>> ALL RIGHT.
GREAT PLEASURE TO BE ABLE TO 
WELCOME RICH LEWIS TODAY.
UM, RICH KID HIS UNDERGRADUATE 
WORK AT YALE AND THEN WENT TO 
WORK WITH GYM HUDSPETH AT KAL 
TECH WHERE HE BEGAN STUDYING ION
CHANNELS IN HAIR CELLS.
HE THEN ACTUALLY MADE A FAIRLY ),"T
HIS POST DOC FELLOWSHIP TO WORK 
WITH MIKE CAHALAN TO WORK AT UC 
IRVINE.
FOR THOSE OF YOU WHO DONATE KNOW
THE STORY, I SEE SOME 
IMMUNOLOGIST IN THE AUDIENCE AND
YOU PROBABLY WOULD FIND IT HARD 
TO BELIEVE THAT PEOPLEt( IN THE 
CHANNEL FIELD WEREN'T INTLALED 
WITH STUDYING BLOOD CELLS AND 
LYMPHOCYTES AND STUFF.ok
MIKE CAHALAN, HIS PLAN WAS TO 
CONTINUE STUDYING THE 
[INDISCERNIBLE] BUT THE SQUID, 
ACTUALLY WENT INTO A DIFFERENT 
PATTERN AND THEY COUNT GET 
SPECIMENS TO STUDY.
SO THEY RESORTED TO FINDING SOME
OTHER PREPARATIONw3 AND DECIDED TO
ACTUALLY START STUDYING BLOOD 
CELLS.
THIS IS ABOUT THE TIME SOON 
THERE AFTER THAT RICH JOINED HIS
LAB AND REALLY, I THINK, KICKED 
OFF A THIS WHOLE EFFORT TO 
CHARACTERIZED THE DIFFERENT 
TYPES OF ION CHANNELS IN 
LYMPHOCYTES AND BEGIN 
UNDERSTANDING THEIR OPERATIONAL 
MECHANISMS AND THEIR 
CONTRIBUTIONS TO DIFFERENT 
PHYSIOLOGICAL PROCESSES.
SO, UM, MIKE ACTUALLY WHEN HE --
WHEN RICH MOVED TO STANFORD TO 
SET UP HIS OWN LAB, HE CONTINUED
STUDYING LYMPHOCYTES AND SHIFTED
GEARS TO WORKING ON THESE 
STORE-OPERATED CHANNEL.
THESE ARE ACTUALLY KA CALCIUM 
CHANNEL ON THE PLASMA MEM RAIN 
THAT BECOME ACTIVATED WITH THE 
ER CALCIUM GETS DEPLETED.
IT'S AN EXCITING PHASE IN THIS 
AREA WITH KIND OF SUB UNITS 
BEING IDENTIFY IN THE THE LAST 
NUMBER OF YEAR AND I LOOK 
FORWARD TO HEARING WHAT RICH IS 
GOING TO TELL THE US ABOUT THE 
MECHANISM OF THESE CHANNEL AND 
HOW THEY ACTUALLY WORK.
SO, RICH, THANKS FOR COMING.ú3ONF
>> WELL, THANKS -- THIS IS 
TIRNED ON NOW?
THANKS AGAIN FOR THE INVITATION.
IT'S BEEN -- IT'S ALWAYS GREAT 
TO VISIT NIH AND SEE HOW MANY 
EXCITE IG THINGS ARE*ñ GOING ON 
AROUND HERE.
JUST TALKING TO TWO PEOPLE THIS 
MORNING, MY HEAD IS ALREADY JUST
COMPLETELY BUZZING, EITHER THAT 
OR IT'S THE JET LAG, I DON'T 
KNOW.
SOMETHING'S MAKING MY HEAD BUZZ 
RIGHT NOW BUT IT'S REALLY BEEN 
GREAT SO FAR.
AND I APPRECIATE THIS CHANCE TO 
SHARE WITH YOU SOME OF THE 
RECENT PROGRESS THAT WE AND 
OTHER LABS HAVE MADE IN 
UNDERSTANDING STORE-OPERATED 
CHANNELS.
THIS IS SOMETHING THAT'S BEEN 
 O
<o FAR WHILE AND IT'S ONLY
IN THE LAST FIVE OR SO YEARS 
THAT BIG%x( 
AND I'LL DESCRIBE SOME OF THOSE 
AS WE GO ALONG.
I THOUGHT I WOULD START OFF JUST
WITH FOR PEOPLE WHO AREN'T 
FAMILIAR WITH THIS CLASS OF O 
CHANNELS WITH A BRIEF 
DESCRIPTION.
STORE-OPERATED CALCIUM CHANNELS 
ARE PROBABLY PRESENT IN EVERY 
CELL TYPE IN THE BODY, AND 
GENERALLY UNDER PHYSIOLOGICAL 
CONDITIONS THEY'RE ACTIVATED BY 
ENGAGEMENT OF CELL SURFACE 
RECEPTORS THAT ARE COUPLE øBç 
PLC.
ONCE ACTIVATED IT GENERATED IP 3
TO RELEASE CALCIUM WHERE FROM 
THE ER.
STORE O-OPERATED CHANNELSç T 
RELEASE OF CALCIUM ER, THE 
DEPLETION OF CALCIUM SENDS A 
SIGNAL TO ACTIVATE, OPEN THESE 
CHANNELS IN THE PLASMA MEMBRANE.
WE KNOW THAT THIS IS THE CASE 
AND IN FACT THE DEFINING 
HALLMARK OF THIS CLASS OF 
CHANNELS IS THAT ONE CAN BY PASSçó
RECEPTOR ACTIVATION COMPLETELY 
AND ACTIVATE THE CHANNELS SIMPLY
BY NUMBER OF MANIPULATIONS THAT 
DEPLETE CALCIUM FROM THE LUMEN 
OF THE ER.
THIS WOULD INCLUDE INHIBITING 
THE CALL IS I YES, I AM AT 
PACES, BY LOADING THE CELL 
CYTOPLASM WITHsA CALCIUM BUFFER,
WHICH PREVENTS CALCIUM GOING 
BACK INTO THE STORES AND SO ON.
THESE, AS I SAID BEFORE, THESE 
STORE-OPERATED CALCIUM CHANNELS 
ARE VERY UBIQUITOUS, PROBABLY 
PRESENT IN EVERY CELL TYPE.
ONE CELL TYPE IN WHICH THEY SEEM
TO BE PARTICULARLY IMPORTANT IN 
WHAT INITIALLY ATTRACTED ME TO 
THIS WHOLE AREA IS IN T 
LYMPHOCYTES, AND THIS IS A MOVIE
I'M GOING SHOW YOU FROM MIKE 
CAHALAN'S LAB THAT SIMPLY SHOWS 
A T CELL LOADED WITH A 
CALCIUM-DEPENDENT DIE DYE 
INTERACTING WITH A CALCIUM 
INTERACTING CELL.
SOON AFTER CONTACT IS MADE E 
WITH THE ANTIGEN PRESENTING 
CELL, THE CALCIUM GOES UP IN THE
T CELL.
THIS IS A CRITICAL EARLY SIGNAL 
BY T CELL ACTIVATION BY ANTIGEN.
THE PROCESS IS INITIATED BE BY 
RECOGNITION OF ANTIAGAIN ON THE 
SURFACE OF ANTIAGAIN CELLS.
THIS CALCIUM DEPLETION FROM THE 
T CELL STORES ACTIVATES A 
STORE-OPERATED CHANNEL THAT WE 
KNOW AS THE CALCIUM-RELEASE 
ACTIVATED CAN KAL IS I YUM 
CHANNEL IN T CELLS.
NOW THE CALCIUM THAT COMES IN 
THROUGH THIS CHANNEL INTERACTS 
WITH OTHER COMPONENTS OF THE 
CELL SUCH AS POE TAS IS I YUM 
CHANNELS, PUMPS ANDi] 
MITOCHONDRIA.
THE CALCIUM SIGNAL, ITSELF, 
OPERATES ON DISTINCT TIME SCALES
TO ELICIT, FOR EXAMPLE, ON A 
SHORT TIME SCALES OF SECONDS TO 
MINUTES, THE RELEASE OF 
CYTOKINES AND CYTOTOXIC GRANULES
FROM T CELLS.
ON LONGER TIME SCALES OF HOURS 
TO DAY TO ACTIVATE GENE 
EXPRESSION PROGRAMS, LARGELY 
THROUGH THE ACTIVATION OF A 
PHOSPHATASE CALL SEW NEURONb
WHICH THEN [INDISCERNIBLE] TO 
CONTROL PROGRAMS OF GENE 
EXPRESSION THAT LEADyM TO T CELL 
ACTIVATION.
THE IMPORTANCE OF THE CRACK 
CHANNEL FOR F CELL NUNGS HUMAN 
HEALTH IS UNDERSCORED BY THE 
FACT THAT LOSS OF FUNCTION IN 
THIS PATHWAY LEADS TO SEVERE 
COMBINED IMMUNODEFICIENCY.
THAT IS A COMPLETE LOSS OF T 
CELL AND B CELL FUNCTION, WHICH 
IS A LETHAL CONDITION UNLESS ONE
RECEIVES A BONE MARROW STEM CELL
TRANSPLANT.
THESE ARE VERY IMPORTANT 
CHANNELS FOR THE IMMUNE SYSTEM.
THEY'VE ALSO MORE RECENTLY BEEN 
SHOWN TO BE IMPORTANT FOR MUSCLE
FUNCTION, PLAIT LET CELL 
FUNCTION AND SO ON.
