THE
ALPHA SUBUNITS, THERE ARE
DIFFERENT CONFIRMATION SAYS AND
THE REST OF THE STATE, THIS HAS
NOT BEEN SEEN BEFORE AND OTHER
NICK O 10IC RECEPTORS OR OTHER
RECEPTORS, SOMETHING FUNNY
PECULIAR TO THE MUSCLE RECEPTOR.
SO THIS SUBUNIT HERE BECOMES
VERY DETEACHED AND DENSITY FOR
THE END OF THIS HELIX
DISAPPEARS, CAN'T MODEL IS,
RIGHT?
OKAY, SO THEN WE CAN EUROPE
IMPOSE RESTING WHICH WE HAVE
OVER HERE IN GRAY, AND OVER HERE
IN GRAY AND THEN DID,-TUBO IN
THIS CASE WILL BE IN GREEN.
WE CAN SEE BINDS IN 1 OF THESE
SITES WHERE THE M-4 HELIX
BECOMES DETACHED AND AGAIN WE'RE
STILL WONDERING IS THIS
MEANINGFUL OR NOT.
SO I CAN SUMMARIZE WHAT I'M
SHOWING YOU HERE.
IN THE RESTING STATE, THESE ARE
THE 2 M4 HELIXES AND THEY'RE
ATTACHED TO THE RECEPTOR, AND
DESENSITIZED STATE 1 OF THEM
OPENS UP A BIT AND THIS
RECALLING 1 OPENS UP A LOT;
OKAY, SO WHY CAN'T D-TUBO BIND
OVER HERE?
WE THINK BECAUSE THEY'RE NOT
RECEPTORS, THE WHO UNITS WHILE
THEY'RE ADENTICAL IN SEQUENCE,
THEY'RE NOT IDENTICAL IN THEIR
NEIGHBORING SUBUNITS IN THE
STRUCTURE SO WE'RE GUESSING THAT
THE FACT THIS 1'S NEXT TO A
DELTA SUBAUTOPSY SERIES CAN
RESTRAIN MOTION, D-TUBE O IS
BIG, THAT'S A GUESS.
SO SO WE WILL LOOK AT IN DETAIL
WHAT'S HAPPENING TO AMINO ACIDS
IN IN CONFIRMATIONAL CHANGE FROM
RESTING TO DESENSITIZED STATE.
SO THE FEENLE AN LEANS, THIS
NEST OF THEM, 3 OF THEM THAT
PACK TIGHTLY DIGNITARY THEY
CHANGE IN CONFIRMATION AND
DURING DESENSITIZATION M4 RIGHT
HERE ADOPTS A ROTE MERCHANGE AND
FOR IT TO RECOVER, THIS IS OUR
IDEA, FOR IT TO RECOVER FROM
DENSE SENSITIZATION TO GO BACK
TO THE RESTING STATE THIS CHANGE
WOULD HAVE TO GOOD LUCK BACK TO
THE RESTING SECTION AND MAYBE
THAT WOULD BREAK ON
DESENSITIZATION, MAYBE, MAYBE,
DON'T KNOW HOW CAN WE TEST THIS,
LET'S MAKE MUTANTS IN THE SIDE
CHAINS SO WE DO THAT, ME MAKE
SINGLE, DOUBLE, TRIPLE MUTANTS
WE DO THE VOLTAGE CLAMP, AND WE
SEE SO ON THE FAR RIGHT SIDE
HERE, THE SOLID CIRCLES, ARE
WILD-TYPE, AND THEN AS WE MAKE
INCREASING NUMBER OF FEIGNLE
ALA19, WE SEE THE EC 50 TO THE
LEFT SO WHEN YOU GET RID OF
THESE BULKY RESIDUES HERE AND
YOU REPLACE THEM WITH
ALANINEs, IT'S EASIER TO
AGGRAVATE THE CHANNEL.
WHY?
CAN WE GO DEEPER?
