>> HELLO, EVERYBODY.
WELCOME TO THE STETTEN LECTURE.
THIS LECTURE IS BEING RECORDED.
MY NAME IS JOHN LO LORSCH.
THIS IS PART OF THE DIRECTOR'S
WEDNESDAY AFTERNOON LECTURE
SERIES.
HE SPENT MOST OF HIS CAREER AT
NIH, SERVING IN TWO INSTITUTES
AS WELL AS THE OFFICE OF THE NIH
DIRECTOR.
AND HE WAS PARTICULARLY
WELL-KNOWN AS THE CO-AUTHOR OF
THE EARLY EDITIONS OF THE
TEXTBOOK, PRINCIPLES OF
BIOCHEMISTRY.
IN RECOGNITION OF HIS MANY
CONTRIBUTIONS TO NIH, BOTH
WITHIN AND OUTSIDE OF THE NIH,
HE HAS THE RARE DISTINCTION OF
HAVING TWO NIH ENTITIES NAMED
AFTER HIM: THIS LECTURE AND THE
DEWITT STETTEN JR. MUSEUM OF
MEDICAL RESEARCH, WHICH YOU
PROBABLY ACTUALLY SAW WHEN YOU
WALKED IN, BECAUSE IT'S
DISTRIBUTED AROUND THE CAMPUS,
MOST OF IT, I THINK ACTUALLY IN
THIS BUILDING.
SO THE EXHIBIT OUT THERE AS WELL
AS THE ONE AROUND THE CORNER OF
MARSHAL NUREMBERG AND THE
DISCOME RETHEDISCOVERY OF THE G.
THE WAY THE LECTURE WORKS IS THE
SPEAKER WILL GIVE A 50 MINUTE
TALK ON HER RESEARCH FOLLOWED BY
A 10 MINUTE QUESTION AND ANSWER
SESSION.
YOU CAN ASK YOUR QUESTIONS BY
USING THE MICS IN THE ROOM.
IF YOU'RE IN THE ROOM.
OR BY SELECTING THE "SEND LIVE
FEEDBACK" BUTTON ON YOUR
VIDEOCAST SCREEN IF YOU'RE
ONLINE.
IF YOU ARE SEEKING A CONTINUING
MEDICAL EDUCATION CREDIT OR CME,
THE CODE IS 57663.
WE INVITE THOSE OF YOU HERE IN
PERSON TO JOIN US OUTSIDE OF THE
AMPHITHEATER FOR A BRIEF
RECEPTION AFTER THE LECTURE,
JUST RIGHT OUT THERE.
SO TODAY'S SPEAKER, SUDHA
CHAKRAPANI, JOINS A VERY
DISTINGUISHED LIST OF STETTEN
LECTURERS, PROFESSOR AND CHAIR
IN THE DEPARTMENT OF
PHARMACOLOGY AT CASE WESTERN
RESERVE UNIVERSITY AND SERVES AS
DIRECTOR OF THE CLEVELAND CENTER
FOR MEMBRANE AND STRUCTURAL
BIOLOGY.
SUDHA HAS DEDICATED HER CAREER
TO INVESTIGATING ION CHANNELS
THAT MEDIATE FAST SYNAPTIC
TRANSMISSION AT THE NEURONAL AND
NEUROMUSCULAR JUNCTION.
SHE LEADS A LABORATORY THAT
UTILIZES CRYOELECTRON MICROSCOPY
TO INVESTIGATE ION TRANSPORT IN
HEALTHY AND DISEASED CONDITIONS
AND HOW MEDICINES CAN TARGET TO
TREAT SPECIFIC DISEASES.
DURING TODAY'S LECTURE, SUDHA
WILL DISCUSS THE CONFORMATIONAL
CHANGES THEY GO THROUGH BASED ON
DIFFERENT FACTORS, THEIR
REGULATION AND THE POTENTIAL TO
DEVELOP SUBTYPE-SPECIFIC
THERAPIES FOR VARIOUS DISEASE
STATES.
SUDHA RECEIVED HER MASTERS IF
BIOMEDICAL ENGINEERING FROM THE
INDIAN INSTITUTE OF TECHNOLOGY
AND A PH.D. IN BIOPHYSICS FROM
THE UNIVERSITY OF BUFFALO IN
NEW YORK.
I'M PLEASED TO NOTE THAT NIGMS
SUPPORTS SUDHA'S WORK THROUGH
OUR MAXIMIZING INVESTIGATORS
RESEARCH AWARD, AND NOW PLEASE
HELP ME WELCOME HER FOR HER TALK
ON MOLECULAR MECHANISMS
UNDERLYING GLYCINERGIC
NEUROTRANSMISSION.
>> ALL RIGHT.
GOOD AFTERNOON, EVERYONE.
AND THANK YOU, JON, FOR THE VERY
KIND INTRODUCTION.
AND FOR THE INVITATION TO
PRESENT THE STETTEN LECTURE HERE
TODAY.
IT'S A TREMENDOUS HONOR FOR ME
TO BE HERE AND TO ALSO PRESENT
SOME WORK THAT WE'VE BEEN DOING
IN THE LAST 10 YEARS OR SO THAT
HAS BEEN CONTINUOUSLY FUNDED BY
NIGMS THROUGH R01 AND R35
AWARDS.
I MUST ALSO MENTION THAT OUR
WORK WHICH HEAVILY RELIES ON
CRYO-EM AS A TECHNIQUE HAS BEEN
SUPPORTED BY THE INFRASTRUCTURE
AT THE NATIONAL CENTERS THAT
FUNDED BY COMMON FUND PROGRAMS
AND NOW THROUGH N IF NIGMS.
SO DELIGHTED TO SHARE THIS WORK
WITH YOU AND THANK YOU ALL FOR
JOINING ME HERE TODAY.
SO WHAT I WANT TO TELL YOU ABOUT
IS SOMEWHAT OF A JOURNEY THAT
I'VE HAD THROUGH STUDYING HOW
CHEMICAL SYNAPSES BEHAVE, AND
WHAT ARE SOME OF THE INTRICATE
UNDERPINNINGS OF RECEPTORS THAT
MEDIATE FAST COMMUNICATION.
SO THESE RECEPTORS, THE ONES WE
STUDY BELONG TO A FAMILY OF ION
CHANNELS.
THEY GOVERN A WIDE RANGE OF
PHYSIOLOGIES, FROM MUSCLE
MOVEMENT TO LEARNING MEMORY,
GUT-BRAIN SIGNALING, PAIN
PERCEPTION AND SO ON.
AMONG THIS CLASS OF CHANNELS IS
GLYCINE RECEPTORS WHICH I WILL
BE TALKING ABOUT TODAY.
AND SINCE I STARTED, MY MASTER'S
DEGREE AND GOING THROUGH MY
GRADUATE EDUCATION, THIS JOURNEY
OF STUDYING THESE RECEPTORS HAS
TAKEN ME THROUGH A WIDE RANGE OF
APPROACHES, STARTED AS A SINGLE
CHANNEL ELECTROPHYSIOLOGIST,
THEN MOVING ON TO STRUCTURAL
BIOLOGY, STUDYING EPR ELECTRON
PARAMAGNETIC RESONANCE
SPECTROSCOPY AND NOW CRYO-EM.
SO IT'S BEEN A FUN RIDE SO FAR.
SO ON TO GLYCINE RECEPTORS.
SO THESE RECEPTORS PLAY A VERY
IMPORTANT ROLE IN REGULATING
SENSORY PATHWAYS AND MOTOR
COORDINATION.
THE SIGNAL THAT COMES FROM THE
PERIPHERAL TERMINALS BROUGHT IN
BY NOCICEPTOR NEURONS OR SENSORY
NEURONS EFFICIENTLY GO TO THE
SOMATOSENSORY CORE TECH, WHERE
IT IS PERCEIVED AS PAIN, AND THE
SIGNAL IS REGULATED AN IN THE
DORSAL HORN OF THE SPINAL CORD
BY THE GLYCINERGIC RECEPTORS
HERE.
SIMILARLY, THE SIGNAL THAT COMES
FROM THE BRAIN THAT GOES TO THE
MOTOR NEURON THAT MEDIATES
MUSCLE CONTRACTION AGAIN IS
REGULATED BY GLYCINE RECEPTORS.
SO LET'S ZOOM IN TO THIS REGION
OF THE SPINAL CORD.
RIGHT HERE.
SO THE SIGNAL BROUGHT IN SO THE
SIGNAL BROUGHT IN BY THE
TERMINALS EVENTUALLY GOES TO THE
DORSAL ROOT PROJECTION ROOT
CAVITY TO THE BRAIN AND THIS
SIGNAL IS MODULATED BY
INHIBITORY INTERNEURONS THAT
RELEASE GLYCINE AND GABA AS
NEUROTRANSMITTERS.
THESE DIFFUSE ACROSS THE
SYNAPTIC CLEFT AND BIND THE
RECEPTORS ON THE MEMBRANE RIGHT
HERE.
AS YOU WILL NOTE, THESE
RECEPTORS ACTUALLY COME IN TWO
FLAVORS.
THEY ARE EITHER ON THE POST
SYNAPTIC MEMBRANE, THEY'RE ALSO
PRESENT IN PRESYNAPTIC AND EXTRA
SYNAPTIC SPACES.
THE GLYCINE RECEPTORS HAVE A
SLIGHTLY DIFFERENT ARCHITECTURE
THAT POPULATE THESE SPACES, THEY
MEDIATE TONIC INHIBITION.
RECEPTORS ON THE POST SYNAPTIC
MEMBRANE APPEAR TO BE MORE
CLUSTERED BY A VERY ECK WHICH IS
SIT MECHANISM I'LL TOUCH ON
LATER AND THESE MAINTAIN THE
POST SYNAPTIC INHIBITION.
SO THE GLYCINE RECEPTORS SH
THERE ARE FOUR GENES THAT CODE
FOR THE ALPHA SUBUNIT AND ONE
GENE THAT CODES FOR THE BETA
SUBUNIT.