I'M GOING TO FOCUS, TODAY, ON 
THE MECHANISMS BY WHICH 
DEPLETION OF THE CALCIUM FROM 
THE STORES ACTIVATE THIS IS 
CLASS OF CHANNEL.B" 
NOW,ñ
STUDIED PROBABLY FOR ABOUT 
NEARLY 20 YEARS WITH VERY LITTLE
PROGRESS BEING MADE ON THE 
MECHANISM THAT COMMUNICATES 
STORE DEPLETION TO CHANNEL 
ACTIVATION, AND THE REASON WAS 
THAT THERE WERE REALFULLY TO 
MOLECULAR COMPONENTS THAT THE 
BEEN IDENTIFIED IN THE PATHWAY 
UP UNTIL ABOUT 2005, AND THIS IS
WHEN RNAI SCREENS CARRIED OUT 
BAY NUMBER OF LABS SUCCEEDED IN 
ISOLATING THE TWO PRINCIPLE 
COMPONENTS OF THIS PATHWAY, AND 
THIS WOULD BE THE STEM PROTEINS,
HERE, WHICH ACT AS THE ER 
CALCIUM SENSORS, AND A, THE AWRY
PROTEINS IN THE PLASMA MEMBRANE 
WHICH CONSTITUTE THESE 
STORE-OPERATED CHANNELS.
EVEN THOUGH BOTH OF THESE 
PROTEINS IN MAMMALIAN SYSTEMS, 
AT LEAST, ARE PARTS OF GENE 
FAMILIES, SO THERE'S A STEM ONE 
STEM TWO AND THERE ARE THREE 
VERSIONS OF AWRY, FOR TODAY I'M 
GOING TO CONCENTRATE ON STEM ONE
AND AWRY ONE AS THESE ARE THE 
MOST DIRECTLY CONNECTED TO CRACK
CHANNEL FUNCTIONS IN T CELLS 
STEM ONE, THE ER CALCIUM SENSOR 
WAS ISOLATED AROUND THE SAME 
TIME BY KEN'S GROUP AND TOBIAS' 
GROUP.
THEY FOUND THIS PROTEIN WHICH 
HAS AN EF HAND IN THE LUMEN OF 
THE ER, AND A NUMBER OF 
COIL-COIL DOMAINS AND OTHER 
DOMAIN WHICH IS I'LL TALK ABOUT 
LATER, INCLUDING A POLL LI BASIC
AT TERMINUS.
AWRY ONE WAS ALSO ISOLATED IN 
RNA ICE SCREENS BY SEVERAL 
GROUPS.
THIS IS A FOUR TRANS MEMBRANE 
PLASMA MEMBRANE PROTEIN WHICH 
ASSEMBLES PROBABLY IN A TE TRA 
AMERICA FORM TO FORM THE 
CHANNEL, ALTHOUGH THERE IS STILL
A LITTLE BIT DEBATE OVER EXACTLY
WHAT THE STOICHIOMETRY IS.
MOST GROUPS WOULD AGREE THE 
ACTIVE CONCENTRATION OF THE 
CHANNEL IS A TET TRA MURAL OF 
AWRY ONE SUB UNITS.
R 91 W MUTATION WAS 
SPONTANEOUSLY OCCURRING HUMAN 
MUTATION THAT LEAD LED TO THE 
SKID PHENOTYPE THAT ONE GROUP 
INITIALLY IDENTIFIED AND USED AS
PART OFeK(D
CHANNEL.
SO THE GREAT ADVANTAGE TO NOW 
HAVING THESE TWO MOLECULES IS 
THAT THEY PROVIDED TOOLS, 
MOLECULARER TOOLS, TO FINALLY 
ADDRESS THE QUESTION OF THE 
MECHANISM, AND THAT BECAME A 
MUCH MORE SPECIFIC QUESTION OF 
UNDERSTANDING HOW IS IT THAT THE
SENSING OF CALCIUM BY STEM ONE 
LEADS TO THE ACTIVATION OF CRACK
CHANNELS OR AWRY ONE IN THE 
PLASMA MEMBRANE?
IT TURNS OUT THAT THE MECHANISM 
INVOLVES THE REDISTRIBUTION OF 
BOTH PROTEINS IN THE CELL ..
BOTH PROTEINS IN THE CELL ..
INITIALLY IN A RESTING CELL IN 
THE PRESENCE OF CALCIUM WHEN THE
ER CALCIUM STORES ARE FULL, AND 
THIS SAY BOUT 400 MICRO MOLAR 
CALCIUM IN THE ERE BOTH STEM ONE
AND IE RYE ONE ARE DEFUSELY 
DISTRIBUTED THROUGHOUT THE CELL.
STEM ONE IN THE ER AS YOU CAN 
SEE BY THIS CHERRY STEM ONE 
TRANSFECTED HEX CELL AND AWRY 
ONE IN THE PLASMA MEMBRANE IS 
SHOWN IN THE SAME CELL GFP AWRY 
ONE IN THE  FINGERPRINT OF THE 
CELL.
IF WE DEPLETE THE STORES, WE CAN
SEE WITHIN MINUTED STEM ONE IS 
REDISTRIBUTE INTO A NUMBER OF 
FLUORESCENT PUNK TA AND AWRY ONE
REDISTRIBUTES AS WELL INTO A 
SERIES OF VERY CLOSELY OPPOSED 
PUNK TA.
THE OVERLAP IS VERY EXACT 
BETWEEN THESE TWO.
WE ARE INTERESTED IN KNOWING 
WHAT THESE PUNK TA CORRESPOND 
TO.
WE TOOK STEM ONE AND GRAFTED A 
HORSE RADISH PROXIMATE DAYS ON 
TO THE END TERMINUS AND USED HRP
TO LOCALIZE STEM ONE AS THE 
OVERSTRUCTURAL LEVEL.
THIS WAS DMON HUMAN T CELL LINE.
WHAT WE FOUND WAS THAT IN 
RESTING CELLSmy AS WE WOULD EXPECT
THE RESTING PRODUCT WAS 
DELIBERATED THROUGHOUT 
CYTOPLASM, BUT THAT FOLLOWING 
TREATMENT AND STORE DEPLETION, 
THAT THE REACTION PRODUCT 
INDICATE WHERE STEM ONE IS 
PRESENT WAS MOSTLY CONCENTRATED 
IN PORTIONS OF THE ER THAT WERE 
CLOSE TO THE PLASMA MEMBRANE.ç
MEASUREMENTS THAT WE MADE ON A 
NUMBER OF CELLS INDICATED THAT 
THE GAP BETWEEN THE ER TUBE 
WALLS AND THE PLASMA MEMBRE
WAS AROUND 10 TO 20 NANOMETERS.
THEY REPRESENT A SPECIALIZED 
SUBSET OF INDOE PLASMIC RA TICK 
LUM.
WHILE MOST OF THESE ER PLASMA 
MEMBRANE JUNCTIONS WERE PLEASANT
BEFORE STORE DEPLETION, THERE 
WAS ABOUT A 50% INCREASE IN THE 
NUMBER OF THESE JUNCTIONS AFTER 
DEPLETION, AND WE COULD DISCUSS 
PERHAPS WHAT MECHANISMS MIGHT BE
INVOLVED IN THAT LATER.
IT WAS REALLY THE GAP BETWEEN 
THESE MEMBRANES THAT INTRIGUED 
US WAS 10 TO 20 NANOMETERS IS 
CLOSE ENOUGH FOR THE STEM ONE TO
COMMUNICATE WITH AWRY ONE IN THE
MEMBRANE.
WE WANTED TO KNOW WHETHER THIS 
IS THE PLACE WHERE THE CALCIUM 
CAME INTO THE CELL.
A GRADUATE STUDENT IN THE LAB 
DID A TURF EXPERIMENT IN WHICH 
SHE TOOK CELLS THAT WERE 
EXPRESSING A CHERRY-LABELED STEM
ONE AND IMAGED THEM USING TURF 
MICROSCOPY AND LOADED THEMB./y WITH 
A CALCIUM-DEPENDENT FLUORESCENT 
DYE ALONG WITH EGTA.
USING THIS METHOD, SHE WAS ABLE 
TO LOCALIZE HOT SPOTS OF CALCIUM
ENTRY INTO THE CELL.
THEN WE COULD JUST CORRELATE THE
SITES OF THE CALCIUM ENTRY TO 
THE SITES OF STEM ONE 
ACCUMULATION.
WE COULD SEE THAT THE TWO 
OVERLAID DEVICE NICELY IF WE 
TAKE, FOR EXAMPLE, A CROSS 
SECTION THROUGH ONE OF THOSE 
PUNK TA IN BOTH THE CHERRY-STEM 
ONE CHANNEL AND THE CALCIUM 
FLUORESCENT CHANNEL.
CALCIUM ISq
FLO RESENSE OF STEM.
P.
IF WE MAKE A DIRECTION 
CORRECTION OF THE DIFFUSION OF 
CALCIUM BOUND TO FLOW FIVE F 
FROM THE FLOW FIVE ENTRY, WE 
COULD SEE THAT THE SPATIAL 
DISTRIBUTION OF THE TWO SIGNALS 
OVERLAYS QUITE EXACTLY.
THIS LED US TO CONCLUDE THAT 
THIS COLOCALIZATION OF STEM AND 
AWRY IS PROBABLY A FUNCTIONALLY 
CONNECTED TO THE FACT THAT THE 
CRACK CHANNELS OPEN PRECISELY AT
THESE SAME LOCATIONS INDICATING 
A VERY LOCAL COMMUNICATION 
BETWEEN THE TWO PROTEINS.
NOW ONE THING I WANTED TO POINT 
OUT IS THAT, UH, THIS PROCESS IS
ACTUALLY RATHER SLOW, AND A ONE 
WAY TO APPRECIATE THAT IS IN 
ANOTHER KIND OF EXPERIMENT WHERE
WE SIMPLY, WE TAKE A JERK OUT T 
CELL EXPRESSING A GFP LABELED 
STEM ONE, USE MICROSCOPY TO 
IMAGE THAT WITHIN ABOUT 100 
NANOMETERS OF THE EM BRAIN AND 
INTREEK THE CELL USING A PATCH 
CONTAINING EGTA.