WE WANT TO LOOK AT, SIMPLE
KINETICS AND THE REAL
ELECTROPHYSIOLOGIST HERE, TRY
NOT TO JUDGE ME--[LAUGHTER]
--I HOPE THIS DOESN'T IRRITATE
YOU TOO MUCH BUT WE WILL SEE, WE
WANTED FASTER EXCHANGE, SO WE
SWITCHED AND TO DO THAT WE HAD
TO SWITCH IN TORPEDO WHICH
DOESN'T EXPRESS AT WARM
TEMPERATURES, TO THE MOUSE
MUSCLE NICK O 10IC RECEPTOR SO
WE MAY SUBSIDIARY TUITIONS THERE
AND LOOKED AT OUR
CERTAINLY--CERTAINLY LECT ROUGH
ATOM PHYSIOLOGIST AND WE'RE
LOOKING AT WILD-TYPE, LONG
APPLICATIONS OF AGONIST AND THEN
SECOND PULSES OF AGONIST TO LOOK
AT RECOVERY FROM
DESENSITIZATION, WE DO THAT WITH
GAIN OF FUNCTION DOUBLE MUTANTS,
SO WE CAN CHANGE THE DURATION OF
RECOVERY FROM--SO WE ACTIVATE
AND WE APPLY FOR AGONIST LONG
ENOUGH TO GET STEADY STATE
CURRENT AND MOST ARE
DESENSITIZED AT THIS POINT SO AS
WE INCREASE THE AMOUNT OF TIME
BEFORE THE SECOND PULSE, WE CAN
GET A ROUGH ESTIMATE OF AN
APPARENT RECOVERY RATE FROM
DESENSITIZATION, WE CAN FIT A
SUM OF 2 EXPONENTIALS FROM THE
RECOVERY RATE.
3.8 SECONDS.
HOW DOES THAT COMPARE TO THE
MUTANT.
SO WE DO THE SAME MUTANT THAT
HAD THE GAIN OF FUNCTION AND WE
SEE THAT MAYBE YOU CAN ALREADY
TELL, THIS THING IS RECOVERY
MUCH FASTER FROM BEING
DESENSITIZED.
SO 5 FOLD FASTER, SOMETHING LIKE
THAT.
SO WE CAN CONNECT THIS GAIN OF
FUNCTION, THESE FEIGN
ALEXANDERRA 9 SIDE CHAINS WE
PRUNED BACK TO ALA19S AND
APPARENT RECOVERY FROM
DESENSITIZATION, SO THAT'S THE
IDEA THAT WHEN THIS M4 HELIX
BECOMES DETACHED THAT'S EITHER
PART OF ACTIVATION OR ACTIVATION
OF DESENSITIZATION, WE DON'T
KNOW WE DON'T HAVE AN ACTIVATED
STATE BUT FOR IT TO RECOVER FROM
BEING DESENSITIZED THAT HELIX
HAS TO COME BACK INTO POSITION
AND THAT IT BE LIMITED BY THE
ROTOMERRIC SHIFTS AND THESE
FENALANINES.
WE HAVE NO IDEA HOW IT REGULATES
BECAUSE IT'S A LOW AFFINITY SITE
COMPARED TO EXTRA CELLULAR
DOMAININNA SITES WHERE IT'S
REALLY DOING ITSAN TAGANIST JOB.
SO HOW DO YOU MEASURE THE
ACTIVITY OF A SECONDAN TAGANIST
SITE, DON'T KNOW SO WE STARTED
LOOKING AT OTHER LIGANDS AND 1
OF THEM WAS ATOM OIDATE WHICH IS
AN INTRAVENOUS ANESTHETIC WITH
THE GABBA A RECEPTOR AND THEY
BIND AT SUBUNIT INTERFACES WHERE
THEY ARE POSITIVE MODULATORS.
ETOMIDATE BINDS TO THE M4, SO
IT'S WHY YOU GET MUSCLE
RELAXATION DURAN CANNING GENERAL
ANESTHESIA.