THE ALPHA SUBUNITS ARE THE ONES
THAT FORM FUNCTIONAL CHANNELS,
THE ALPHA-1, 2 AND 3 SUBTYPES.
THESE SUBUNITS CAN ASSEMBLE ON
THEIR OWN TO FORM A HOMO PEN TOE
MEERK CHANNELS FOUND IN THE
PRESYNAPTIC AND EXTRA SYNAPTIC
MEMBRANES.
WHEREAS THE BETA SUBUNIT CANNOT
FORM A PEN TOE MER ON ITS OWN SO
IT ASSEMBLES WITH ONE OF THE
ALPHA SUBUNITS.
AND THE BETA SUBUNIT IS PART OF
THE ASSEMBLIES OF GLYCINE
RECEPTORS IN THE POST SYNAPTIC
MEMBRANE.
AND THE REASON FOR THAT IS THAT
THE BETA SUBUNIT HAS A SEQUENCE
THAT BINDS TO A PROTEIN CALLED
DP. EFFRIN, A POST SYNAPTIC
SCAFFOLDING PROTEIN.
THIS IS ESSENTIAL FOR THE POST
SYNAPTIC ARCHITECTURE.
SO LOOKING AT IT AT THE
INDIVIDUAL LEVEL, EACH OF THE
ALPHA SUBUNITS SHARE A VERY
STRONG SEQUENCE SIMILARITY, AND
INCLUDING THE BETA SUBUNIT, THIS
IS ABOUT 85 TO 90% SEQUENCE AND
IN THE BETA, YOU HAVE ABOUT 50%
SEQUENCE INVERITY TO THESE.
SO EACH OF THESE SUBUNITS AS YOU
CAN SEE HAS THREE MODULAR
DOMAINS.
THE EXTRACELLULAR DOMAIN WHICH
HOUSES THE NEUROTRANSMITTER
BINDING POCKET, IT IS A SET OF
BETA STRANDS THAT FOLD TO FORM
THE SCAFFOLD.
THE TRANSMEMBRANE REGION HAS
FOUR HE HELICES, IT'S A SITE FOR
SELECTIVITY AND ITS GATING
MECHANISM.
THE REGION WE DON'T SEE HERE IN
THE SCAFFOLD IS THE
INTRACELLULAR DOMAIN.
WE THINK THAT THIS IS A VERY
INTERESTING REGION, BUT
STRUCTURALLY, REALLY HARD TO
WORK WITH BECAUSE THIS IS HIGHLY
UNSTRUCTURED AND WHAT YOU WILL
SEE IN THE SLIDES THAT I'M GOING
TO SHOW ARE WE HAVEN'T REALLY
BEEN ABLE TO GET A HANDLE ON THE
DOMAIN.
BUT WHAT MAKES THEM VERY
INTERESTING IS THAT THIS
INTRACELLULAR DOMAIN IS THE
REGION THAT BINDS TO GEFFRIN
THAT I MENTIONED, THE POST
SYNAPTIC SCAFFOLDING PROTEIN BUT
ALSO INTERACTS WITH SEVERAL
OTHER PROTEINS THAT MODIFY THESE
RECEPTORS AND MODULATE THEIR
FUNCTION.
SO THERE IS A LOT THAT WE NEED
TO STILL DO TO UNDERSTAND THIS
DOMAIN.
SO ALSO IN THIS -- I WANTED TO
MENTION THAT THE ALPHA SUBUNIT,
ALPHA-1 TYPE, IS EXPRESSED
THROUGHOUT THE GREY MATTER OF
THE SPINAL CORD WHEREAS THE
ALPHA THREE SUBTYPE IS ONLY
PRESENT IN THE DORSAL HORN.
THE ALPHA-1 BRINGS ABOUT THE
MOTOR FUNCTIONS, WHEREAS THE
ALPHA 3 IS INVOLVED IN PAIN
PERCEPTION, SO THEY HAVE
DISTINCT EXPRESSION PATTERNS AND
FUNCTIONAL BEHAVIOR.
SO THE ALPHA 2 IS THE FIRST OF
THE GLYCINE ALPHA TYPES TO BE
EXPRESSED.
THEY ARE EXPRESSED BEFORE THE
SYNAPSES ARE FORMED, AND THEY
PLAY A VERY IMPORTANT ROLE IN
THE INTERNEURON MIGRATION, AND
THESE, IF ALPHA 2 IS KNOCKED
OUT, THE NUMBER OF DORSAL ROOT
PROJECTION NEURON NUMBER IS
PRETTY LOW.
AND SO THEY PLAY AN IMPORTANT
ROLE IN HAVING SYNAPSES DEVELOP
AND ANOTHER INTERESTING FEATURE
IS THAT DURING DEVELOPMENT, THE
CHLORIDE GRADIENT IS ACTUALLY
OPPOSITE, SO WHEN THESE CHANNELS
OPEN, THEY DEPOLARIZE THE
MEMBRANE.
SO THEY DEPOLARIZE THE MEMBRANE,
ACTIVATE CALCIUM CHANNEL AND
EVENTUALLY CAUSES THE ACTIN
MYOSIN ENGAGEMENT LEADING TO
NEURON MIGRATION.
BUT AS THE SYNAPSES DEVELOP, YOU
HAVE THIS EXQUISITE ARCHITECTURE
THAT I WAS TALKING ABOUT, WHICH
IS AN INTERACTION, YOU HAVE
GLYCINE RECEPTORS WHICH IN TURN
INTERACTS WITH OTHERS SO THIS IS
A NETWORK OF PROTEIN THAT
MAINTAINS SYNAPTIC ARCHITECTURE.
ALSO LS WHAT YOU SEE IS ACTIVITY
DEPENDENT SO IF YOU BLOCK THE
FUNCTIONALITY OF GLYCINE
RECEPTORS, YOU SEE THAT THESE
RECEPTORS DIFFUSE AWAY IN THE
SYNAPTIC ARCHITECTURE IS
MODIFIED.
SO THERE ARE A NUMBER OF
MUTATIONS THAT ARE KNOWN IN
GLYCINE RECEPTORS THAT IMPAIR
GLYCINERGIC NEUROTRANSMISSION,
AND MANY OF THESE ARE IN THE
ALPHA ONE SUBUNIT.
IT'S DYSFUNCTION IS ASSOCIATED
WITH A DISEASE CALLED
HYPEREKPLEXIA.
IN THIS SITUATION, THE BABIES
EXPERIENCE AN EXAGGERATED
STARTLE REFLEX AND SEIZURE-LIKE
SYMPTOMS.
AND IF YOU LOOK AT WHERE THESE
MUTATIONS ARE, THEY ARE LOCATED
THROUGHOUT THE PROTEIN, AND MOST
OF THE IMPAIRMENT THAT YOU SEE
COMES FROM A DECREASED GLYCINE
RECEPTOR EXPRESSION AND INCREASE
IN DESENSITIZATION RATE.
YOU SEE A REDUCTION IN GLYCINE
SENSITIVITY AND CHLORIDE
CONDUCTANCE AND ALSO A LOSS OF
ZINC POTENTIATION.
ANOTHER KIND OF NEUROLOGICAL
DISORDER IS THE STIFF PERSON
SYNDROME DISORDER AND ANOTHER
ONE WHICH IS THE PROGRESSIVE
ENCEPHALOMYELITIS, WHICH IS
SEVERE FORM OF THE STIFF PERSON
SYNDROME DISORDER.
IN THIS CASE, THE PERSON MAKES
ANTIBODIES TOWARDS GLYCINE
RECEPTORS, AND AS A RESULT, YOU
HAVE A DROP IN GLYCINE RECEPTOR
DENSITY IN THE POST SYNAPTIC
MEMBRANE.
SO IN PATHOPHYSIOLOGY, THEY ARE
ASSOCIATED WITH ALPHA 3 GLYCINE
RECEPTORS, COME FROM THEIR ROLE
IN INFLAMMATORY PAIN STATES.
SO DURING INFLAMMATION,
CYCLOOXYGENASE, EVENTUALLY
RELEASE OF PROSTAGLANDIN
ACTIVATES PROSTAGLANDIN
RECEPTORS, WHICH ACT VAIK
CYCLIC -- ACTIVATES FOR PROTEIN
KINASE A WHICH PHOSPHORYLATES
ALPHA 3 GLYCINE RECEPTORS.
PHOSPHORYLATION OF THESE
RECEPTORS THEN DECREASES OR
DIMINISHES GLYCINE RECEPTOR
FUNCTION.
AS A RESULT, YOU HAVE
HYPERACTIVATION OF THESE NEURONS
AND TRANSITION TO PAIN STATE.
TO SUPPORT THIS MODEL OF
PHOSPHODEFICIENT MUTANT THAT
DOES NOT CARRY THIS
PHOSPHORYLATED BY PK, YOU CAN
SEE THE IMPACT BY THESE MICE
LOSE SENSITIVITY TO
PROSTAGLANDINS.
SO IN TERMS OF PHARMACOLOGY,
GLYCINE RECEPTORS HAVE A VERY
DIVERSE AND RICH PHARMACOLOGY TO
TAKE A LOOK RIGHT HERE.
SO THERE ARE A NUMBER OF KNOWN
BINDING POCKETS, WHICH ARE
MODULATED BY A RANGE OF
ENDOGENOUS AND EXOGENOUS
LIGANDS, AND MANY OF THESE MAY
HAVE THERAPEUTIC VALUE, SO THERE
ARE LIGANDS THAT WERE PURSUED BY
AM GENERGEN AND PFIZER THAT BINO
THE EXTRACELLULAR DOMAIN THAT
ARE CANNABINOIDS, NEUROSTEROIDS,
ALCOHOLS, IVERMECTIN AND
ANESTHETICS BIND TO THESE
VARIOUS POCKETS AND ALSO
MODULATED BY ENDOGENOUS
REGULATORS LIKE PH AND ZINC.