IT WILL DEPLETE THE CALCIUM 
STORES AND OVER SOME TIME COURSE
ACTIVATE THE CRACK CHANNELS AND 
WE CAN MONITOR THE CRACK CHANNEL
ACTIVATION ELECTRICALLY USING 
THE PATCH CAMP WHILE MONITORING 
USING TURF MICROSCOPY.
HERE'S A TIME LAPSE SEQUENCE 
WHERE WE'REIÑ MEASURING THE 
CURRENT OVER RANGE OF VOLTAGES 
WHILE MEASURING GFP 
FLUORESCENCE.
THIS IS ONE MEASUREMENT TAKEN 
EVERY THREE SECONDS JUST TO GIVE
YOU AN IDEA.
INITIALLY, THERE'S NO CURRENT, 
BUT THEN AS STEM ONE STARTS TO 
ACCUMULATE IN THE PUNK TA, WE 
SEE THE CURRENT STARTS TO TURN 
ON GIVING THIS TYPICALLY INWARD 
RECTIFYING VOLTAGE RELATION THAT
WE KNOW AND EXPECT FROM THE 
CRACK CHANNEL.
IF WE SIMPLY MEASURED THIS 
ACCUMULATION OF THE GFP STEM ONE
FLUORESCENCE AT THE CELL 
FINGERPRINT AND PLOTTED ALONG 
WITH THE DEVELOPMENT OF THE 
CRACK CURRENT, WE CAN SEE THAT 
STEM ONE ACCUMULATE IN THESE 
PUNK TA SLIGHTLY IN ADVANCE OF 
THE ACTIVATION OF THE CHANNELS.
IS ABOUT TEN SECONDS IN THIS 
CELL T LAG BETWEEN THEM, AND THE
# A K CURRENT DEVELOPS OVERú
TIME COURSE OF TENS OF SECONDS 
NAP GIVES YOU AN IDEA OF HOW 
SLOW THIS IS TO OPERATE.
INTERESTINGLY, WE FOUND THAT 
THERE GENERALLY WAS çABOUT A TEN 
SECOND LAG BETWEEN THE ARRIVAL 
OF STEM AND OPENING OF THE 
CHANNELS.
THIS OCCURRED WHETHER OR NOT WE 
ACTIVATED PASSIVELY OR MORE 
ACTIVELY BY STIMULATED THE T 
CELL RECEPTORS ON THE SURFACE 
WITH ANTIBODIES.
THERE'S SOME DELAY BETWEEN THExD 
TWO, AND THIS IS AGAIN SOMETHING
WE COULD TALK ABOUT LATER IF 
PEOPLE ARE INTERESTED.
BUT JUST TO SUMMARIZE WHAT I'VE 
TOLD YOU SO FAR; THE OVERALL 
COOR YOG FI OF THIS CRACK 
CHANNEL ACTIVATION IN THE CASE 
MECHANISM IS REALLY WHAT WE 
WOULD THINK OF AS ASSEMBLY ON 
DEMAND KIND OF SYSTEM WHERE THE 
COMPONENTS ARE INITIALLY 
DISTRIBUTED RAN DOLLY THROUGHOUT
THE CELL BUT AFTER THE STIMULUS 
ARRIVES TO RELEASE CALCIUM FROM 
THE ER, IT CAUSES THExD 
ACCUMULATION OF STEM AND AWRY IN
THESE SPECIALIZED JUNCTIONS, AND
SOMETHING ABOUT THAT 
COLOCALLIZATION LEADS TO THE 
OPENING OF THE CHANNELS IN THE 
PLASMA MEMBRANE.
FOR PEOPLE WHO ARE FAMILIAR WITH
MUSCLE EXCITATION CONTRACTION 
COUPLING THIS MIGHT LOOK 
FAMILIAR, MIGHT REMIND YOU OF 
THE TRIAD MUSCLES.
ONE IMPORTANT DISTINCTION 
BETWEEN THESE TWO KINDS OF 
SYSTEMS IS THIS SYSTEM, WHEREAS 
IN MUS IT WILL TRIADS ARE SET UP
PRIOR TO ARRIVAL OF THE 
STIMULUS, IN THIS q
WHOLE COLOCALIZATION OF THE TWO 
PROTEINS AND THEIR COMMUNICATION
ONLY OCCURS OVER TENS OF SECONDS
AFTER THE ARRIVAL OF THE 
STIMULUS.
THIS LEADS TO LAGS BETWEEN 
STIMULUS AND RESPONSE, WHICH, 
AGAIN, CAN LEAD TO INTERESTING 
DYNAMICS, CALCIUM OSCILLATIONS 
AND THINGS OF THAT SORT.
SO WHAT I'D LIKE TO DO NOW IS 
JUST SORT OF TAKE YOU THROUGH 
SOME OF THESE STEPS IN A LITTLE 
BIT GREATER DETAIL AND ASK 
SPECIFICALLY WHAT INITIATES 
THESE REORGANIZATIONS THAT WE 
SEE TAKING PLACE FOLLOWING STORE
DEPLETION, AND HOW DO THE 
CHANNELS AND THE STEM ONE 
SENSORS ACTUALLY FIND EACH 
OTHERS AT THESE JUNCTIONS AND 
WHAT LEADS TO THE OPENING OF THE
CHANNELS ONCE THEY FIND EACH 
OTHER?
SO STEM ONE IN ITS RESTING STATE
PROBABLY EXISTS AS A DIMER WHEN 
THE ER STORES ARE FULL AND 
CALCIUM IS BOUNDç TO THE EF HAND.
THIS IS STILL A LITTLE BIT 
DEBATABLE, BUT THERE IS EVIDENCE
THAT IT'S PROBABLY A DIMER, AND 
THERE IS ALSO EVIDENCE FROM A 
COUPLE OF SOURCES THAT FOLLOWING
STORE DEPLETION, THESE STEM ONE 
DIMERS LIG MAR RISE TO FORM 
HIGHER ORDER LIG MERS.
THE EVIDENCE FOR THAT WAS 
TWO-FOLD.
ONE WAS FROM A GROUP WHO STUDIED
THE ISOLATED FRAGMENT, THE 
LOOMAL FRAG CHLT OF STEM ONE IN 
VITRO AND FOUND THAT IN THE 
PRESENCE OF CALCIUM THAT THESE 
FRAGMENTS WERE MONO AMERICA BUT 
THE REMOVAL OF THE CALCIUM 
CAUSED THEM TO FORM 
[INDISCERNIBLE].
THIS WAS FOLLOWED BY A GROUP WHO
ATTACHED FRET PROBES TO THE 
INTERMINUS OF STEM FWHUN LIVING 
CELLS AND FOUND THAT STORE 
DEPLETION CAUSED AN INCREASE IN 
FRET INDICATING THE FORMATION OF
THESE HIGHER-ORDER LIG MERS.
SO BASED ON THESE RESULTS, WE 
THOUGHT IT AN IMPORTANT QUESTION
OF WHETHER THIS HIGHER ORDER OF 
LIG MER RYIZATION PLAYED A ROLE 
IN TRIGGERING IF CALCIUM ENTRY 
AND WHAT SORT OF A ROLE IT WOULD
PLAY.
WHAT WE DID WAS TO O TRY TO 
THINK OF A WAY TO LIG MA RISE 
STEM ONE INDEPENDENT OF CALCIUM 
DEPLETION OF THE ER.
WHAT WE DECIDED TO DO WAS TAKE 
ADVANTAGE OF HETEROu!Ko DIMER 
RYIZATION SYSTEM IN WHICH THE 
PROTEIN MODULES FRB AND FKBP CAN
BE CROSS LINKED WITH A MEMBRANE 
PERM YENT SMALL KUL RAP MYOSIN.
BY REPLACING WITH FRD, WE 
CREATED A SYSTEM IN WHICH WE 
COULD ADD AN ANALOG OF RIPE 
MYOSIN AND CREATE HIGHER ORDER 
OF LIG MERS INDEPENDENTLY OF 
STORE DEMREELGS AND THEN ASK 
WHAT EFFECT DOES THIS HA HAVE ON
THE CELL?
WHAT WE FOUND WAS THAT THE RAP 
LOG CAUSED THE FORMATION OF PUNK
TA OF A CHERRY-LABELED STEM ONE 
IN JERK OUT CELLS, AND THAT THIS
WAS ALSO ACCOMPANIED BY 
ACTIVATION OF THE ENDOGENOUS 
CRACK CHANNELS IN THESE JERK OUT
CELLS.
FROM THE MOMENT OF BREAKING IN 
TO ONE OF THESE CELLED THAT BEEN
PRETREATED, THE CURRENT, THE 
CRACK CURRENT IS ACTIVE SHOWN 
HERE IN THIS RECORDING.
WHEREAS IN UNTREATED CELLS 
EXPRESSING THESE TROE PROTEINS 
THERE'S THE USUAL TIME-DEPENDENT
DEPLAY FOLLOWING BREAK-IN AND 
STORE DEPLETION.
SHOWED THAT THE LIG MER 
RYIZATION OF STEM ONE IS A 
SUFFICIENT SIGNAL TO O TRIGGER 
THIS ENTIRE SEQUENCE OF EVENTS 
THAT WE SHOWED EARLIER LEADING 
FROM FIRST FORMATION OF STEM ONE
PUNK TA AND ALL THE WAY THROUGH 
TO THE ACTIVATION OF THE CRACK 
CHANNEL.
SO THE WAY ONE COULD THINK OF 
DELIG MARIAHIZATION OF O STEM 
ONE AS BEING A SWITCH THAT ONCE 
IT'S FLIPPED RELEASES AN AUTO 
SELF-ORGANIZING SEQUENCE OF 
EVENTS THAT CULMINATES IN CRACK 
CHANNEL ACTIVATION.
SO ALL YOU HAVE TO DO IS LIG MA 
RISE STEM AND EVERYTHING ELSE 
TAKES PLACE AUTOMATICALLY.