SO ETOMIDATE BINDS IN THE SAME
SIGHT THAT IT BINDS IN AND IT
ONLY BINDS THERE, AND DOESN'T
BIND IN OTHER PLACES AND WE HAVE
STRUCTURES NOW WITH BOTH
CARBOCOLAND CHOLINE BOUND HERE
AND THESE 2 WORK TOGETHER TO
STABILIZE DESENSITIZED STATES OF
THE CHANNEL, SO WE CAN LOOK AT
THE ETOMIDATE SITE HERE AND WHEN
WE LOOK AT AFTIO CHOLINE AND
INCREASING CONCENTRATIONS OF
D-TUBO WE SEE WHAT LOOKS LIKE A
RATE IN THE FAST
DESENSITIZATION, SO ETOMIDATE IS
SITTING IN THERE THROUGH
ENHANCING DESENSITIZATION.
SO THIS WAS A RELIEF BECAUSE WE
WEREN'T SURE WHAT TO MAKE OUT OF
THIS D-TUBO SITE, WAS IT AN
ARTIFACT OF USING TOO MUCH
D-TUBO, WE THINK IT WAS CLUEING
US INTO SOMETHING
MECHANISTICALLY AND NOW WE HAVE
A DRUG THAT'S USED ALL THE TIME
IN THE CLINIC THAT BINDS THERE
AND ONLY THERE.
OKAY, SO TO SUMMARIZE I'VE SHOWN
YOU THE RESTING STATE OF THE
REPRESENTOR WHERE THE PORE IS
CLOSED, VERTICALLY, AND THE
DESENSITIZED STATE, THE TOP,
IT'S--THE PORE IS FUNNEL SHAPED
SO THE TOP OF IT OPENS UP, BUT
THE BOTTOM STAYS CLOSED, 1 OF
THE M4S POPS OPEN, WHEN D-TUBO'S
BOUND IT FITS IN THE SITE IN
ADDITION TO THESE OTHER SITES
AND THEN ETOMIDATE TAKES THE
CONFIRMATION IN THE DESENSITIZED
STATE AND SIT THERE IS TO
PREVENT SLOW RECOVER FREE
RADICALS DESENSITIZATION.
OKAY, SO I'VE SHOWN YOU THE
ARCHITECTURE OF APO RECEPTOR AND
LIPID INTERACTIONS
CONFIRMATIONAL CHANGES
UNDERLYING THE STATE TRANSITIONS
BUT NOT ALL, DISTINCT MECHANISMS
UNDERLYING RESUPPORT
ORANTAGONISM, AND D-TUBO
DESTABILIZED THE END STATE.
THE SAME RESULT IS THE SAME.
YOU GET MUSCLE PARALYSIS, THE M4
HELIX DETACHES DURING
DESENSITIZATION AND SOME LIGANDS
CAN TAKE ADVANTAGE OF THIS TO
ALOESTERICALLY INHIBIT THE
RESOPTOR.
THERE'S STILL A LOT TO DO WE
DON'T HAVE AN ACTIVATED STATE
AND WIRE TRYING TO GET THIS IS
WE HAVE CAGED AGONIST THAT
MICHAEL STOLE HAS SYNTHITIZED
AND WE HAVE A LIGHT PIPE COMING
INTO THE SAMPLE LOADING WIND
UPON OS IN THE [INDISCERNIBLE].
SO AS THE SAMPLE DROPS THROUGH
THAT AND UNCAGES THE AGONIST AND
MAYBE WE WILL GET AN ACTIVATED
STATE.
SO FAR WHAT WE'VE GOTTEN IS THE
CAGED AGONIST BOUND WHICH IS NOT
INFORMED TO ANYTHING BUT WE'RE
MAKING PROGRESS SLOWLY.
STRUCTURAL PHARMACOLOGY OF
MODERN NEUROMUSCULAR JUNCTION
BLOCKERS, SO IF YOU NEED TO HAVE
GENERAL ANESTHESIA, THEY ARE
GOING TO GIVE YOU A COMBINATION
OF A GENERAL ANESTHETIC WHETHER
IT'S PROFOFOL OR KETAMINE THAT
ACTS ON NMDA RECEPTORS AND A
NEUROMUSCULAR BLOCKER, SOMETHING
LIKE THOSE TO PARALYZE SKELETAL
MUSCLE OPEN SOURCE SOFTWAREY
THEY CAN INTUBATE YOU AND USE A
LOWER DOSE OF GENERALLANCE
ESTIMATE THADTHETIC THESE 2
THINGS ARE USED TOGETHER.