THE ISSUES THAT WE FACE WITH
USING ANY OF THESE RIGHT NOW IS
THAT THESE COMPOUNDS ARE FAIRLY
NON-SELECTIVE FOR GLYCINE
RECEPTORS, THEY TARGET A WIDE
RANGE OF RECEPTORS, SO THIS IS A
GOOD STARTING POINT.
IT'S A GREAT STARTING POINT TO
UNDERSTAND THE FUNDAMENTAL
MECHANISM OF HOW THESE
MODULATORS AFFECT FUNCTION, BUT
WE REALLY ARE ON THE LOOKOUT FOR
BETTER MODULATORS OF THESE
RECEPTORS.
SO WHAT DO WE KNOW ABOUT THE
DIFFERENT SUBTYPES THAT WE CAN
HARNESS TO TARGET THEM?
SO THE ALPHA -- EVEN THOUGH THEY
HAVE A PRETTY STRONG SEQUENCE
SIMILARITIES, YOU CAN SEE THAT
THEY HAVE DIFFERENCES IN
SENSITIVITY TO GLYCINE AS YOU
CAN SEE ALPHA-1 IS MUCH HIGHER
SENSITIVE TO GLYCINE AND ALSO TO
PARTIAL AGONIST COMPARED TO THAT
OF THE ALPHA 3 SUBUNIT SO YOU
CAN SEE THAT IN THE SHIFT IN THE
DOSE-RESPONSE.
YOU ALSO SEE THE ASSEMBLIES ALSO
HAVE DIFFERENCES IN SENSITIVITY
TO GLYCINE AS YOU CAN SEE IN THE
SHIFT IN THE DOSE-RESPONSE, NOT
ONLY THAT, THEY ALSO DISPLAY
VERY DISTINCT SINGLE CHANNEL
BEHAVIOR.
YOU CAN SEE THE ALPHA-1
POPULATES AT LEAST FIVE
DIFFERENT SUBCONDUCT TANT
STATES, WHEREAS THE ALPHA-1 BETA
RECEPTOR HAS A SHIFT TOWARDS
LOWER SUBCONDUCTANT STATES.
SO THE QUESTIONS THAT THE LAB
HAS BEEN EXPLORING IN THE LAST
FEW YEARS IN AREAS WHERE WE WANT
TO HEAD TOWARDS, FIRST OFF, WE
WANT TO UNDERSTAND THE STRUCTURE
AND DYNAMICS THAT UNDERLIES
FUNDAMENTAL MECHANISM IN THEE
REP TORE FAMTHESERECEPTOR FAMIL.
WE WANT TO REALLY UNDERSTAND
WHAT GOES ON AT THE MOLECULAR
LEVEL.
WE WANT TO UNDERSTAND, WHEN WE
STUDY THESE BY CRYO-EM AT THIS
TIME WE'RE STILL LOOKING AT A
MINIMAL UNIT, PURIFIED
RECEPTORS, BUT THESE RECEPTORS
SELDOM WORK ALONE.
THEY ARE A PART OF SUPER
MOLECULAR COMPLEXS THAT MODULATE
FUNCTION SO WE WANT TO BE ABLE
TO EVENTUALLY ACHIEVE
UNDERSTANDING HOW THESE
RECEPTORS ARE ASSEMBLED IN THEIR
FUNCTIONAL ENTITIES.
AND FINALLY, WE WANT TO BE ABLE
TO LOOK AT THEM IN THEIR NATIVE
CELLULAR ENVIRONMENT IN THIS
CASE IN THE CONTEXT OF A
SYNAPSE, THIS IS A STRETCH GOAL
AT THIS TIME, BUT HOPEFULLY WE
AS A FIELD WILL GET THERE
SOMETIME.
SO WHAT DO WE KNOW ABOUT THE
STRUCTURES OF THESE RECEPTORS?
SO THE PIONEERING WORK IN THE
GLYCINE STRUCTURE FIELD WAS DONE
IN THE LABS OF ERIC GEUL, THIS
WAS A CRYO-EM STRUCTURE GOING
BACK TO 2015 OF ALPHA-1 GLYCINE
RECEPTORS, AND TET, THE GROUP OF
PAUL SCHAEFER FROM AMGEN, THEY
PUBLISHED THIS STRUCTURE OF THE
ALPHA 3 GLYCINE RECEPTORS.
SO TRUNCATING THE INTRACELLULAR
DOMAIN AS YOU CAN IMAGINE, THE
PROTEIN WITH THAT, SO THESE WERE
THE INITIAL EFFORTS AND WITHIN
THESE STRUCTURES THEY WERE ABLE
TO CAPTURE MULTIPLE
CONFORMATIONS.
THERE ARE SOME LINGERING
QUESTIONS THAT WERE EVENTUALLY
ANSWERED WITH STRUCTURED
PUBLISHED BY OUR GROUP, SO THESE
ARE FULL LENGTH RECEPTOR
STRUCTURES, THIS IS IN THE
RECONSTITUTED AND LIPID NANODISK
AND THIS ONE IN SMA POLYMERS.
THESE ARE BOTH FULL LENGTH
STRUCTURES BUT AS YOU CAN SEE WE
HAVEN'T STILL GOTTEN INITIAL
VIEW OF THE INTRACELLULAR
DOMAIN.
AND MORE RECENTLY, LAST COUPLE
OF YEARS, THERE ARE A NUMBER OF
HETEROMERIC GLYCINE RECEPTOR
STRUCTURES AT SOUTHWESTERN AND
THIS IS A STRUCTURE OF RECEPTORS
AND THIS IS FROM OUR LAB.
AT THIS POINT, HOW MANY Bs AND
HOW MANY As CONSTITUTE THESE
RECEPTORS WAS STILL UNCLEAR.
THREE LABS, THREE INDEPENDENT
APPROACHES, THREE EXPRESSION
SYSTEMS, ARRIVING AT THE SAME
CONCLUSION OF A 4 IS TO 1 --
4 ALPHA AND 1 BETA, I THINK THAT
WAS QUITE REMARKABLE.
ANOTHER POINT, THEY USED GFP SO
THEY REMOVED THE INTRACELLULAR
DOMAIN FROM BETA SUBUNIT AND
REPLACED IT WITH A GFB SO THAT
ALLOWED THEM TO ANNOTATE WHICH
ONE IS AN ALPHA AND BETA
SUBUNIT.
IN THIS CASE, THEY USED AN
ANTIBODY WHICH RECOGNIZES THE
ALPHA SUBUNIT AGAIN USED AS A
MARKER FOR ANNOTATING SUBUNITS
AND IN OUR CASE, WE ACTUALLY
USED GEFFRIN AS A CO-COMPLEX AND
WE WERE ABLE TO IMAGE THIS
WITHOUT ADDITIONAL MARKER AND
PROAT TEEN HAD ENOUGH SIGNATURES
WITHIN IT THAT ALLOWED US TO
SOLVE THE STRUCTURE
INDEPENDENTLY.
SO THERE ARE INTERESTING
QUESTIONS THAT ARE UNIQUE TO
EACH OF THESE SUBTYPES, SO I
WANT TO SHOW YOU THREE VIGNETTES
THAT SHOWS YOU HOW ARE THESE
CAPTURED -- I CHOSE TO TALK
ABOUT TWO ENDOGENOUS MOJ LAY
TORES, ZINC AND PH IN THE
CONTEXT OF PAIN.
I WANT TO SHOW YOU CANNABINOID
MODULATION IN THE ALPHA-1
SUBTYPE AND I WANT TO FINISH OFF
WITH MODULATION OF ALPHA-1 BETA
BY EYE VER ME IVERMECTIN, WHICHA
POTENTIATOR OF THIS CHANNEL, TO
SHOW YOU A GLIMPSE OF HOW WE CAN
SORT OF TACKLE SUBTYPE
SPECIFICITY.
SO LET'S SEE.
AT A FUNDAMENTAL LEVEL, THESE
RECEPTORS WILL CYCLE THROUGH
THESE GATING SCHEMES.
SO THERE'S THE RESTING STATE
GETTING ACTIVATED BY AN AGONIST,
THE CHANNEL IS TRANSIENTLY OPEN,
THEN IT DESENSITIZES SO IT GETS
INTO THE DESENSITIZED STATE.
BUT THIS IS A PRETTY BIG
OVERSIMPLIFICATION BECAUSE AS
YOU CAN SEE IT IN THE SINGLE
CHANNEL, THERE ARE A NUMBER OF
STATES THAT THESE CHANNELS
POPULATE BUT AT LEAST THIS IS A
STARTING MODEL FOR US TO GO.
WHAT DO MODULATORS DO?
THEY ESSENTIALLY SWITCH THE
EQUILIBRIUM BETWEEN THESE
STATES.
SO IF YOU'RE USING A
POTENTIATOR, IT PUSHES THE
EQUILIBRIUM TOWARDS THE OPEN
CONFORMATION.
IF YOU'RE USING AN INHIBITOR, IT
PERHAPS SHIFTS IT TOWARDS A
RESTING STATE, AND THIS IS SORT
OF A THEME THAT YOU WILL SEE IN
THE DATA THAT I'M GOING TO
PRESENT.
SO THIS IS WORK THAT WAS DONE BY
A GRADUATE STUDENT, A VERY
TALENTED GRADUATE STUDENT IN THE
LAB, KAYLA, AND A REALLY
TALENTED POSTDOC, ERIC GIBBS, IN
COLLABORATION WITH PHIL B IGG
IN'S GROUP, ALL WERE DONE IN
PHIL'S GROUP.
SO WE WANTED TO STUDY ZINC
MODULATION IN ALPHA 3 GLYCINE
RECEPTORS.
THEY HAVE A NUMBER OF MODULATORY
EFFECT ON SYNAPTIC RECEPTORS.
THEY GO THROUGH ALL OF THE
MODULATIONS AND ALSO THROUGH
THIS DIFFUSION OF ZINC ACROSS TO
OTHER SYNAPSES.