SO HOW DOES THIS WORK?
WELL, WE THINK THAT THE 
MECHANISM IS ACTUALLY A TWO-PART
DIFFUSION TRAP.
NOW THIS, IS BASED FIRST ON THE 
IDEA OR THE EVIDENCE THAT WE AND
MANY OTHER GROUPS HAVE FOUND 
THAT BOTH STEM AND AWRY IN THE 
CELL ARE FREELY DIFFUSABLE, HIGH
LIMO BILE.
YOU CAN SEE THAT IN A FRAP 
EXPERIMENT.
HERE'S A CROSS EXPRESSION AND 
WE'LL BLEACH OUT THIS PORTION OF
THE CELL INDICATE BID THE BAR 
AND YOU UH CAN SEE THAT IT GETS 
BREACHED AND THEN FLUORESCENCE 
RECOVERED OVER A PERIOD OF TENS 
OF SECONDS.
THE SAME THING TRUE WITH GFP IN 
THIS FINGERPRINT OF A HEX CELL 
THAT IF WE BLEACH IT THERE, 
OVERTIME THE FLO RENS RECOVERED 
SMOOTHLY.
THESE PROTEINS ARE FREE LIMO 
BILE, SO WHAT CAUSED STEM TWOUN 
BECOME TRAPPED AT THESE ER 
PLASMA MEMBRANE JUNCTIONS?v:
WELL, IT TURNS OUT THAT STEM ONE
EXPRESSED ON ITS OWN WITHOUT 
AWRY ONE IN THE HEX CELL WILL 
BECOME TRAPPED AND FORM PUNK TA 
ONCE STORES ARE DEPLETED, BUT 
THEN IF THAT LYSINE RICH DOMAIN 
OF ABOUT TEN AMINO ACIDS AT THE 
C TERMINUS IS DELETED FROM THE 
PROTEIN, STEM CAN NO LONGER 
ACCUMULATE ON ITS OWN.
THIS AND ORE RESULTS HAVE 
SUGGESTS THAT THIS POLLY BASIC 
DOMAIN IS IMPORTANT FOR THE 
TARGETS OF STEM ONE ON ITS OWN 
TO THE PLASMA MEMBRANE 
JUNCTIONS.
THERE'S EVIDENCE SFR SEVERAL 
GROUPS THAT THIS POLLY BASIC 
DOMAIN MAY INTERACT WITH 
NEGATIVELY CHARGES PHOSPHOLIPIDS
IN THE PLASMAt( MEMBRANE AND LEAD 
TO THE ENRICHMENT AND TRAPPING 
OF THESE PROTEINS HERE.
4eEé$AS BEEN EVIDENCE PRESENTED 
AGAINST THIS HYPOTHESIS, SO IT'S
POSSIBLE THAT THERE IS SOME 
OTHER INTERACTION INVOLVED AND 
THIS IS SOMETHING THAT STILL 
REMAINS TO BE FIRMLY 
ESTABLISHED.
IT IS TRUE THAT STEM ONE CAN 
ACCUMULATE HERE IN THE ABSENCE 
OF AWRY ONE.
THERE'S SOME SORT OF 
AWRY-INDEPENDENT INTERACTION 
THAT CREATES A CLUSTER OF STEM 
AT THE JUNCTION.
WHILE STEM CAN FORM THESE PUNK 
TA INTD PENT OF AWRY ONE, THE 
SAME IS NOT TRUE OF AWRY.
IF AWRY IS EXPRESSED ALONE IN A 
HEX CELL AND THEN THE STORES ARE
DEPLETED P IT WILL NOT FORM PUNK
TA.9
WE HAD ACCUMULATED SOME.óu EVIDENCE
THAT THE AWRY IS ACTUALLY, IT 
NEEDS STEM ONE TO 
THAT IT'S PROBABLY INTERACTING 
IN SOME WAY WITH STEM ONE TO DO 
THIS.
AT THIS POINT, WE BEGAN A 
COLLABORATION WITH SOME GUYS AT 
STANFORD.
WHAT YOUNG HAD BEEN DOING IS 
TAKING OFF ON A DISCOVERY MADE 
BY A GUY WHO'S NOW HERE AT NIH, 
WHO FOUND THAT THE CAR BOX XI 
TERMINUS OF STEM T CYTOSOLIC 
PORTION OF THIS PROTEIN WHEN 
EXPRESS BID ITSELF HAD THE 
ABILITY TO ACTIVATE CALCIUM 
INFLUX THROUGH CRACK CHANNELS, 
ANDç CHEN WAS WHITTLING DOWN THIS
CYTOSO LICK DOMAIN TO TRY TO 
FIND THE SMALLER POSSIBLE 
FRAGMENT KAIBç CAPABLE OF 
SOLICITING INFLUX.
HE FOUND A SEGMENT OF ABOUT A 
HUNDRED AMY KNOW ASEDES WHICH WE
CALL CAD WHICH WHEN P,PRESSED BY
ITSELF OR ALONG WITH AWRY WITH N
HEX CELLS CAUSED A RISE IN 
INTERCELLAR CALCIUM AND WHEN 
XCHLED WITH PATCHok CLAMP 
TECHNIQUE CAUSED ACTIVATION OF 
CRACK CURRENT INDICATED HERE AND
BY THESE RAMPS HERE.
AROUND THE SAME TIME, TWO OTHER 
GROUPS FOUND ESSENTIALLY THE 
SAME FRAGMENT.
THERE WAS A FRAGMENT CALLED SORE
AND ANOTHER GROUP FOUND A 
SIMILAR FRAGMENT CALLED CCB 9.
THERE SEEMS TO BE A BROAD 
AGREEMENT THAT THERE'S ABOUT 100
A
AMINO ACID SEGMENT THAT'S 
SUFFICIENT FOR ACTIVATION OF THE
CHANNELS.
WE FOUND THEfá EXPRESSION OF THIS 
CAD WITH AWRY CAUSED SAME -- 
WHEREAS WE WERE AT SIMILAR 
LEVELS OF EXPRESSION, WE FOUND 
THAT THE ENTIRE CAR BOX XI 
TERMINUS OF STEM HAD MINIMAL 
ACTIVITY.
ONLY WHEN GREATLY OVEREXPRESSED 
COULD WE GET ACTIVITY.
AN EXPLANATION FOR THIS, 
EVIDENCE THAT THIS CYTOSOL LICK 
FRAGMENT MAY BE FOLDED OVER ON 
ITSELF UNDER MOST CONDITIONS TO 
OBSCURE OR HIDE THE CAD DOMAIN 
AND IT'S ONLY AFTER VARIOUS 
TREATMENTS ARE DONE TO SUCH AS A
LIG MARIAHIZATION TO BRING THESE
TOGETHER THAT MIGHT CAUSE AN 
UNFOLDING THATok ENABLE THIS IS 
CAT DOMAIN FOR THIS ACTIVITY.
WE NEED TOWNS THE CHANGES OF THE
CAD DOMAIN TO THE CRACK CHANNEL.
WE WANTED TO KNOW HOW THIS 
ACTIVATION WORKS AND IT TURNS 
OUT THAT IT WORKS BY DIRECTLY 
BINDINGç TO AWRY.
WE CAN SEE THAT P IF WE EXPRESS 
YFP-LABELED CAD ALONE IN HEX 
CELLS, IT'S LARGELY CYTOE 
PLASMIC, BUT THAT WHEN 
COEXPRESSED WITH AWRY, IT 
BECOMES RECRUITED TO THE PLASMA 
MEMBRANE THROUGH SOME SORT OF 
BINDING REACTION.
ONE PIECE OF EVIDENCE COMES FROM
GST PULLDOWNS WITH A GST LABELED
CAD IS ABLE TO PULL DOWN HIS TAG
AWRY BUT NOT GST ALONE.
YOUNG DID SOME ADDITIONAL 
EXPERIMENTS TO SHOW THAT THE CAD
BINDS TO BOTH.
HE FOUND THAT THE FLAG TAGGED 
CAD PROTEIN CAN PULL DOWN THE N 
TERMINUS OF AWRY AND THE C 
TERMINUS OF AWRY BUT NOT THE 
TWO-THREE LOOP.
THERE SEEMS TO BE AN INTERACTION
WITH BOTH END OFTHS PROTEIN BUT 
NOT THE INTRACELLAR LOOP, AND I 
SHOULD JUST SAY THAT IN TERMS OF
THE TRAPPING OF THE CRACK 
CHANNEL IN PUNK TA, IT SEEMS 
THAT THE C TERMINUS PROBABLY 
PLAYS THE DOMINANT ROLE IN THAT 
TRUNCATING THE C TERMINUS 
PREVENTS THE PROTEIN FROM 
COLOCALIZING WITH STEM WHEREAS 
TRUNCATING THE N TERMINUS 
DOESN'T SEEM TO AFFECT IT BUT 
DOES PREVENT OPENING OF A 
CHANNEL.
SO ONE THING THAT THE CAD DOES 
NOT DO EVEN THOUGH IT ACTIVATES 
THE CHANNEL, IT DOES NOT 
APPARENTLY SUPPORT ENACT VEGAS 
ENACT VEGAS.
IT'S SUBJECT TO 
CALCIUM-DEPENDENT FEEDBACK 
INHIBITION THROUGH A FAST ENACT 
VEGAS PROCESS WHEREok CALCIUM 
ENTERING THE CELL BINDS TO SITES
WITHIN A FEW NANO METERS OF THE 
PORE.
THIS IS SHOWN HERE IN THIS 
EXPERIMENT USING WILD-TYPE STEM 
AND AWRY EXPRESS IN THE A HEX 
CELL.
WE CAN SEE THAT HYPER POE  
POLARIZING WHICH ACTIVATING.
WE DO THE SAME EXPERIMENT 
COEXPRESSING THE CAD FRAGM)nu 
AND AWRY ONE.