SO NEUROMUSCULAR BLOCKERS AND
GENERAL ANESTHETICS AND IT'S
INTERESTING THEY CAN ACT SINNER
GESTICLY OR TOGETHER AT THE SAME
TIME ON THE SAME CHANNEL.
AND THEN THE BIG NEW AREA IN THE
LAB IS LOOKING AT NATIVE
RECEPTOR STRUCTURES WHETHER IT'S
BY TOMOGRAPHY OR PURIFYING
RECEPTORS AND COMPLEXES FROM
TISSUE.
LRLT, SO THANK YOU VERY MUCH
THIS IS MY GROUP.
MICHAEL HAS BEEN GREAT IN
CHEMISTRY, STEVE PROVIDING GENES
AND DISCUSSIONS, ARTHUR
PREGNANTED OUT HOW TO PURIFY
THIS THING AND FUNDING AND BE
HAPPY TO TAKE QUESTIONS.
[ APPLAUSE ]
>>YES BUT YOU'RE SUPPOSED TO
USE THE MICROPHONE.
>>SO LOVELY STUFF, RYAN, I
GUESS, I HAVE A LOT OF QUESTIONS
ABOUT YOU THE 1 THAT SORT OF
KEEPS COMING BACK IS THE IDEA OF
THE POLYPHARMACOLOGY, THE WHYED
THAT THE SAME DRUG ACTS AS AN
INHIBITOR IN 1 SITE IN THE GATE
KEEPER BA RECEPTORS AND
ACTIVATOR IN THESE.
AND I MEAN, IS THERE AN
EVOLUTIONARY BASIS OF THIS OR
DOES IT FIT IN SPACES THAT ARE
IN EVERY PROTEIN?
I MEAN, HOW DOES THAT COME ABOUT
AND WHERE DO THESE MOLECULES
COME FROM?
>>SURE.
I DON'T KNOW.
[LAUGHTER]
GABBA RECEPTORS AND NICOTINIC
RECEPTORS HAVEN'T BEEN
MAINTAINED FOR THOSE SITES THAT
WE SINGICIZED BUT THOSE SITE
WHEN IS THEY'RE NOT ACPYING THEM
ARE CERTAINLY OCCUPIED BY LIPIDS
AND NEUROSTEROIDS ARE THINGS FOR
EXAMPLE, GABBA A RECEPTORS THAT
POTENT, IMPORTANT, ENDOGENOUS
MODULATORS AND OTHER
CHOLESTEROL, PROBABLY DOING THE
SAME THING IN BOTH THE GABBAA
RECEPTOR, NICOTINIC RECEPTOR AND
ANYONE'S FAVORITE CHANNEL SO
THESE, YOU KNOW MAYBE THE ANSWER
IS PARTLY THAT THESE COMPOUNDS
ARE--WELL THEY'RE CERTAINLY
REPLACING LIPIDS, WHAT LIPIDS
ARE DOING, YOU KNOW MORE WITH
THE DIFFERENT KIND OF ACTIVITY,
BUT I'M NOT ANSWERING YOUR
QUESTION.
>>TO SOME EXTENT YOU ARE
BECAUSE IT MAKES YOU THINK ABOUT
IT, AND THE AGONIST BUT ARE
THERE--IF YOU LOOK AT THE
NONSYSTEM RECEPTORS, DO THE
DRUGS AFFECT THEM TOO OR JUST
THE RECEPTORS?
>>YEAH, SOME OF THEM DO.
SO, THERE ARE POTASSIUM CHANNELS
THAT ARE AFFECTED BY THESE
DRUGS, NMDA RECEPTORS ARE A
MAJOR TARGET OF SOME OF THESE
DRUGS.