SO GLYCINE RECEPTOR HAS BEEN NOW
KNOWN TO BE MODULATED BY ZINC,
BUT WHAT IS INTERESTING IS THAT
UNLIKE OTHER RECEPTORS WHERE
THEY INHIBIT -- THE EFFECT OF
GLYCINE ON ALPHA 3 IS BIPHASIC
SO AT VERY LOW CONCENTRATION AND
CLOSE TO 1 TO 10 MICROMOLAR UP
TO 1 TO 10 MICRO, YOU HAVE THIS
POTENTIATION, WHEREAS BEYOND
THAT, YOU START TO SEE THIS
INHIBITION.
SO RIGHT OFF YOU KNOW THAT THERE
ARE TWO BINDING SITES, ONE THAT
IS POTENTIATING HIGH AFFINITY,
THE OTHER ONE IS LOW AFFINITY
INHIBITORY SITES.
SO MU MUTAGENESIS HAVE GIVEN UA
GLIMPSE OF WHERE THEE SITES MAY
BE BUT THERE ARE MANY SITE
OUTSIDE OF THESE THAT ALSO
IMPACT ZINC MODULATION.
AND SO WHAT ARE ALLOSTERIC AND
WHAT ARE REALLY BINDING SITES
ARE STILL QUESTIONS TO BE
ASSESSED.
THE OTHER THING FROM A DISEASE
PHYSIOLOGY THAT MUTATIONS THAT
ELIMINATE ZINC POTENTIATION HAVE
HYPERREFLEXIA PHENOTYPES,
SUGGESTING THAT ZINC MODULATION
MIGHT BE PHYSIOLOGICAL MECHANISM
OF HOW THESE RECEPTORS FUNCTION.
SO TO DO THIS, WHAT WE EXPRESSED
ALPHA 3 FULL LENGTH HUMAN ALPHA
3 GLYCINE RECEPTORS, WE PURIFIED
THEM USING AFFINITY
CHROMATOGRAPHY, WE RECONSTITUTE
THEM IN A BELT OF PEPTIDES THAT
WRAP AROUND THE TRANSMEMBRANE
REGION SO THEN WE IMAGE THESE
AND THEN YOU CAN SEE THESE
IMAGES THAT ARE ALIGNED SHOWING
SIDE VIEWS, TOP VIEWS OF THE
PROTEIN, YOU CAN START TO SEE
SOME HELIC ES.
THIS IS WHERE YOU START TO GET
EXCITED BECAUSE YOU SEE THEM AT
THESE POSITIONS, THEN CAN YOU
TAKE THESE PARTICLES AND
CLASSIFY THEM AND YOU WILL SEE
IN THIS DATASET THAT WITHIN THE
SAME DATASET WE START TO SEE
MULTIPLE CONFORMATIONS THAT ARE
THERE, SO OUR STRATEGY WAS TO
IMAGE THIS LOWER THAN THE EC50
FOR GLYCINE STHOO YOU ARE
CAPTURING SOME NON-ACTIVATED AND
ACTIVATED POPULATION.
SO WHAT WE SAW, WHICH IS WHAT
WE'RE SEEING HERE, SO THIS IS
THE CURRENT TRACES FOR GLYCINE
AND HERE WE IMAGE IT UNDER 100
MICROMOLAR GLYCINE
CONCENTRATIONS, AND ONCE YOU SAW
THE PARTICLES, WE SEE THAT THE
PARTICLES BELONG TO TWO
DIFFERENT CORE FORMAT CONFORMAT,
ROUGHLY AT 50% OF OCCUPANCY INTO
THESE TWO STATES.
SO THIS IS THE MAP OF THOSE TWO
CONFORMATIONS, THIS IS THE MODEL
WE BUILT AND WHAT I'M SHOWING
YOU HERE ARE TWO SUBUNITS.
THIS IS SLICED THROUGH THE
CHANNEL TO SEE THE ION
PERMEATION PATHWAY.
SO THIS IS A RESTING
CONFORMATION.
YOU CAN SEE THAT THE CHANNEL
IS -- SO THE GREEN PATCH RIGHT
THERE IS WHERE AN OCCLUSION
OCCURS OF ION PERMEATION.
I CAN SEE THAT IT A CONSTRICTED
RADII PLOTTED ALONG THE LENGTH
OF THE CHANNEL.
AND THIS IS A RING OF LEUCINE
RESIDUES THAT -- SO THIS FORMS
THE ACTIVATION GATE OF THE
CHANNEL.
IN THE DESENSITIZED CORE
CONFORMATION, SO THIS GATE HERE
IS WIDE OPEN WHEREAS THIS GATE
IS CONSTRICTED, IT'S A RING OF
PROTEIN RESIDUES THAT CAUSES
CONSTRICTION IN THE ACTIVATED --
ONCE THE CHANNEL IS ACTIVATED.
SO ZOOMING INTO THE BINDING
POCKET RIGHT HERE, THE INTERFACE
BETWEEN THESE TWO SUBUNITS, WE
CAN SEE IN THE RESTING
CONFORMATION OR THE CLOSED
STATE, YOU SEE NO DENSITY, IT'S
AN EMP EMPTY POCKET, WHEREAS HEE
YOU HAVE A NICE DENSITY AND YOU
CAN ACT OUT ALL THE INTERACTING
PARTNERS.
SO WHAT HAPPENS IS GLYCINE BINDS
HERE AND THIS LOOP REFERRED TO
AS LOOP C CLOSES IN ON TO THE
BINDING POCKET, AND CAUSES A
TWISTING MOVEMENT THAT IS
EVENTUALLY CONVEYED TO THE PORE
OF THE CHANNEL SO THAT'S THE
ACTIVATION MECHANISM, A
MECHANISM THAT IS VERY CONSERVED
AMONGST THIS FAMILY OF CHANNELS
SHOWN BY OUR LAB AND SEVERAL
OTHER LABS.
SO THEN WHAT WE DID IS INCREASED
THE CONCENTRATION OF GLYCINE, SO
WE WENT UP TO SATURATING LEVELS,
AND WHAT WE SEE IS THE
EQUILIBRIUM SHIFT TOWARDS THE
ACTIVATED STATE RIGHT HERE, AND
THE STATE IS VERY SIMILAR TO
THAT OF THE STATE CAPTURED HERE.
SO HERE IS A MOVIE THAT
SUMMARIZES THE MOVEMENTS THAT WE
SEE UPON GLYCINE ACTIVATION.
SO WHAT YOU ARE SEEING HERE IS
THE GLYCINE POCKET, YOU SEE THIS
INWARD MOVEMENT THAT I TALKED
ABOUT OF LOOP C AND THAT'S A
LITTLE GLYCINE DENSITY AT THE
BACK END, AND ONCE GLYCINE
BINDS, SO GLYCINE BINDS IN THIS
POCKET RIGHT HERE, SO THESE ARE
THE INTERACTING RESIDUES.
AND THAT'S THE INWARD MOVEMENT
OVER THE GLYCINE.
AND THIS IS AN IRIS-LIKE
MOVEMENT OF THE CHANNEL PORE, SO
THIS ALLOWS FOR IONS NOT TO PASS
THROUGH.
SO HOW IS THIS BASIC MECHANISM
OF GATING OR GLYCINE ACTIVATION,
AFFECTED BY ZINC AT LOW
CONCENTRATION?
SO HERE IS THE ZINC POTENTIATION
THAT YOU CAN SEE AT LOW
CONCENTRATION, IT TAPERS OUT AND
STARTS TO MOVE INTO THE
INHIBITION MODE RIGHT HERE AT
HIGHER CONCENTRATION.
SO THEN WE IMAGE THIS IN THE
SAME CONDITION WHAT WE ARE
LOOKING AT, SO THIS IS IN THE
ABSENCE OF GLYCINE, AGAIN, YOU
HAVE THESE DISTRIBUTION, AND
THEN WHEN WE ADD ONE MICROMOLAR
OF GLYCINE WHICH IS WHEN YOU'RE
POTENTIATING, WE ARE SIMPLY
ELIMINATING THIS CLOSED
CONFORMATION.
YOU REALLY HAVE ALL OF THE
PARTICLES UP HERE TO BELONG TO
THE DESENSITIZED STATE RIGHT
HERE.
THEN IF WE GO TOWARDS THE HIGHER
CONCENTRATION OF GLYCINE, WE
SHIFT THE EQUILIBRIUM TOWARDS
THE CLOSED CONFORMATION SO YOU
CAN SEE THESE STATES ARE VERY
SIMILAR TO GLYCINE-BOUND STATES
THAT WE SAW BEFORE.
THE ZINC'S ROLE HAS BEEN IN
SHIFTING THIS EQUILIBRIUM.
SO WE ASKED THE QUESTION WHAT IS
DRIVING THIS SHIFT IN
EQUILIBRIUM BETWEEN THESE
POPULATION.
SO TO SUMMARIZE ACTUALLY, SO
HERE WE HAVE THE TWO, WE HAVE
IDENTIFIED TWO ZINC BINDING
SITES.
THE POTENTIATING OR THE HIGH
AFFINITY ZINC SITES THAT ARE
OCCUPIED AT LOW CONCENTRATIONS
OF GLYCINE IS RIGHT AT THE
INTERFACE BETWEEN THE
INTRACELLULAR AND TRANSMEMBRANE
DOMAIN AND YOU WILL SEE IN THE
NEXT SLIDES THAT THIS POSITION
IS OPTIMALLY PLACED TO INTERACT
OR REGULATE THE MOVEMENT OF LOOP
C.
SO THIS ALLOWS FOR LOOP C
MOVEMENT TO HAPPEN THAT
FACILITATES GLYCINE MEDIATED
ACTIVATION.
AND THE SECOND BINDING SITE FOR
GLYCINE, WHICH IS THE LOW
AFFINITY INHIBITORY SITE IS AT
THE BACK END OF THE POCKET.
SO THIS IS THE HIGH AFFINITY
BINDING SITE, WE CAN CLEARLY SEE
AT THIS INTERFACE.
THERE ARE FOUR AMINO ACIDS THAT
COORDINATE THE ZINC TO AS PER
TATE GLUED MATE AND THEN THE WHO
HISTIDINES SO THEY BELONG TO
DIFFERENT BETA STRANDS HERE AND
THE M4 HELIX, SO AT AN OPTIMAL
POSITION TO COUPLE MOVEMENTS IN
THE EXTRACELLULAR AND
TRANSMEMBRANE DOMAINS.