WE DON'T SEE ANY EVIDENCE OF 
ENACT
THERE'S A SMALL POE TENUATION OF
THE CURRENT DURING THESE HYPER 
POLARIZING PULSES.
THIS LED US TO CONSIDER WHAT WAS
MISSING IN CAD.
WHAT WERE WE MISSING THAT WOULD 
BE PRESENT IN WHOOILD WILD TYPE 
STEM THAT WOULD ALLOW ENACTç 
VEGAS?
SO WE DID UH A SEERDZ OF 
EXPERIMENT IN WHICH HE EXPRESSED
FIRST A TRUNCATED STEM ONE, 
TRUNK KATED AT THE END OF THE 
CAD REGION AND FOUND THAT JUST 
AS IN CELLS EXPRESSING ONLY THE 
CAD FRAGMENT ALONE, THERE WAS NO
ENACT
CURRENT, AND ADDING BACK 20 
AMINO ACIDS TO THIS STILL DID 
NOT RESTORE, BUT THE NEXT 20 
RESTORED A FULL ENACT VEGAS 
PROFILE.
WE CALL THIS 20 AMINO ACIDS HERE
T ENACT VEGAS DOMAIN OF STEM, 
AND IT WAS AN INTERESTING DOMAINñr
PARTLY BECAUSE OF A CLUSTER OF 
ACIDIC RESIDUES WE FOUND DURING 
NEAR TERMINUS.
BIENNIAL GI TO THE 
NEGATIVELY-CHARGED CALCIUM BOWL 
REGION OF POE TA IS I YUM 
CHANNELS, WE GUESSED THAT 
PERHAPS SOMETHING LIKE THIS 
MIGHT ACT AS A CALCIUM CENTER AN
SOME HOW PARTICIPANT IN ENACT 
VEGAS.
WE STARTED TO O NEUTRALIZE THESE
E RESIDUES AND SEE WHAT EFFECTS 
THEY WOULD HAVE.
IT TURN OUT THAT THERE IS A 
PRETTY GOOD CORRELATIONw3 BETWEEN 
NEW RALIZATION AND LOSS OF ENACT
VEGAS.
WE TAKE PAIRS OF ACIDIC RESIDUES
AND IF WE TAKE SIX OUT OF THE 
SEVEN AND NEUTRALIZE THOSE, 
QUESTION PLEATTY MUCH COMPLETELY
BLOCK ENACT VEGAS.
IT'S NOT AS SIMPLE AS JUST 
SAYING THAT -- AND THIS IS A, 
YOU KNOW, A DEFUSED LOCALIZED 
NEGATIVE CHARGE THAT DETERMINES 
INACTIVATION AND THEN CALCIUM 
OVERLAY EXPERIMENTS WE DID, IT 
WASN'T CLEAR THAT THERE WAS A 
PERFECT CORRELATION BETWEEN THE 
ABILITY TO BIND CALCIUM AND THE 
EXTENT OF INACTIVATION.
SO THIS IS STILL SOMETHING THAT 
WE NEED TO UNDERSTAND IS JUST 
HOW THIS REGION IS INVOLVED, BUT
IT'S CLEARLY IMPORTANT IN ONE OF
THE DETERMINANTS IN THIS 
PROCESS.
THIS MAKES US THINK THAT THE 
CRACK CHANNEL, ITSELF, CAN'T 
REALLY BE VIEWED AS JUST AWRY 
ONE IN THE PLASMA MEMBRANE WITH 
STEM ACTING AS A LIE  LIE BEGAN 
TO [INDISCERNIBLE].
IT'S PROBABLY BETTER VIEWED AS 
MOBILE SUB UNIT OF THE CHANNEL 
THAN AS A LIE BEGAN.
WHERE IS THE CALCIUM SENSOR?
THE BEST CANDIDATE IS ACTUALLY 
CALMODULIN.
CAME ABOUT INITIALLY FROMyM A 
BIOINFORMATIC PREDICTION THAT 
AWRY SOMEONE A CALMODULINE-BIND 
PROGRESS TEEN.
 PROTEIN.
PARK DISSECTED OUT THE DIFFERENT
PARTS OF THE ENTERMINUS TO 
ISOLATE A MINIMAL FRAG CHLT, 
68-91, JUST RIGHT ADJACENT TO 
THE INSERTION INTO THE PLASMA 
MEMBRANE WHERE CAM A WAS ABLE TO
PRECIPITATE THAT FRAGMENT OF THE
END TERMINUS IN THE PRESENCE OF 
CALCIUM BUT NOT IN THE ABSENCE 
ç
OF IT.
TO TRY TO UNDERSTAND WHAT 
FUNCTIONAL EFFECT THAT MIGHT 
HAVE ON INACTIVATION, WE MUTATED
THAT REGION.
FIRST, WE TOOK THAT 68-91 
SEQUENCE AND PUT IT ON A HELICAL
WHEEL TO SEE WHETHER THERE MIGHT
BE A HYDRO PHOBIC FACE OF THE 
HELIX THAT WOULD BE EXPECTED TO 
BIND CALMODULINE.
ON THIS FACE, WE PICKED SEVERAL 
RESIDUES FOR MUTATION.
WE TOOK THESE RESIDUES AND MADE 
CHARGED SUBSTITUTIONS.
THE SUBSTITUTIONS THAT WE MADE 
THAT PREVENTED CALMODULINE 
BINDING ALL GOD GOT RID OF FAST 
INACTIVATION AS YOU CAN SEE 
HERE.
WE TAKE SOME OF THESE RESIDUES 
AND FOUND SUBSTITUTIONS THAT 
PRESERVES CALMODULINE BINDING.
IT'S HARD FOR ME TO SEE, I THINK
IT'S Y 80 A AND Y 80 S INSTEAD 
OF Y 80 E.
WE FIND INACTIVATION NOT OLD Lh
PRESERVED BUT ACCELERATED 
RELATIVE TO WILD TYPE.
THERE SEEMS TO BE A GOOD 
CORRELATION BETWEEN MUTANTS THAT
COMBINE CALMODULINEw3 AND CALCIUM 
ACTIVATION AND MUTANTS THAT HAVE
LOST THAT ABILITY TO INACTIVATE.
OUR WORKING HYPOTHESIS THEN IS 
THATu! CALMODULINE BINDING TO THIS
REGION IS ONE OF THE EVENTS THAT
LEADS TO CALCIUM-DEPENDENT
INACTIVATION.
NOW, THERE'S A LOT MORE WORK TO 
BE DONE TO UNDERSTAND WHY WE GET
SOME OF THESE CHANGES AND WHAT 
ACTUALLY DETERMINED THE RATE AND
EXTENT OF THAT INACTIVATION 
PROCESS.
SO JUST TO SUMMARIZE, UH, THIS 
PART OF THE TALK, I THINK THE 
BASICALLY, THE UPSHOT IS THAT WE
CAN'T REALLY VIEW THE CRACK 
CHANNEL AS JUST A TETkO TRAq
AWRY ONE THAT'S ACTIVATED IN 
SORT OF ALY GAND-DEPENDENT 
FASHION IN THE ER MEMBRANE, BUT 
INSTEAD T CRACK CHANNELS SHOULD 
BE PROBABLY VIEWED AS PROTEIN 
COMPLEX CONSISTING OF CERTAIN 
NUMBER OF O STEM ONES, COMPLEXED
TO THE PROTEIN INTERACTING 
THROUGH THE CAD DOMAIN AND ALSO 
THROUGH THIS INACTIVATION DOMAIN
AS WELL AS BINDING OF CALCIUM 
CALMODULINE TO THE END TERMINUS.
I'M SURE THIS IS JUST SCRATCHING
THE SURFACE.
IN FACT, THERE'S EVIDENCE FROM 
WOK AND OTHERS THAT THERE ARE 
ADDITIONAL PROTEINS THAT ARE 
STARTING TO APPEAR THAT 
INTERACT, BIND, TO STEM AND/OR 
AWRY AND MAY INFLUENCE THE WAY 
IN WHICH THEY COMMUNICATE ANDç 
THE EFFICIENCY WITH WHICH THEY 
COMMUNICATE WITH EACH OTHER.
THIS IS ANOTHER AREA THAT'S JUST
GROWING AT THIS POINT AND IS 
SURE TO REVEAL SOME NEW 
SURPRISES IN THE FUTURE.
WHAT I WANT TO NOW JUST COVER IN
THE LAST PART OF THE TALK IS 
 BEEN RECENT
LY TRYING 
WHAT WE'VE)
TO UNDERSTAND, WHICH IS HOW DOES
STEM IN THE ER MEMBRANE TRAP AND
ACTIVATE AWRY ONE IN THE PLASMA 
MEMBRANE?
AND SPECIFICALLY, WHAT WE WANTED
TO KNOW WAS HOW MANY STEMS ARE 
NEEDED TO TRAP THE CHANNEL AT A 
MINIMUM, AND ARE THESE, IS THIS 
SUFFICIENT TO OPEN THE CHANNEL 
OR DO WE NEED MORE STEMS TO 
ACTUALLY OPEN THE CHANNEL?
AND AS WELL TO CAUSE 
INACTIVATION IN RESPONSE TO 
CALCIUM INFLUX.
THIS IS WHAT I'M GOING TO TELL 
YOU ABOUT.
THESE ARE EXPERIMENTS DONE BY 
PAUL HOOVER WHO'S A STU DENT IN 
THE LAP.
WHAT PAUL DECIDEDED TO DO WAS A 
FAIRLY SIMPLE EXPERIMENT BUT IT 
TURNS OUT TO BE PRETTY DIFFICULT
IN TERMS OF CARRYING OUT.
WHAT HE DECIDED TO DO WAS TO 
EXPRESS A CONSTANT AMOUNT OF 
STEM ONE END CELLS, AND, UH, TO 
EXPRESS A LOW AMOUNT OF AWRY OR 
HIGH AMOUNT OF AWRY, BUT WITH 
THE SAME AMOUNT OF STEM.