AND THEN IT'S A QUESTION OF IS
THE CONCENTRATION ARRANGED
THAT'S HITTING THE KINASEIS ARE
ACTIVATED OR MODULATES BY SOME
OF THE GENERAL ANESTHETICS
ESPECIALLY THE VOLATILE
ANESTHETICS ARE LIKE HOW THOSE
WORK, THEY DO ALL KINDS OF
THINGS TO ALL KINDS OF PROTEINS.
SO IT'S NOT A--OH IT'S
INHIBITING THIS CHANNEL AND
THAT'S DOING WHAT IT DOES IS NOT
THE WAY TO LOOK AT IT.
YEAH.
>>THANKS.
THANK YOU.
>>BEAUTIFUL TALK.
TWO QUESTIONS.
SO THE D-TUBO, THE NEW SITE, YOU
DIDN'T SHOW THE D-TUBO MODEL IN
THERE SO I'M GUESSING YOU DON'T
HAVE THE MODEL DENSITY TO MODEL
IT CANNED WHAT?
IS THAT RIGHT?
>>IT'S ACTUALLY QUITE GOOD, THE
DENSITY IS QUITE GOOD.
WE DID MODEL IS IT.
IT'S BETTER THAT THE LOWER
AFFINITY EXTRA CELLULAR DOMAIN
SITE.
>>SO IS IT DEMONSTRATING A
HYDROPHOBIC INTO THE BILAYER AND
THAT'S--
>>YES, YES, YES.
>>DOING IT?
>>YES.
>>SO THE SECOND QUESTION WAS
THE TOXIN STRUCTURE, I COULD BT
HAPPEN THINKING, LOOKINGA THE
STRUCTURE AND HOW EXTRACTING
SENTIA WILY THE TOXIN IS
ESSENTIALLY WHAT IT'S ENGAGING
TO THE RECEPTOR, CAN YOU USE
THAT TO NOW, FOR A MINIMAL
VERSION OF THE TOXIN THAT THE IT
WILL ESSENTIALLY TRIM IT DOWN TO
WHAT YOU NEED?
HAVE YOU EVER THOUGHT ABOUT
THAT?
>>PEOPLE HAVE DONE THAT.
YEAH.
THERE ARE--YOU CAN TAKE THAT--I
DON'T KNOW, I FORGET HOW MANY
RESIDUES BUT IT'S NOT VERY MUCH,
SOMETHING LIKE, I DON'T KNOW 10
OR 12 RESIDUES OF THE TIP OF THE
LOOP AND TELL BIND AND INHIBIT.
YEAH.
>>THANK YOU.
>>YEAH, YEAH.
>>RYAN 1 OF THE THINGS THAT'S
PUZZLED ME ABOUT YOUR ALPHA 7
STRUCTURE AND I DIDN'T REALIZE
THAT THIS IS THE SAME THERE IN
THE NICKEE TEENIC RECEPTORS AS
WELL, HAVE YOU THE GLUTAMATE,
DID YOU SEE EVIDENCE OF CAN THE
WHAT ON THAT?
>>YEAH, RIGHT.
SO IN THE MUSCLE NICOTINIC, SO
THE VERY BOTTOM OF THE ION
CHANNEL CONTAINS DEPENDING ON
THE RECEPTOR TYPE MOSTLY OR
EXCLIEWKSIVELY GLUTAMATE SIDE
CHAINS SO THEY FORM A RING AT
THE BOTTOM, AND THEY ARE THE
MAJOR DETERMINE NABT OF CAT ION
SELECTIVITY.
IF YOU NEUTRALIZE THOSE OR
SELECT THE CHARGE, CAN YOU
SELECT THE CHANNEL TO AN ION
SELECTIVITY IN SOME CASES IT'S
THAT SIMPLE SOPHISTICATEDY
WHAT'S HAPPENING TO THOSE SIDE
CHAINS IN THE DESENSITIZED STATE
OR THE RESTING STATE OR
WHATEVER.
AND WHAT WE SEE IN THE TORPEDO
RECEPTOR IS THAT, THEY ACTUALLY
ROTATE AWAY FROM THE CENTRAL
AXIS AND THESE NONCONDUCTING
STATES AND THEY CAP THE END TERM
NIGH OF THE HELIXES OF THE
ADJACENT SUBUNITS.