AND THIS IS THE INHIBITORY SITE
THAT WE SEE AT THE BACK END OF
THIS POCKET.
UNLIKE THE HIGH AFFINITY SITE
WHICH HAS A TETRACOORDINATION,
THE LOW AFFINITY SITE IS
COORDINATED BY A HISTIDINE AND A
GLUTE PLATE ANGLUTAMATE AND THEO
LIKELY THROUGH WATER MOLECULES.
SO THIS IS A SITE WHOSE
ACCESSIBILITY IS NOT -- DOES NOT
UNDERLIE LOW AFFINITY, IT'S JUST
THAT THE COORDINATION IS WEAKER
THAN THE HIGH AFFINITY SITES.
SO HERE IS A MOVIE THAT
HIGHLIGHT THE ZINC SITES IN BOTH
THE CLOSED AND THE DESENSITIZED
CONFORMATION.
SO THAT IS A ZINC COORDINATION
FOR THE HIGH AFFINITY SITE.
SO THE TRACES IN ORANGE ARE THE
RESTING CONFORMATION AND THE
BLUE IS THE ACTIVATED STATE.
SO THERE IS A SLIGHT
REARRANGEMENT BETWEEN THE TWO
STATES THAT SEEM TO SUGGEST THAT
ZINC IN THE ACTIVATED STATE IS
BETTER COORDINATED, AND THIS IS
EVENTUALLY TRANSLATED TO LOOP C
MOVING INWARD AND THIS IS THE
BACK END.
SO THIS WAS A TRICKY SITE TO
UNDERSTAND WHY THIS WOULD LEAD
TO AN INHIBITION AT THIS POCKET.
IT DOESN'T SEEM TO BE VERY CLOSE
TO -- IT'S BEHIND THE BINDING
POCKET AND CANNOT DIRECTLY
UNFLEUNS THE BINDING SITE, BUT
DIGGING DEEPER INTO THE
LITERATURE, IT WAS CLEAR THAT
THIS ARGININE AND GLUTE MADE,
THEY MAKE AN INTERACTION VERY
CRUCIAL FOR GLYCINE ACTIVATION
AND MUTATION TO THESE RESIDUES
HAVE BEEN ASSOCIATED WITH
HYPERREPLEXIA.
SO THIS INTERACTION NEEDS TO BE
IN PLACE FOR GLYCINE MEDIATED
ACTIVATION, AND WE THINK THAT
HAVING A ZINC IN THIS
COORDINATION TRIAD IS WEAKENING
THE INTERACTION BETWEEN ARGININE
AND GLUTAMATE, AND THAT MIGHT
MEDIATE INHIBITION.
SO WE COLLABORATED WITH PHIL
BIGGINS GROUP, THEY EVALUATED
THE STABILITY OF THESE
INTERACTIONS BY SIMULATIONS AND
THEY FIND THAT ZINC THROUGHOUT
THE -- IN THE POTENTIATED SITE
STAYS PRETTY CLOSE.
THESE RESIDUES INTERCHANGE IN
TERMS OF THEIR INTERACTIONS, BUT
THE ZINC ITSELF STAYS FIRMLY
BOUND HERE.
THEY ALSO SAW THAT THE GLUTAMATE
WAS AN IMPORTANT INTERACTING
RESIDUE FOR THE INHIBITORY SITE,
AND AGAIN, STABLE INTERACTIONS
WHEN ORDERED FOR THE GLUTAMATE
AND ZINC RIGHT HERE.
SO AGAIN COMES TO OUR PROPOSED
MECHANISM THAT YOU HAVE THIS
INTERFACE INTERACTION THAT
MEDIATES LOOP C MOVEMENT AND
WHICH MIGHT BE STABILIZING
GLYCINE IN THE POCKET AND
INHIBITORY SITE MIGHT BE
IMPACTING GLUED GLEUT MATE
ARGININE INTERACTION.
SO TO FURTHER LOOK INTO OW MUCH
THIS PROPOSAL -- THIS IDEA
HOLDS, SO PHIL BIGGINS GROUP
LOOKED AT THE INTERACTION DURING
THE SIMULATION THAT COWARDS
NAITS THOSE WITH NO ZINC VERSUS
THOSE WITH ZINC.
THE INTERACTION IS VERY STABLE
WHEN YOU ADD ZINC AND -- AND SO
WE THINK THIS COULD BE A
STARTING MECHANISM.
WE ALSO -- STRUCTURES, THESE ARE
STATIC IMAGES, SO THESE
MECHANISMS NEED TO BE VALIDATED.
IN THIS CASE, SO THIS IS LOOKING
AT ZINC MODULATION, SO THIS
IS -- IN BLUE IS THE
POTENTIATION WINDOW AND ORANGE
IS THE INHIBITION.
WE TESTED THE HYPOTHESIS BY
MAKING MUTATION ESSENTIALLY
DELETING THIS END OF M4, WHERE
THERE ARE MULTIPLE RESIDUES THAT
WERE PARTICIPATING IN THIS
COORDINATION AND WE SEE THAT
ZINC POTENTIATION COMPLETELY
GOES AWAY.
SIMILARLY WE MAKE A MUTATION TO
THIS GLUTAMATE, AND WE SEE THAT
THE INHIBITION GETS SHIFTED TO
MUCH HIGHER CONCENTRATION, SO
THIS SORT OF GAVE US SOME IDEA
ON WHAT'S HAPPENING WITH ZINC
MODULATION.
SO ANOTHER MODULATORY FACTOR
THAT IS IMPORTANT IN PAIN
SENSATION IS PH.
DURING NEUROPATHIC PAIN AND
HYPOXIA.
SO YOU HAVE THE EXTRACELLULAR PH
MOVES TOWARDS ACIDIC DIRECTION,
EVEN TO SOME EXTENT TO ONE PH
UNIT, AND OTHER STUDIES HAVE
SHOWN GLYCINE SENSITIVITY IS
RIGHT-SHIFTED.
ALSO ITS ACTIVITY TO PARTIAL
AGONISM IS DECREASED, ALTHOUGH
IT IS VERY INTERESTING TO NOTE
THAT WHAT WE ARE ESSENTIALLY
SEEING IS A REDUCED GLYCINERGIC
RESPONSE BASED ON THE PEAK
AMPLITUDE, AND AT THE SINGLE
CHANNEL LEVEL UNDER ACIDIC
CONDITIONS, YOU HAVE THESE VERY
SHORT LIVED CLOSURES, SO THE
CHANNEL TENDS TO MOVE INTO THE
CLOSED CONFORMATION, MULTIPLE
TYPES OF CLOSED CONFORMATION SO
YOU HAVE ONE COMING FROM REDUCED
AMPLITUDE BUT ALSO THESE FAST
FLICKERS THAT ARE CONTRIBUTING
TO LOWER OPEN PROBABILITY.
SO WE THEN LOOKED AT HOW ALPHA E
CONDITIONS.
SO HERE IS ELECTROPHYSIOLOGY.
BUT LIKE OTHER -- YOU DIMINISH
THE EFFECT OF THIS MODULATOR.
SO WHAT WE NOTICED IS UNDER
ACIDIC PH, THIS IS AT NORMAL
CONDITIONS, PH -- LOWER PH
POPULATED A NUMBER OF STATES
THAT HAD DISTINCT FEATURES THAT
WERE DIFFERENT FROM THAT OF
THESE CONFORMATIONS.
SO THE SHIFT WAS -- THE FIRST
THING WE NOTICED WAS A SHIFT
TOWARDS MORE CLOSED AND MORE
CONDUCTING CONFORMATIONS, AND
ALSO APPEARANCE OF A NEW
INTERMEDIATE STATE.
SO ALL OF THESE STATES SHIFT
TOWARDS A DESENSITIZED STATE.
SO HERE IS A MOVIE THAT SHOWS
YOU WHAT THESE CHANGES ARE SO
THE CLOSE CONFORMATION THAT WE
STABILIZED HAD A DIFFERENT
ORIENTATION FOR LOOP C, IT ALSO
INTERESTING HAS GLYCINE IN THE
POCKET SO YOU HAVE GLYCINE YET
THE CHANNEL IS NOT ACTIVATED.
AND YOU CAN SEE SOME AUTOMATIC
DIFFERENCES, AGAIN, YOU HAVE
GLYCINE IN THE POCKET, THE
CHANNEL YOU WILL SEE -- SO THIS
IS MOVING TOWARDS GLYCINE
ACTIVATED STATE.
AND HERE IS A DESENSITIZED
CONFORMATION, HERE IS THE IPT
IMMEDIATE CONFORMATION, AND THEN
MOVING TO THE CLOSED
CONFORMATION.
SO WE HAVE THE PORE OF THE
CHANNEL IS STABILIZED IN THREE
DISTINCT STATES THAT CONTRIBUTES
TO NON-CONDUCTING STATES AND WE
HYPOTHESIZE THAT THIS
INTERMEDIATE STATE MIGHT BE
CORRESPONDING TO THE
FLICKERING -- FREQUENTLY
FLICKERING STATES THAT WAS
OBSERVED IN ELECTROPHYSIOLOGY.
SO SWITCHING GEARS, I WANT TO
TELL YOU -- SO THESE WERE THE
ENDOGENOUS REGULATORS OF THE
CHANNELS.
WHAT ABOUT SOME EXOGENOUS
MODULATORS, ESPECIALLY LIPID
MODULATORS.
SO THIS WORK WAS DONE BY ARVIN
KUMAR, A POSTDOC IN THE LAB
ALONG WITH KAYLA KINDIG,
AFRODITI AND PHIL BIGGIN'S
GROUP.