BE F ONE EXPRESSES -- ONE WOULD 
EXPECT THAT ALL OF THE CRACK 
CHANNELS AT THIS PUNK TA WOULD 
BE SATURATED WITH STEM AND WE'D 
GET SORT OF A MAXIMAL BINDING OF
STEM TO THE CHANNEL.
WHEREAS, IF AWRY IS IN EXCESS OF
STEM, AWRY WILL SORT OF DEPLETE 
THE FINITGç POOL OF STEM ONE 
MOLECULES SO THAT EACH CRACK 
CHANNEL THAT'S IMMOBILIZED OR 
TRAPPED AT THIS JUPGS WILL END 
UP BEING BOUND TO THE MINIMAL 
NUMBER OFsSTEMS THAT'S NEEDED TO
TRAP IT.
OKAY.
SO BY DOING THIS EXPERIMENT, BY 
EXPRESSING HIGHER AND HIGHER 
LEVELS OF AWRY AND LOOKING AT 
THE RATIOñr OF STEM TO AWRY IN 
THESE JUNCTIONS, ONE COULD 
ACTUALLY TRY TO MEASURE THAT 
MINIMAL BINDING NEEDED FOR 
TRAPPING.
SO TO DO THIS EXPERIMENT, HE 
EXPRESSED A GFP-LABELED AWRY 
ALONG WITH d8CHERRY-LABELED STEM, 
AND AS I SAID, STEM IS ALWAYS 
EXPRESSED AT AROUND THE SAME 
LEVEL, AND THIS IS VERIFIED BY 
SELECTING CELLS FOR ANALYSIS 
THAT HAVE A CONSTANT AMOUNT OF 
STEM ONE FLUORESCENCE AT THE 
PLASMA MEMBRANE AND NEAR IT.
AFTER STORE DEPLETION TO USEw3 
MICROSCOPY TO MEASURE THE RATIO 
OF GFP FLUORESCENCE TO CHERRY 
FLUORESCENCE IN THESE PUNK TA.
M
í
THIS CELL EXPRESSES A LOWsAMOUNT9LáD8ñúv
çL8 <DqDe!N
OF AWRY AND RELATIVELY HIGH 
STEM-TO-IE RYE RATIO.
WHEREAS THE STEM EXPRESSING HIGH
AWRY HAS A RELATIVELY LOW 
STEM-TO-AWRY RATIO FOP GET THESE
RATIO, WE MEASURE THE CHERRY TO 
GFP RATIO BUT HE DESIGNED A5a 
CONSTRUCT THAT'S BASIC LAY 
MODIFIED STEM PROTEIN WITH A 
SINGLE CHERFULLY THE LUMEN OF 
THE ER JUST AS IT IS HERE AND A 
SINGLE GFP GRAFTED ON TO THE 
CYTOPLASMIC REGION JUST AS IT 
WOULD BE EXPOSED ATTACHED TO 
AWRY.
THIS STEM ONE PROTEIN WAS 
MODIFIED BY DELETE LEETED THE 
CO
COIL-COIL DOMAIN.
ONE CAN PLAYED OUT CELLS 
EXPRESSING THIS CONSTRUCT IN THE
SAME FIELD OF VIEW TOGETHER WITH
CELLSCñryMEXPRESSING THESE CON 
TRUKTDS AND VERY SIMPLY IDENTIFY
THE CALL BRAY TOR CELL BIS THE 
FACT THEY DON'T FORM PUNK TA.
HERE'S THE RED-GREEN OVERLAY OF 
THE CELL, ER LOCALIZED AND THAT 
CALL BRAY TOR CONSTRUCT GIVES A 
CONSTANT RATIO VALUE WHICH 
CORRESPONDS TO ONE-TO-ONE.
WEç CAN USE THESE TO CALIBRATE 
THESE EXPERIMENTS TO GET A 
STEM-TO-AWRY RATIO.
HE DID THIS EXPERIMENT FAR 
NUMBER OF CELLS EXPRESSING 
DIFFERENT AMOUNT OF STEM AND 
AWRY.
THIS IS THE RESULT.
SO AS ONE INCREASES THE AMOUNT 
OF AWRY EXPRESS IN THE THE CELL 
RELATIVE TO STEM, THAT 
STEM-TO-AWRY RATIO IN THE PUNK 
TA DECLINES NOT TO ZERO BUT TO A
KONS TRANT SFAL VALUE.
THIS WHICH WHAT WAS PREDICTED 
THAT THIS KONS ABOUT THE VALUE 
WOULD INDICATE THE MINIMAL 
STOICHIOMETRY THAT'S NECESSARY 
TO TRAP THE CHANNELS.
I SHOULDd8 SAY THAT THE RED DOTS 
ARE EXPERIMENTS IN WHICH STEM 
ONE WAS HELD AT A CONSTANT LEVEL
AND ONLY AWRY WAS CHANGED, AND 
IN SOME EXPERIMENTS HE ACTUALLY 
ALLOWED BOTH PROTEINS TO VARY, 
BUT NOT GOT THE SAME RESULT, 
WHICH IS IMPORTANT BECAUSE IT 
INDICATES THAT THIS FLA TOE 
LEVEL IS NOT SIMPLY DETERMINED 
BY THE LEVEL OF AWRY, BUT REALLY
THE RATIO BETWEEN THE TWO 
PROTEINS THAT DETERMINES THIS 
LEVEL.
NOW, THAT PLATEAUñr OCCURS AT 
ABOUT.3 STEMS P PER AWRY.
WE WANTED TO THINK ABOUT WHAT 
SOURCES OF ERROR COULD CREEP 
INTO THIS.
ONE OF THE SOURCES WAS ACTUALLY 
THE FACT THAT IN THESE PUNK TA, 
WE DON'T REALLY KNOW HOW MUCH 
FREE SPACE THERE ISok IN-BETWEEN 
THESE CHANNELS THAT ARE TRAPPED.
ONE COULD IMAGINE THAT 
FLUORESCENT CHANNELS ARE 
DIFFUSING THROUGH THESE PUNK TA 
LEADING TO A HIGH GREEN 
BACKGROUND THAT WOULD TEND TO 
DEPRESS THE STEM-TO-AWRY RATIO 
THAT WE MEASURE.
WE TRY TAD FEW WAYS OF TRYING TO
MEASURE THIS EXACTLY AND FAILED,
SO WE DECIDED WE COULD JUST 
ESTIMATE THE MAXIMUM POSSIBLE 
ERROR THAT WOULD ARISE FROM THIS
SORT OF BACKGROUND WHICH WOULD 
BE ASSUMING THAT THE FREE AWRY 
IN THESE PUNK TA WAS ACTUAL THE 
DENSITY OF FREE AWRY WAS EQUAL 
TO THE FREE AWRY OUTSIDE THE 
PUNK TA.
WE KNOW THAT IT CAN'T BE 
ENTIRELY CORRECT BECAUSE THERE'S
SOME SPACE TAKEN UP BY THE 
LABELED BY THE BOUND AWRY, BUT 
THIS WOULD INDICATE A MAXIMUM 
POSSIBLE ERROR FROM THIS 
BACKGROUND.
WHEN WE APPLY THAT BACK GROUND 
CORRECTION, WE GET THESE POINTS 
INDICATED BY THE OPEN SYMBOLS.
THIS SORT OF INDICATES AN UPPER 
LIMIT TO THIS ESTIMATE T STEM-TO
O-AWRYç RATIO ABOUT.6.
BETWEEN.3 AND.6 IS WHERE WE 
WOULD PLACE THE RESULT, AND 
BASED ON THE FACT THAT THE CRACK
CHANNEL IS THOUGHT TO BE BEyM A TE
TRA MER OF AWRY, THIS WOULD 
IMPLY THAT ONE TO TWO STEMS 
BINDING TO THAT TE TRA MER IS 
SUFFICIENT TO TRAP THE CHANNEL 
AT THE ER PLASMA MEMBRANE 
JUNCTION.
SO THE QUESTION IS, IS THIS 
ENOUGH?
IT TRAPS THE CHANNEL, BUT THIS 
MIM MINIMAL BINDING ENOUGH TO 
ACTIVATE THE CHANNEL?
I THINK IT'S HELPFUL TO CONSIDER
A COUPLE OF SITUATIONS.
IF THE MINIMAL BINDING IS ABLE 
TO ACTIVATE THE CHANNEL, THEN IF
CURRENT IN THE CELL AS A 
FUNCTION OF AWRY EXPRESSION, YOU
EXPECT THAT CURRENT TO GET 
BIGGER AND BIGGER AND BIGGERER 
AS YOU ADD MORE CHANNELS AND 
VENTLY TO PLATEAU AT A MAXIMUM 
WHICH IS SET REALLY BY THE 
AVAILABLE SUPPLY OF STEM ONE IN 
WHICH ALL THE CHANNELS WOULD BE 
IN THIS MINIMALLY-BOUND STATE 
BUT ACTIVE.
WHEREAS THIS MINIMALLY BOUND 
STATE DOES NOT OPEN THEN ONE 
EXPECTS A RISE IN CURRENT WHEN 
MOST OF THE CHANNELS AREçç 
SATURATED WITH STEM, BUT THEN AT
SOME POINT THE CURRENT SHOULD 
DROP OFF TO A MINIMAL LEVEL WHEN
THESE CLOSED CHANNELS ARE 
FORMED.(
EXPERIMENT MONITORING CRACK 
CURRENT DENSITY AS A FUNCTION OF
AWRY EXPRESSION ON A WHOLE 
NUMBER OF CELLS AND FOUND 
EVIDENCE THAT THIS MINIMAL 
STOICHIOMETRY IS NOT SUFFICIENT 
TO ACTIVATE.