SO THESE BUILD UP IN-CHARGESOT
THEY'RE POSITIVE PARTIAL CHARGES
AND PARTIAL NEGATIVE CHARGES AND
THESE HELIXES LIKE TO BE
STABILIZED BY CAPPING
INTERACTIONS AND LOGICAL THAT
NEGATIVELY CHARGED SED CHAIN
WOULD CAP THE END TERMINUS,
THAT'S WHAT WE SEE.
VERY STABLE, VERY CLEAR DENSITY.
SO IN ALPHA 7, THE DENSITY IN
THE EARLIER STRUCTURES WE
PUBLISHED, THE DENSITY FOR THOSE
SED CHAINS YOU COULDN'T SEE THEM
REALLY MAYBE YOU COULD SEE
DENSITY FOR 1, AND IN THAT CASE,
EITHER THAT WAS JUST LOCALLY BAD
OR POOR RESOLUTION, OR WHAT
THEY'RE DOING IS THIS, AND
THEY'RE ADOPTING MULTIPLE
CONFIRMATIONS, AND IT DOESN'T
MAKE SENSE TO ME THAT THEY WOULD
BE FORMING A DESENSITIZATION
GATED UNLESS, WHAT YOU'RE DOING
IS KEYALATING SOME SOMETHING
LIKE CALCIUM, RIGHT?
DOWN IN THE RING AND WE DO SEE
IN ALPHA 3 FOR 4, AND WHEN WE
ADD DDTA, WE CAN DEPLETE 1 OF
THOSE SITES AND SO AS WE KNOW,
CALCIUM DOES ALL KINDS OF
THINGS, THERE'S USUALLY A
POSITIVE MODLATTORY EFFECT AND
THE OTHER SUBTYPES CALCIUM, CAN
INCREASE THE--INCREASE IN
CALCIUM CONCENTRATION CAN
INCREASE THE DESENSITIZATION
RATE AS WELL.
SO IT COULD BE DOING THAT BY
STABILIZING THESE GLUTAMATES
ORIENTED INTO THE CENTRALAXIS,
THERE'S PIECES THAEVER ARE
COMING TOGETHER, IT'S A LOGICAL
IDEA.
>>O A RELATED FOLLOW UP AND
IT'S GOOD TO BE THE CONTROVERSY
OF THE POLAR GATE AND WHETHER OR
NOT IT'S LEAKING IS STILL VERY
MUCH RAGING THAT'S EXCITING I
WAS WONDERING WHY, IF YOU THINK
YOU COULD GIVE MORE INFORMATION
ABOUT THE PROXIMITY IN THE
BOTTOM PART WHICH DOESN'T SEEM
TO BE CHANGING IN ANY OF YOUR
MODELS.
DOES 1 GET INFORMATION ON
WHATEVER PERMEABILITY THERE IS
BY LOOKING AT THESE DIFFERENT
REAGENTS [INDISCERNIBLE]?
>>YEAH, SORT OF A HOW CAN WE
HAVE A RULER FOR WHAT THOSE ARE
USING SOME ORTHOGONAL APPROACH
THAT'S NOT STRUCTURAL BIOLOGY.
I DON'T KNOW.
IF YOU DON'T HAVE A CONDUCTING
CHANNEL, YOU CAN'T MEASURE THE
SIZE OF WHAT'S GOING THROUGH
BECAUSE NOTHING'S GOING THROUGH,
I DON'T KNOW, I MEAN.
>>IT'S NOT NOTHING, THAT'S
RIGHT, IT'S GOOD TOO BUT THE
STUDIES WITH THESE PERMEATION
SERIES S&POOSE NOT LIKE ONCE YOU
GOET TO 7 AND HALF ANGSTROMS IT
GOES TO FULLY CONDUCTING 0, IT
TAPERS OFF AND THEY HAVE SOME
WAY OF SAYING OKAY, THIS IS THEN
THE DIAMETER THAT I DIDN'T
UNDERSTAND, YEAH ALL WE'VE BEEN
DOING IS LOOKING AT SIMULATIONS
GROUPS WITH ERIC MENDALL'S
GROUPS AND CAN WE CORRELATE IT?