ONE THING THAT REALLY POPS OUT
IS THESE CAVITIES THAT I SHOWED
IN THE FIRST SLADE WHICH ARE
SITES FOR BINDING OF VARIOUS
MODULATORS, SO THESE CAVITIES
CHANGE VOLUMES BETWEEN CLOSED
AND DESENSITIZED CONFORMATION
THAT MIGHT EXPLAIN FOR SOME OF
THE STATE-DEPENDENT BINDING OF
THESE LIGANDS.
SO HERE I WANT TO SHOW YOU, SO
AGAIN, THESE ARE THE CALFITIES
WHERE ACAVITIESWHERE A NUMBER OE
MODULATORS BIND, SO I WANT TO
HIGHLIGHT ONE HERE IN THIS CASE
THE GLYCINE RECEPTORS ARE
MODULATED BY ENDOCANNABINOIDS,
THESE ARE POLYUNSATURATED -- POE
10 SHAITS GLYCINE 1 CURRENTS,
AND ALSO THIS SORT OF AN EFFECT
IS MIMICKED BY THC OR DELTA-9
TETRAHYDROCANNABINOL WHICH IS
THE PSYCHOACTIVE INGREDIENT IN
THE CANNABIS PLANT.
SO YOU CAN SEE AGAIN
POTENTIATION, IT HAS PREVIOUSLY
BEEN PROPOSED THAT T HC BINDS
WITHIN THESE TRANSMEMBRANE
HELIXES TO BRING ABOUT THIS
POTENTIATION.
SO AGAIN, SO THERE IS ANOTHER
MODULATOR THAT I'LL TALK ABOUT
IS IVERMECTIN, WHICH BINDS INTO
A SITE THAT IS BETWEEN THE TWO
SUBUNITS.
SO THC HAS THIS VERY NICE AND
STRONG POTENTIATION THAT YOU CAN
SEE AND THIS POTENTIATION IS
FULLY DIVERSIBLE AND YOU CAN
RECAPTURE THE ACTIVITY.
LIKE OTHER ALLOSTERIC MODULATORS
AT SATURATING CONCENTRATION YOU
HAVE THIS POTENTIATION EFFECT GO
AWAY.
SO WE SAW THE NUMBER OF
STRUCTURES IN THE PRESENCE OF
VARYING CONCENTRATIONS OF
GLYCINE, SO UNLIKE IN THE ALPHA
3, WHERE AT SUBSATURATING
CONCENTRATIONS WHERE AT
MICROMOLAR WE CAPTURED -- THE
SENSITIVITY TO GLYCINE OF
ALPHA-1 IS HIGHER SO AS YOU CAN
SEE, AT 100 MICROMOLAR WE SIMPLY
HAVE ONE DESENSITIZED
CONFORMATION.
SO IT WAS A CHALLENGING TASK FOR
US TO CAPTURE AN OPEN LIFE STATE
IN THIS CASE, OR PARTIALLY
ACTIVATED STATE SO IN THIS CASE,
WHEN WE USE THC SO HERE WE ARE
REPEATING THIS ENTIRE TITRATION,
BUT IN THE PRESENCE OF 32
MICROMOLAR THC, WE START TO
POPULATE STATES, THERE ARE SOME
IN BETWEEN THE RESTING AND THE
DESENSITIZED CONFORMATION, AND
IN THIS PURSUIT, WE ACTUALLY
CAPTURED AN OPEN -- THE
TRANSIENT OPEN CONFORMATION.
SO THIS IS A STATE THAT WE HAVE
SEEN BEFORE, IT'S DESENSITIZED,
HERE IS THE DESENSITIZATION GATE
CLOSED IN AND MOLECULAR DYNAMIC
SIMULATIONS WHICH ALLOW YOU TO
LOOK AT PERMEATION EVENTS ACROSS
THE MEMBRANE, SO YOU CAN SEE
THAT HERE THIS WHITE PATCH
SUGGESTED THERE ARE A FEWER
NUMBER OF IONS PASSING THROUGH
THIS DESENSITIZED GATE OF THE
CHANNEL.
SO THIS IS IN THE PRESENCE OF
THC THE CHANNELS OPENING WIDENS
UP AND THEN YOU START TO SEE A
MORE PERMEATING CHANNEL.
SO AGAIN, THE SHIFT IN
EQUILIBRIUM TOWARDS A MORE
CONDUCTING CONFORMATION.
SO WHERE IS THC BINDING HERE?
WE HAVE AN ADDITIONAL DENSITY
THAT WE SEE LIKELY CORRESPONDS
TO THE HTC MOLECULE, THE SAME
SITE THAT WAS PREVIOUSLY
PROPOSED, AND IT WAS PROPOSED
THAT THC INTERACTS WITH THE --
RESIDUE IN THE TM3 OR THE THIRD
HELIX.
THIS HAS BEEN SHOWN BEFORE AND
ALSO WE CAN SEE IT IN OUR AND
THAT WHEN YOU GET RID OF THIS
CHAIN THEN YOU LOWER THC
POTENTIATION.
SO WHEN WE COMPARE THOUGHT THC,
WE NOTICE THAT YOU HAVE THE
BINDING POCKET BUT THE
STRUCTURAL CHANGES ARE ALSO SEEN
ALONG OTHER TM HEE LISTS SUCH
THAT IT WAS SORT OF GOING
TOWARDS THE DESENSITIZED
CONFORMATION.
PARTICULARLY NOTEWORTHY ARE
THESE REORIENTATIONS OF THESE
SITE CHAINS WHICH EVENTUALLY
PHYSICALLY COUPLE TO THE
DESENSITIZED GATE.
SO WE MAKE NOTATIONS TO EACH OF
THESE POSITIONS AND WE NOTICE
THAT THC POTENTIATION GOES AWAY
AND IN SOME CASES PRETTY
DRAMATIC THAT THERE IS NO
POTENTIATION TO THC.
SO THE MECHANISM THAT WE ARE
PROPOSING IS THC BINDING RIGHT
HERE, EE VENGSLY -- AUTOMATIC
RESIDUES TO COMMUNICATE TO THE
DESENSITIZATION GATE WHICH
STABILIZES THE OPEN
CONFORMATION.
SO -- AND PHIL BIGGINS' GROUP,
THEY TOOK THE THC COORDINATES,
THEY DOCUMENTED ON THE GLYCINE
ALONE STATE AND THEY LOOKED AT
THE STABILITY AND YOU CAN SEE
THREE OUT OF FOUR OR FIVE
SUBUNITS HOLDS ON TO THC AND IN
FACT THE CONFORMATIONAL CHANGES
THE PROTEIN UNDERGOES WERE
SIMILAR TO WHAT WE HAD OBSERVED
UNDER THE CRYO-EM STRUCTURES, TO
THE POINT THAT THC DOCKED
GLYCINE STRUCTURES MOVED FROM
THESE INITIAL COORDINATE TO
START OPENING IT UP AT THE
DESENSITIZED COP FORMATION.
COULD BE FORMATION.
CONFORMATION.
SO TO THE LAST PART I WANT TO
TELL YOU ABOUT ANOTHER MODULATOR
BUT IN THE CONTEXT OF A
HETEROAMERICA GLYCINE RECEPTOR.
SO THIS WAS A CHALLENGE, SO THIS
WORK WAS DONE BY ERIC GIBBS AND
EMILY KRUEGER, AN MD PH.D.
STUDENT IN THE LAB.
SO AS WE WERE WORKING ON THIS
FOR SEVERAL YEARS, WE NAIVELY
THOUGHT WE COULD USE GEF RIN
WHICH BINDS TO THE DOMAIN.
WE COULD CAPTURE A COMPLEX IN
THE PRESENCE OF SCAFFOLDING
PROTEIN BUT ALSO GEF RIN WOULD
ACT AS A IF I DURABLE MARKER
ALLOWING FOR THE IDENTIFICATION
OF BETA SUBUNIT IN THESE.
SO WE TOOK A BIT OF A SHORTCUT,
WE USED DOMAIN E, A DIMERIZATION
DOMAIN THAT HAS PREVIOUSLY BEEN
SHOWN TO BIND TO THE BETA
PEPTIDE REGION RIGHT HERE IN THE
INTRACELLULAR DOMAIN.
SO WE WORKED OUT A SYSTEM TO
EXPRESS ALL THE THREE PROTEIN
AND YOU CAN SEE WE GOT DOWN TO A
PRETTY DECENT BEAUTIFICATION
STRATEGY.
WE COULD ALSO SEE IN GEL
FILTRATION THAT WE HAVE A NICE
SHIFT THAT INDICATES HIGH
MOLECULAR WA WEIGHT AND WE CAN O
ESTIMATE THE WEIGHT BY MASS FLOW
TOM TRI.
IN THE BEGINNING WE START TODAY
SEE SOME ADDITIONAL DENSITIES WE
HAD NOT SEEN IN THE
INTRACELLULAR DOMAIN BEFORE, BUT
AS WE STARTED TO PROCESS IT, WE
REALIZED THAT THE INTRACELLULAR
DOMAIN IS SO FLEXIBLE THAT IT
SWINGS LIKELY WITH THE BIG BULKY
GEPHYRIN AND WE REALLY COULDN'T
CAPTURE MUCH, BUT IN THE PROCESS
WE WERE ABLE TO GET SOME
GLIMPSES OF THE INTRACELLULAR
DOMAIN THAT I'LL SHOW YOU.
BUT WHAT WAS PRETTY NEAT IS THAT
THE ALPHA AND THE BETA SUBUNITS
HAVE DIFFERENT GLYCOSYLATION
SITES, SO THESE HAVE -- THESE
EXTENSIONS THAT YOU SEE IN THE
BETA SUBUNIT ALSO HAS AN END
TERMINAL EXTENSION AND THE
M4 LENS AT ALPHA AND BETA
SUBUNIT ARE DIFFERENT -- TO
ASSIGN THE SUBUNITS, THE ALPHA
AND BETA SUBUNITS.
SO HERE IS A MOVIE THAT GOES
THROUGH MULTIPLE CONTOURING SO
THAT YOU CAN SEE, SO THIS IS THE
ALPHA SUBUNITS ARE IN LIGHT
PURPLE AND THE VIOLET IS THE
BETA SUBUNIT.