AS YOU ADD MORE CHANNELS TO THE 
CELL, YOU GET MORE AND MORE 
CURRENT, BUT THEN AT A CERTAIN 
POINT THERE'S THIS PRECIPITOUS 
DROP NAUF CURRENT LEVEL DOWN TO 
ESSENTIALLY ZERO CURRENT AT THE 
VERY HIGHEST LEVELS OF AWRY.
SO THIS IS CONSISTENT WITH THE 
IDEA THAT, UH, THAT A HIGH LEVEL
OF BINDING TO STEM ONE ACTIVATES
A CHANNEL BUT THAT THE MINIMAL 
LEVEL OF BINDING IS NOT 
SUFFICIENT, AND WE WANTED TO 
KNOW, SORT OF, WHERE THIS CUTOFF
WAS, THIS QUITE STEEP CUTOFF.
IN A TERM NUMBER OF CELLS PAUL 
WAS ABLE TO MEASURE THE g#
STEM-TO-AWRY RATIO IN THE PUNK 
TA, AND IN THESE RED SYMBOLS 
HERE.
FOUND IN THESEçó CELLS THERE WAS 
AN AVERAGE OF ABOUT 1.8 STEMS 
PER AWRY.
WHEN YOU MULTIBLIE MROOI BY FOUR
YOU GET SEVEN OR SO STEMS PER 
AWRY, STEMS PER CHANNEL IN THIS 
REGION WHERE THE SKIRT IS 
STARTING TO DROP OFF.
UNDER CONDITIONS WHERE THERE'S 
AN EXCESS OF STEM, WHEN THERE'S 
ABOUT EIGHT STEMS AVAILABLE TO 
BIND TO THE CHANNEL, CHANNELS 
ARE MAXIMALLY ACTIVE, BUT ONCE 
THE RATIO OF STEMS PER CHANNEL 
FOLLOWS BELOW THIS LEVEL T 
CHANNEL ACTIVITY DROPS VERY 
RAPIDLY.
SO WE WANTED TO ACTUALLY -- SO 
NOW I'M GOING TO TELL YOU A 
LITTLE ABOUT THESE 
CHARACTERISTIC OF THE CURRENTS 
THAT HE FOUND IN THIS REGION OF 
AWRY EXPRESSION BECAUSE IT'S 
SORT OF INTERESTING AND IT GIVES
EVIDENCE FOR ACTIVITY OF 
CHANNELS THAT HAVE LESS THAN 
EIGHT STEMS PER AWRY, LESS THAN 
EIGHT STEMS BOUND PER CHANNEL.
WHAT WE'RE SHOWINGsHERE ARE THE 
INDIVIDUAL RESPONSES TO HYPER 
POLARIZING PULSES OF CELLS 
EXPRESSING INCREASING AMOUNTS OF
AWRY.
q
I HOPE YOU CAN SEE THAT AS"y
INCREASE THE AMOUNT OF AWRY, THE
LEVEL OF CALCIUM-DEPENDENT 
INACTIVATION DECLINES AND AT THE
HIGHEST LEVEL IS REPLACED BY 
THIS POE TEN SHAGS OF THE 
CURRENT.
BY MEASURING THE CURRENT 
REMAINING AT THE END OF EACH 
STEP AND PLOTTING THAT, YOU CAN 
 REGION 
SEE THAT IN THE LOW AWRYX
WHERE THERE'S PLENTY OF STEM 
AROUND, THESE CHANNELS ARE ALL 
INACTIVATING, ABOUT HALF AIR, 
BOUT 50%.
THEN AS WE REACH THIS THRESHOLD 
THERE,'S A LOSS OF INACTIVATION 
AND THEN IT'S REPLACED BY THIS 
POE TEN SHAGS PHENOMENON WHERE 
THE CURRENTS GET BIGGER 
OVERTIME.
TO TRY TO RELATE THIS TO THE 
ABILITY OF THE CHANNELS TO 
ACTIVATE, WE TOOK THE 
MEASUREMENTS OF PEAK CURRENT FOR
EACH OF THESE CELLS AND 
COLOR-CODED EACH E POINT 
ACCORDING TO THE LEVEL OF 
INACTIVATION OR POE TEN SHAGS 
ALONG THIS SCALE.
OVER THIS ENTIRE REGION OF AWRY 
EXPRESSION,ER KURNTSS ARE ALL 
INACTIVATING CURRENTS, BUT AS WE
EXCEED THAT
CURRENTS START TO DROP THIS, IS 
ALSO THE REGION WHERE THE 
CALCIUM-DEPENDENT INACTIVATION 
STARTS TO DECLINE AND IT 
ACTUALLY GETS TO A POINT OUT 
HERE AT VERY HIGH AWRY LEVEL 
WHERE IS THE CURRENTS ARE 
ACTUALLY POTENTIATING.
WE KNOW FL THIS KIND OF 
EXPERIMENT THAT THESE CHANNELS 
OUT HERE AT HIGH AWRY HAVE TO BE
LIE GANDED TO A DIFFERENT NUMBER
OF STEMS THAN THESE CHANNELS 
BECAUSE THEY HAVE A DIFFERENT 
PROPERTY.çGANDED TO A DIFFERENT NUMBER 
OF STEMS THAN THESE CHANNELS 
BECAUSE THEY HAVE A DIFFERENT 
PROPERTY.
THERE'S NO WAY YOU CAN GENERATE 
THAT TO A CHANNEL THAT WAS 
LIGANDED TO THE SAME NUMBER OVER
HERE.
THAT THIS GIVES US DIRECT EFTD 
FOR A DIFFERENT POPULATION OF 
CHANNELS THAT'S LIMITED TO A 
NUMBER -- SMALLER NUMBER OF 
STEMS.
WE WANTED TO UNDERSTAND, THEN 
WHASHG IS THE RELATIONSHIP 
BETWEEN THE NUMBER OF STEMS 
BOUND AND THE ACTIVATION AND THE
ACTIVATION OF THE53
AND IT LOOKS LIKE THIS 
RELATIONSHIP IS HIGHLY 
COOPERATIVE, BUT THERE'S QUITE A
SHARP DROP-OFF WITH JUST A 
TWOFOLD INCREASE IN AWRYzV LEVELS.
THERE'S A VERY LARGE DROOP DROP 
IN THE AMOUNT OF CURRENT.
SO SORT OF DESCRIBE THIS 
COOPERATIVITY AS A FIRST STEP WE
z MODEL THIS USING A MANO
T:GJ
MODEL, NWC-TYPE MODEL WHICH WAS 
CHOSEN REALLY BECAUSE OF ITS 
SIMPLICITYu! IN THIS CASE.
THIS MODEL BASICALLY JUST 
ASSUMED TWO CONFIRMATIONS OF THE
PROTEIN; A CLOSED CONFIRMATION 
AND AN OPEN CONFIRMATION.
AND THE STEM BINDS TO FOUR 
BINDING SITES AND WHEN IT'S 
BOUND TO EACH SITE, IT'S 
INDICATE BADE BLACK SQUARE OR 
STEM STABILIZES THE OPEN STATE 
BY A CONSTANT FACTOR, F.
AND SO THERE'S A COOPERATIVITY 
IN OPENING THAT'S CONFER BID THE
STABLELIZATION OF THE OPEN STATE
BY STEM ONE BINDING.
SO THIS MODEL WAS FIT TO OUR 
DATA OF CRACK CURRENT DENSITY 
VERSUS AWRY WITH A NUMBER OF 
CONSTRAINTS THAT ACTUALLY VERY 
HIGHLY LIMITED THE FLEXIBILITY 
OF THE MODEL.
FLUSH SFLOUSH THE FIRST THING IS
THAT WE KNOW THAT THE 
SPONTANEOUS OPENING RATE OF THE 
CHANNEL INDICATED BY L, IS VERY,
VERY LOW, AND WE CHOSE VALUES OF
L OF TONE THE MINUS FOURTH OR 
BELOW, AND YET, WE KNOW FROM 
NOISE ANALYSIS OF THE CURRENT 
THAT THE PROBABILITY T OPEN 
POSSIBILITY OF THE CHANNEL IN 
ITS FULLY ACTIVATED STATE IS 
ABOUT.8, AND THAT WOULD 
CORRESPOND TO THIS STATE IN THE 
MODEL.
SO WE KNOW BASED ON THIS THAT IT
CONSTRAINS THE VALUE OF F THAT, 
BINDING STABLELIZATION 
PARAMETER.
THE ONLY OTHER FREE PARAMETERS 
ARE THE TOTAL AMOUNT OF STEM 
THAT WE ASSUME IN THE MODEL AND 
THIS AMOUNT OF STEM WAS SET TO 
3.2 BECAUSE THAT SETS THE 
POSITION OF THIS PEAK ALONG THE 
AWRY AXIS.
FINALLY T ASSOCIATION CONSTANT 
FOR STEM ONE BINDING WAS SET TO 
100, AND THAT WAS TO TRY TO FIT 
THE STEEPNESS OF THIS PARTç OF 
THE CURVE, AND FINALLY, THERE 
WAS A BINDING COOPERATIVITY 
FACTOR IN A STANDARD MWC MODELmy 
IT'S GENERALLY ASSUMED THAT THE 
BIND PROGRESS SAYS IS 
NON-COOPERATIVE AND INDEPENDENT.
THE ONLY COOPERATIVITY COMES 
FROM STABILIZING THE OPENING 
TRANSITION.
WHEN WEç TRIED THAT, WE WERE 
UNABLE TO GET A GOOD FIT FOR 
THIS PART OF THE CURVE, AND IT 
ALSO DID NOT PREDICT THE 
PARTIALLY LIGANDEDCñWNN2i[e0vh
BEING THE DOMINANT STATES OUT IN
THIS REGION OF FOUR TO SEVEN 
AWRY UNITS THAT I SHOWED IN THE 
PREVIOUS SLIDE.