IT MAKES SENSE BUT STILL MESSY.
>>JUST A FOLLOW UP ON THAT
ISSUES THE POTASSIUM CHANNELS
WHERE THERE'S MULTILE FORMS OF
ACTIVATION, THERE IS' 1 OF THE
CORE, DILATES THE FILTER AND
DIMINISHES POTASSIUM BUT
INCREASES TOADIUM SO THERE ARE
PARALLELS, SO FURTHER IN THAT
THE OTHER QUESTION I HAVE IF YOU
GOES IT KIND OFIOURE
INTERPRETATIONS ABOUT IT SEEPS
LIKE ALL YOUR LIGANDS HAVE SOME
PROBABILITY OF COURSE AND YOU'RE
NO LONGER, YOU KNOW DEALING WITH
THE MEMBRANE BUT I WAS CURIOUS
ABOUT THAT?
>>YEAH, DIFFERENT PEOPLE IN THE
FIELD HAVE INTERPRETED THIS IN
DIFFERENT WAYS.
HIEWBENER READ, AND THEY HAD AN
AGONIST BOUND STATE THAT BASED
ON ELECTROPHYSIOLOGY WE WOULD
SAY THEY WOULD ALSO SAY THIS
SHOULD BE DESENSITIZED BUT THEY
DO MD AND THEY SEE IT'S LEAKY
AND THEY SAY MAYBE IT'S A
PREOPEN STATE.
THE PREOPEN STATE SHOULD NOT
REALLY BE POPULATED IN A REAL
MEMBRANE, BUT WE HAVE RIPPED
THIS THRING OUT OF THE MEMBRANE
WITH DETERGENT AND THEN
RECONSITUTE IT INTO NANO DISKS
AND EVERY SCAFFOLD YOU USE WILL
HAVE SOME EFFECT ON
CONFIRMATIONALLY EQUILIBRIUM 2.
>>SO--
>>WHAT?
>>[INDISCERNIBLE] REOPEN STATE
DOES EXIST NORMALLY?
>>THE REOPEN STATE EXISTS WHAT?
>>DOES EXIST.
YEAH.
I THINK THE PREOPEN STATE EXISTS
BUT WHETHER YOU WOULD EXPECT IT
TO BE ABUNDANT YOU SHOULD
SATURATING AGONIST
CONCENTRATION, I DON'T KNOW,
YEAH; YEAH.
>>THAT WAS REALLY BEAUTIFUL, I
AM INTERESTED IN THE PARTICLES
THAT YOU THROW AWAY, BECAUSE THE
HELIXES ON THE INSIDE ARE NOT
VISIBLE, ARE THEY VISIBLE, IS IT
POSSIBLE IF YOU ACTUALLY
CLASSIFY THEM IN LOOK INTO THE
THEM IN MORE DETAILS THAT THE
HELIXES ARE RUNNING PARALLEL TO
THE SURFACE OF THE NANO DISK AND
ON THE MEMBRANE LIKE LITERALLY
CHANGING CONFIRMATION RATHER
THAN DISAPPEARING?
HAVE YOU CONSIDERED THAT.
>>OH, I EXPECT THAT IN REALITY
THEY ARE THAT THEY DO SOMETHING
LIKE THAT BUT IN A WAY THAT
VARIES FROM PARTICLE TO
PARTICLE.
SO THEY AVERAGE OUT.
WE HAVE TRIED TO INCLUDING THOSE
PARTICLES IN OVERALL REFINEMENT
AND REFINING THAT SUBSET AND WE
GET THE EXACT SAME CONFIRMATION
THAT'S PRESENT WHEN THE
INTRACELLULAR DOMAIN IS THERE,
JUST MISSING THE INTRACELLULAR
DOMAIN; YEAH.
>>YEAH.
>>ALL RIGHT, THANK YOU ALL SO
MUCH.