AND SO THIS PARTICULAR
CONFORMATION WAS CAPTURED IN THE
PRESENCE OF STRICK NEEN WHICH IS
A HIGHLY TOXIC INHIBITOR OF
GLYCINE RECEPTORS DERIVED --
HERE IS THE CLOSED CHANNEL AND
YOU CAN SEE THE SITE CHAINS THAT
FORM THE ACTIVATION AND THE
DESENSITIZATION GATE RIGHT
THERE, THIS IS THE PROLINE.
SO AS WE WERE LOOKING THROUGH
THESE STRUCTURES, WE NOTICED
THAT THE DENSITY FOR THE
INTRACELLULAR DOMAIN
INTERESTINGLY IS NOT PLACED AT
THE CENTER OF THE PORE AXIS BUT
SHIFTED AWAY FROM THE BETA
SUBUNIT.
WE SOLVED CONFORMATIONS OF
MULTIPLE -- I MEAN, WITH
MULTIPLE MODULATORS IN THIS AND
WE REALIZED THAT THE
INTRACELLULAR DOMAIN, THE ROLE
TO PLAY BESIDES ALL THE OTHER
ROLES AT THE GATING PART OF IT,
IT INFLUENCES THE OVERALL
MOVEMENT OF THE TRANSMEMBRANE
HEE LISTS.
SO THIS IS THE INITIAL VIEW, YOU
CAN SEE THE BETA SUBUNIT AND
THIS IS PART OF THE
INTRACELLULAR DOMAIN SO WE ARE
REALLY WORKING VERY HARD TO
TEASE THIS OUT AND ACTUALLY
CAPTURE MORE OF THIS DOMAIN.
BUT HERE IS THE POSITIONAL
DIFFERENCE THIS, IS THE BULK OF
THE INTRACELLULAR DOMAIN AND IF
YOU LOOK AT THE MOVEMENTS WITHIN
THE TRANSMEMBRANE HELIXES, THE
EXTENT OF MOVEMENTS OF HELIXES
THAT ARE AWAY FROM THIS BULKY
INTRACELLULAR DOMAIN ARE MUCH
MORE THAN THE MOVEMENTS THAT YOU
SEE IN THE HEE LISTS THAT ARE
RIGHT BENEATH IT.
AND THE CONSEQUENCE OF THAT IS
AN ASYMMETRIC PORE MOVEMENT.
AND ALSO WE THINK THAT THE AN
IONIC RECEPTORS HAVE A DIFFERENT
MOLD OF GATING WITHIN THE
INTRACELLULAR DOMAIN.
THIS IS BECAUSE PREVIOUS WORK
FROM OUR LAB AND OTHERS HAVE
SHOWN THIS IS FROM SEROTONIN
RECEPTORS AS A CHANNEL GATES,
YOU HAVE THE OPENING OF THESE
INTRACELLULAR DOMAIN LEADING TO
CREATION OF THESE PORTALS
THROUGH WHICH IONS GO THROUGH.
IN THIS CASE IT'S NOT A DIRECT
PORTAL AND THIS IS A PRETTY
INTERESTING ARCHITECTURE THAT WE
WANT TO DIG FURTHER INTO.
AND SO THIS IS THE ASYMMETRY OF
THE PORE THAT I'M TALKING ABOUT.
SO HAVING AN ICD RIGHT HERE,
THIS IS NO MORE A SYMMETRICAL
MOVEMENT OF THE PORE HE HELIXES.
THERE WERE DIFFERENCES IN THE
LIPID DENSITIES, PREVIOUSLY IT
HAS BEEN SHOWN THAT CERTAIN
MODULATORS DO NOT BIND TO THE
BETA SUBUNIT.
AND IVERMECTIN WAS PROPOSED TO
BE ONE OF THEM SO IVERMECTIN
IS -- IT'S A PRETTY STRONG
POTENTIATION OF -- POTENTIATOR
OF GLYCINE RECEPTORS, POE 10
SHAITS LOW GLYCINE
CONCENTRATIONS, UNIQUE IN THAT
AT HIGHER CONCENTRATION IT CAN
ACT AS AN ACTIVATOR.
SO IT'S KNOWN THAT IT BINDS
WITHIN THE TRANSMEMBRANE DOMAIN
PREVIOUSLY AND THE LAB HAS SHOWN
THIS.
SO THIS IS WHERE IVERMECTIN
BINDS.
IT BINDS AT THE INTERFACE
BETWEEN THE TWO SUBUNITS AND
DIRECTLY CONTACTS M2 WHICH FORMS
THE PORE OF THE CHANNEL.
AND THEN IT SORT OF ACTIVATES
THE CHANNEL.
SO IT WAS PREVIOUSLY SUGGESTED
THAT SO AS IT INSERTS, THIS IS
LOOKING AT IT FROM THE TOP,
THERE IS AN ALANINE RES DUAL IN
THE ALPHA SUBUNIT WHEREAS THIS
RESIDUE IS ISOLEUCINE IN THE
BETA SUBUNIT.
SO BECAUSE OF THIS BULKINESS, IT
WAS PROPOSED THAT IT LIKELY
DOESN'T BIND THIS BETA SUBUNIT,
AND AGAIN, I THINK A NICE
FEATURE ABOUT THIS IS THAT
IVERMECTIN IS AN ANTIPARASITIC
DRUG AND IN NEMATODES WHERE
THERE IS A CHANNEL THAT IS
GLUTAMATE GATED, CHLORIDE
CHANNEL ACTIVATED VERY STRONGLY
BY IVERMECTIN AND THIS RIGHT
HERE IS A GLYCINE, SO YOU MAKE
THIS RESIDUE SMALLER, YOU HAVE A
GREATER POTENTIATION WITH
IVERMECTIN.
SO AGAIN, BUT WHAT WE FOUND IS
THAT IVERMECTIN BINDS INTO ALL
OF THE INTERFACES, BUT HAS A
UNIQUE ORIENTATION IN THE BETA
SUBUNIT INTERFACE BECAUSE OF
THIS PUSHING OVER IT AND VEGING
OUT THE IVERMECTIN MOLECULE.
SO HERE IS A TOP VIEW THAT YOU
CAN SEE, THIS IS IN THE ALPHA
VERSUS BETA SUBUNIT.
THIS BINDING MOLD HAS A WEAKER
AFFINITY FOR GLYCINE.
AND SO WE THEN ASKED, OKAY, IF
THIS IS THE CASE, IF IVERMECTIN
IS A LIGAND THAT HAS ITS OWN
ACTIVATION PATHWAY, SO INSTEAD
OF ACTIVATION ORIGINATING AT THE
LEVEL OF THE EXTRACELLULAR
DOMAIN MOVING INTO THE
TRANSMEMBRANE REGION, NOW YOU
HAVE A PATH THAT GOES FROM
TRANSMEMBRANE DOMAIN, SO CAN WE
USE THIS TO UNDERSTAND WHETHER
THERE ARE BINDING AFFINITY
DIFFERENCES, ARE THERE -- I
MEAN, CAN WE CAPTURE
INTERMEDIATES BECAUSE WE'VE BEEN
ONLY TO GET THESE STATES EITHER
CLOSED, OPEN OR DESENSITIZED.
SO WHAT WE DECIDED TO DO IS WE
ADDED -- THEN WE ADD IVERMECTIN
TO BASICALLY LOOK AT BINDING
MODES AND ALSO LOOK AT
INTERMEDIATES IF POSSIBLE.
SO IN THIS PURSUIT, SO WE HAVE
IVERMECTIN WITH STRYCHNINE, THIS
IS THE BENCHMARKING, THIS IS IN
THE PRESENCE OF GLYCINE
IVERMECTIN, YOU SEE THESE
BINDING MODES, YOU CAN SEE THIS
DISTINCT MODE HERE.
SO AS YOU START TO DECREASE
IVERMECTIN CONCENTRATION, WE
START TO LOSE ON THE OCCUPANCY
FIRST IN THE BETA INTERFACE BETA
ALPHA INTERFACE, THEN IN ALPHA
BETA, AND THEN EVENTUALLY IN THE
ALPHA-ALPHA INTERFACES,
SUGGESTING HIGH AFFINITY BINDING
MOLD WHICH SHIFTS DOWN AS WE GO
TO BINDING INTERFACES.
AND THEN WE CAN ALSO LOOK AT THE
CONFORMATION SO THIS IS SIMPLY
LOOKING AT PROJECTION, THIS IS
RESTING, THIS IS ACTIVATED
STATE, AND THESE HEE LISTS AND
STRANDS MEANS THAT THEY LOOK
MORE LIKE STRICK NEEN AS THEY
BECOME MORE RED AND THICKER,
THEY LOOK MORE LIKE THE OPEN
CONFORMATION.
AS YOU INCREASE CONCENTRATIONS,
YOU START TO MOVE TOWARDS THE
ACTIVATED STATE RIGHT HERE.
BUT WHAT'S INTERESTING IS MOST
OF THE CONFORMATIONS WITHIN THE
TRANSMEMBRANE DOMAIN HAS ALREADY
HAPPENED SO EVEN WITH PARTIAL
IVERMECTIN OCCUPANCIES, YOU HAVE
A FULLY ACTIVATED STATE.
BUT THE RESPONSE IN THE
EXTRACELLULAR DOMAIN IS MORE
NUANCED AS YOU CAN SEE SOME OF
THESE REGIONS ARE ACTIVATED,
SOME OF THEM ARE NOT, LIKELY
SUGGESTING INTERMEDIATES WITHIN
THE GATING PATHWAY.
SO HERE IS A MOVIE THAT
SUMMARIZES THIS.
SO YOU HAVE THIS MOVEMENT, THERE
IS NO IVERMECTIN OCCUPANCY YET.
YOU CAN SEE THERE'S NOT MUCH
CHANGE, THEN AS YOU MOVE, YOU
CAN SEE THE FIRST IVERMECTIN
COMES IN AND THEN AS YOU START
TO SEE HIGHER BINDING AFFINITY
MODES, THEN THE CHANNEL MOVES TO
IT.
SO IF YOU ALIGN THEM, YOU EITHER
SEE THEY'RE ACTIVATED OR THEY'RE
NOT, SO THERE IS FULL
COOPERATIVITY IN THIS CASE.
SO THIS IS STRYCHNINE AND YOU
CAN ALSO SEE AS YOU INCREASE
IVERMECTIN STRYCHNINES,
ORIENTATION STARTS TO CHANGE AND
MOVE TOWARDS LOW AFFINITY MODES.
SO AGAIN, THESE ARE SOME OF THE
DIFFERENT, WE BELIEVE, GATING
INTERMEDIATES IN THIS ACTIVATION
PATHWAY.
ANOTHER WAY TO REPRESENT HERE,
THIS IS JUST ALONG THE LENGTH OF
THE SEQUENCE, SO CHANGES HERE IN
THE TRANSMEMBRANE DOMAIN ARE
ALMOST ALL OR NONE WHEREAS HERE
YOU SEE VERY MANY GREY
INTERMEDIATES WITHIN THE
EXTRACELLULAR DOMAIN.
SO THIS IS SORT OF -- CONCLUDES
WHAT I WANTED TO SAY HERE.
WE ARE JUST STARTING TO
UNDERSTAND THE BASIS FOR THESE
SUBTYPE SPECIFICITY, WE ARE
STILL SCRATCHING THE TIP OF THE
ICEBERG HERE.
AND HOPEFULLY WE CAN USE THESE
UNDERSTANDING TO LOOK AT OTHER
MODULATORS.
SO A GENERAL SUMMARY IS THAT
THESE CRYO-EM STRUCTURES REVEAL
THESE INTRICATE MOVEMENT THAT
UNDERLIE FUNCTIONAL BEHAVIORS
AND MODULATIONS, AND WE CAN
START TO TEASE OUT ASYNCHRONOUS
MOVEMENTS THAT ARE PART OF THESE
INDIVIDUAL DOMAINS, AND I THINK
THE BIG PART OF -- WE WANT TO
UNDERSTAND MODULATION, WE WANT
TO UNDERSTAND HOW THEY ARE
MODULATED BY LIPIDS AND LIPID
MODULATORS AND COMPOUNDS THAT
HAVE GOOD THERAPEUTIC PROFILES.
SO WITH THAT, I WANT TO
ACKNOWLEDGE THE PEOPLE WHO HAVE
DONE ALL OF THIS WORK,
PARTICULARLY ALL THE PAST
MEMBERS WHO HAVE MOVED ON AND
THIS IS OUR CURRENT TEAM AND
HAVE ACKNOWLEDGED INDIVIDUAL
PEOPLE AS WE WENT THROUGH THIS
IS THE WORK OF -- THAT I DIDN'T
TALK ABOUT, CRYO-EM ACCESS, WE
ARE FORTUNATE TO HAVE LOCAL
ACCESS BUT WE RETEENLY USE
NATIONAL SENATONATIONAL SENATOR,
THANK YOU FOR LISTENING AND
HAPPY TO TAKE QUESTIONS.
>> I ENCOURAGE PEOPLE TO TAKE
THE MICROPHONES.
THERE ARE NO QUESTIONS ONLINE
YET.
WE DO HAVE A ROBUST AUDIENCE.
THE CME CODE, 57663.
PLEASE USE THE MICS.
>> BEAUTIFUL TALK.
I WAS VERY INTRIGUED BY SORT OF
THE CHANGE IN THE CAVITY VOLUMES
THAT YOU ALLUDED TO.
AND I'M IMAGINING THAT AS THESE
PROTEINS IN GENERAL UNDERGO
CONFORMATIONAL CHANGES NOT ONLY
DO YOU CHANGE THE VOLUMES OF
THESE CAVITIES, YOU ALSO
POTENTIALLY CHANGE THE SURFACE
CHEMISTRY OF THESE CALFITIES.
SO IN PRINCIPLE, THAT MEANS THAT
OPENS UP THE POSSIBILITY OF
THESE PROTEINS BEING REGULATED
BY PHOSPHOLIPIDS THAT ARE IN THE
MEMBRANE.
IS THERE ANY WAY TO TEASE THIS
APART, DO YOU THINK THIS IS JUST
A FANTASY?
CAN YOU SORT OF EDUCATE ME ON
THIS?
>> I MEAN, ABSOLUTELY
FASCINATING QUESTION.
AND ALSO A CHALLENGE FOR A
STRUCTURAL BIOLOGIST.
THIS IS OUR FIELD AND WE
STRUGGLE TO TEASE THIS APART,
RIGHT?
SO THESE, I MEAN, WHAT YOU POINT
OUT IS VERY IMPORTANT.
IT'S NOT JUST THE VOLUME, IT'S
THE CHEMISTRY OF THIS.
SO SOME OF THESE POCKETS ARE
HYDROPHOBIC AS THEY MOVE OUT AND
REORIENT THE HELICES, AND ONCE
THE CHANNEL AT A NORMAL GATING
CONDITIONS AS THEY OPEN AND
THESE CAVITIES OPEN UP, IT'S NOT
IN VACUUM.
YOU HAVE LIPIDS GETTING THERE
AND THOSE LIPIDS ARE NATURAL
REGULATORS OF CHANNEL FUNCTION.
AND NOW YOU PUT A MODULATOR THAT
LOOKS LIKE SOME OF THESE LIPIDS,
HOW DO YOU DISTINGUISH BETWEEN
THAT?
SO I THINK THAT'S A CHALLENGE
AND ALSO WHEN YOU WORK WITH THE
MINIMALISTIC SYSTEM, WE REALLY
DON'T HAVE THOSE LIPIDS, THEN WE
PUT THE MODULATOR IN, SO THEIR
AFFINITYS ARE PROBABLY
DIFFERENT.
SO I THINK THOSE ARE SOME OF THE
CHALLENGES, CAN WE USE MASS SPEC
TO START IDENTIFYING WHAT THOSE
LIPIDS ARE SO THEN CAN YOU START
INCORPORATING THEM INTO OUR
STRUCTURAL STUDIES.
BUT I THINK INTERESTING AND
CHALLENGING █PURSUIT THERE.
>> A COUPLE QUESTIONS ARE COMING
IN ONLINE.
OKAY.
SO DO YOU THINK THE PH
DEPENDENCE OF ZINC ACTIVATION ON
THE GLYCINE RECEPTOR IS DUE TO
THE EYE ONIZATION STATE OF
HISTIDINE WHICH COORDINATES TO
THE HIGH AFFINITY ZINC STATE AND
TO THE LOW AFFINITY ZINC STATE?
>> SO GREAT QUESTION.
SO ABOUT THE PH EFFECTS, AND THE
IONIZABLE RESIDUES AND WHAT
IS -- WHAT ARE THE RESIDUES THAT
ARE MEDIATING THIS, IT'S UNCLEAR
AT THIS POINT.
BUT THERE ARE SEVERAL RESIDUES
THAT HAVE BEEN IMPLICATED IN PH
DEPENDENCE.
SO HISTIDINES IN THE
POTENTIATING SITES ARE LIKELY TO
BE SOME OF THEM BECAUSE WHEN YOU
HAVE ACIDIC PH ZINC MODULATION
GOES AWAY.
ZINC POTENTIATION GOES AWAY.
SO IT IS LIKELY THOSE HISTIDINES
PLAY A ROLE.
ALSO INTERESTINGLY, SOME --
THREE ANINES HAVE BEEN
IMPLICATED, THAT THEY ELIMINATE
PH -- OR ACIDIC PH INHIBITION.
IT'S UNCLEAR WHAT THEY'RE DOING
THERE BUT IT'S ALSO POSSIBLE
THAT IT'S AN ALLOSTERIC EFFECT
THAT YOU MAKE A MUTATION, IT IS
CHANGING THE WAY THE HISTIDINE
IS SEEN IN OTHER INTERACTIONTION
AND IT COULD BE POSSIBLE BECAUSE
OF THAT.
BUT FOR SURE THE POTENTIATING
RESIDUES HAVE A ROLE DIRECTLY.
>> ANOTHER QUESTION.
IS THE BINDING CONSTANT KD KNOWN
FOR THE ZINC OR OTHER LIGANDS?
>> YEAH, SO THE BINDING -- THE
BUY LYE GANDZ THAT I WAS TALKING
ABOUT, AGAIN, THE KDs VARY
QUITE A BIT, SO FOR THE ZINC, AT
THE LOWER -- SO FOR SURE THERE
ARE LOW MICROMOLARS FOR THE
POTENTIATING SITES, AND HIGHER
FOR THE INHIBITORY SITE.
AGAIN IT DEPENDS ON THE TYPE OR
SUBTYPE THAT YOU'RE LOOKING AT,
THE ALPHA 3s AND THE 300
MICROMOLAR RANGE FOR THE
INHIBITORY ONE.
>> PROFOUND CLOSING COMMENTS?
>> ONE LAST QUESTION.
SO DO HEAVY METALS BIND TO THE
ZINC SITES?
>> ALSO, YES.
>> DOES THAT -- DOES THAT HAVE
ANYTHING TO DO WITH LEAD OR
MERCURY NEUROTOXICITY?
>> YEAH.
I MEAN, IT IS POSSIBLE.
AGAIN, THE SITE IS PRIME FOR
BINDING OTHER -- AND IT'S KNOWN
THAT SOME OF THESE HAVE, BUT IN
TERMS OF WHETHER THEY LEAD TO
NEUROTOXICITY AND THAT KIND OF
THING UNCLEAR, BUT IT IS
POSSIBLE, IT'S NOT EXCLUSIVE TO
ZINC, BUT ITS DIRECT ROLE, NOT
SURE.
>> FANTASTIC.
IF THERE ARE NO OTHER QUESTIONS,
THEN LET'S THANK SUDHA FOR A
FANTASTIC TALK.
[APPLAUSE]
OH, AND DON'T FORGET THERE'S A
RECEPTION OUTSIDE.
SO PLEASE JOIN US.