IN ORDER TO PREVENT THE FULLY 
LIGANDED STATE FROM DOMINATING 
IN THIS REGION, WE NEEDED TO 
INTRODUCEOh
COOPERATIVITY OF STEM BINDING 
USING A FACTOR OF.5 FOR EACH OF 
THE STEM BINDING REACTIONS.
ONCE WE DID THIS, WE GET A 
FAIRLY GOOD FIT TO THE cu'4áB&L 
SHAPE OF THIS CURVE.
THE MODEL CAN PREDICT WHAT THE 
DIFFERENT SPECIES ARE AS A 
FUNCTION OF AWRY, AND WE CAN SEE
PARTICULARLY AS WE
HIGH AWRY LEVEL THE FULLY 
LIGANDED STATE REALLY DROPPED 
OFF SUDDENLY.
IF WE ZOOM IN ON JUST THAT 
REGION WHERE WE FOUND THE LOSS 
OF CALCIUM-DEPENDENT 
INACTIVATION AND BLOW THAT UP, 
WE CAN SEE THAT IN THIS FOUR TO 
SEVEN REGION, THAT FULLY 
LIGANDED STATE IS REALLY NEAR 
ZERO AND WHAT WE'VE REPLACED IT 
WITH ARE THESE TRIPLY-BOUND AND 
DOUBLY-BOUND CHANNELS.
SO OUR -- THIS IS OBVIOUSLY NOT 
A UNIQUE DESCRIPTION OF THE 
CHANNEL GAINING MECHANISM, BUT 
IT'S A STARTING POINTíR FOR TRYING
TO UNDERSTAND WHAT'S GOING ON 
BETWEEN BINDING AND ACTIVATION 
AND INACTIVATION.
AND IT MAKES A NUMBER OF 
TESTABLE PREDICTIONS.
ONE PREDICTION THAT IT MAKES IS 
IT PREDICTS THE OPEN 
PROBABILITIES OF THE DIFFERENT 
STATES, WHICH WE CAN NOW TEST 
USI
USING CHANNELS WITH DEFINED 
NUMBERS OF BINDING SITES.
IT ALSO PREDICTS THAT THE 
INACTIVATION PROPERTIES OF THE 
CURRENTS SHOULD DEPEND ON WHICH 
STATE, HOW MANY STEMS ARE BOUND 
TO THE CHANNEL AND WE CAN NOW 
TEST THAT AS WELL.
AND FINAL LYRIC IT MAKES A 
PREDICTION THAT THE BINDING OF 
STEM TO THE CHANNEL IS 
NEGATIVELY COOPERATIVE, AND THIS
INITIALLY WAS A LITTLE BIT OF A 
SURPRISE TO US, BUT WE -- IT'S 
QUITE POSSIBLE THAT THE BINDING 
OF STEM TO THE CHANNEL EITHER 
INTRODUCES CHANGES IN THE 
CHANNEL OR PERHAPS JUST 
EXPERIENCES STESHG CONSTRAINTSv: 
THAT MAKE IT MORE DIFFICULT TO 
BIND EACH SUCCESS SIEVE STOEM 
THAT CONFINED SPACE BENEATH EACH
CHANNEL.
THIS IS, AGAIN, SOMETHING WE'RE 
IN THE PROCESS OF TESTING 
THROUGH DIRECT BINDING 
EXPERIMENTS TO SEE WHETHER THAT 
BINDING IS NEGATIVELY 
COOPERATIVE.
SO THIS IS JUST INTENDED AS A 
SORT OF A TEASER TO SEE, YOU 
KNOW, HOW FARsWE'VE COME AND 
WHERE WE HAVE TO GO, AND I THINK
THERE'S GOING TO BE A LOT MORE 
TO DO TO TRY TO UNDERSTAND MORE 
DETAIL HOW THE BINDING OF STEM 
TO THE CHANNEL IS ACTUALLY ABLE 
TO O CAUSE IT TO OPEN AND ALLOW 
IT TO INACTIVATE.
SO I'LL STOP THERE AND JUST 
ACKNOWLEDGE SOME OF THE PEOPLEç 
THAT REALLY SPEAR HEADED THIS 
WORK.
REEN A&M -- REENA, AND MINNIE.
FRANKLIN MULLINS.
PAUL HOOVER, I SAID WAS THE MD 
Ph.D. STUDENT WHO JUST FINISHED 
HIS THESIS AND DID ALL THE WORK 
ON THE STOICHIOMETRY THAT I 
TALKED ABOUT.
FINALLY OUR COLLABORATORS AT 
STANFORD CONTRIBUTED A LOT TO 
THE CAD PART OF THE STORY.
STEFON AND TOM AT HARVARD DID 
SOME SINGLE PARTICLE EM WHICH I 
DIDN'T HAVE TIME TO TALK ABOUT 
BUT ISD PART OF THIS STUDY.
I'LL STOP THERE AND TAKE 
QUESTION PS.
THANK PS.
[APPLAUSE]
>> [LOW AUDIO].
>> THAT'S A GOOD QUESTION.
SO THE PREDICTION WOULD BE IF 
YOU ALLOWED -- IF STEM WERE 
ACCUMULATING VERY SLOWLY IN THE 
PRESENCE OF AN EXCESS OF AWRY, 
YOU WOULD EXPECT THESE CHANNELS 
TO SLOWLY GO THROUGH A SERIES OF
STATES FROM MINIMALLY BOUND TO 
MAXIMALLY BOUND AND YOU SHOULD 
BE ABLE TO FOLLOW THAT WITH 
INACTIVATION.
WE DON'T KNOW WHAT THE ANSWER TO
THAT IS.
I MEAN, THAT'S AN EXPERIMENT 
THAT WE'RE TRYING TO SET UP TO 
DO.
ALLñr WE KNOW IS THAT WE DID AN 
EXPERIMENT UNWITTINGLY DID AN 
EXPERIMENT WITH JERK OUT T CELLS
YEARS AGO LOOKING AT FAST 
INACTIVATION WHERE WE FOLLOWED 
IT AS THE CRACK CURRENT 
DEVELOPED DURING STORE DEPLETION
AND THEN INACTIVATED DURING 
STORE REMREEGS.
UNDER THOSE CONDITIONS, THE 
CURRENT, THE INACTIVATION LEVEL 
WAS PRETTY CONSTANT THROUGHOUT.
SO AT LEAST IN THE ENDOGENOUS 
CELLS, YOU KNOW, JERK OUT T 
CELLS WITH WHICH AMOUND OF STEM 
AND AWRY, IT DOESN'T SEEM TO 
SPEND A LOT OF TIME IN A STATE 
WHERE THE CHANNELS ARE SUB 
MAXIMALLY BOUND WITH STEM.
>> [LOW AUDIO].
>> YEAH.
>> [LOW AUDIO].
>> THAT'S RIGHT.
THAT'S RIGHT.
SO WE DON'T -- SO I MEAN THE 
BOTTOM LINE IS THAT THERE COULD 
BE SOME INTERESTING SITUATIONS 
DEPENDING ON HOW A CELL 
EXPRESSES AWRY WHERE IT MIGHT 
FAVOR THE FORMATION OF THESE 
OTHER STATES OR NOT.
ALL I CAN SAY IN THE JERK OUT T 
CELL T CHANNELS ALWAYS SEEM TO 
BE MAXIMALLY BOUND WITH STEM, 
AND WE WOULD EXPECT, YOU KNOW --
>> [LOW AUDIO].
>> YEAH.
SO YOU MIGHT HAVE MORE OF A 
CHANGE THERE.
>> [LOW AUDIO].ç
>> SO THE FIRST QUESTION IS, 
COULD HOW DOES STEM ACTUALLY 
MAKE THE CONNECTION WITH AWRY 
AND COULD THERE BE OTHER THINGS 
INVOLVED?
AND, UM, THERE CERTAINLY COULD 
BE OTHER PROTEINS INVOLVED.
IN FACT, QAWK, I THINK I 
MENTIONED PULLED OUT A PROTEIN 
CALLED CRACKER 2 A AND B, WHICH 
BINDS TO BOTH STEM AND AWRY, AND
IN THEIR EXPERIMENTS WHEN THEY 
KNOCKED THAT PROTEIN DOWN, THEY 
COULD DISRUPT THE COMMUNICATION 
BETWEEN STEM AND AWRY.
IT HASN'T BEEN SHOWN BY OTHER 
PEOPLE YET, AND IN FACT, PATRICK
HOGAN HAS DONE EXPERIMENTS USING
A YEAST SYSTEM WHERE IT'S PRETTY
MUCH A REKONS INSTITUTED SYSTEM 
WHERE HE CAN GET ACTIVATION OF 
THE CRACK CHANNEL WITH 
RECONSTITUTED OR PURIFIED STEM 
AND AWRYç PROTEINS WITHOUT THIS 
OTHER PROTEIN.
SO IT'S NOT EXACTLY CLEAR, YOU 
KNOW, WHETHER OR NOT THERE COULD
BE OTHER PROTEIN.
IT WOULDN'T SURPRISE ME IF OTHER
PREE PROE TEENS CAN MODULATE 
THAT INTERACTION, BUT FOR THE 
TIME BEING, I THINK THE SIMPLEST
MODEL SUPPORTED BY HOGAN'S DATA 
AS WELL ASñr OURS IS THAT IT'S 
SIMPLY THE DIFFUSION TRAP THAT 
ONCE STEM ACCUMULATES AS THESE 
JUNCTIONS IT'S SITTING THERE 
PRETEND PRESENTING A LIGAND FOR 
THE CRACK CHANNELS.
SO ONCE AWRY DIFFUSIONS JUST 
RANDOM LAY CROSS THAT JUNCTION, 
IT WILL BIND
ACCUMULATE OVER A CERTAIN TIME 
l!x÷> WE'RE 
PERIOD, AND THAT'S HOW
TREATING IT NOW.
AND THAT CAN BE TESTED, YOU 
KNOW, AND OBVIOUSLY SHOULD BE 
TESTED FURTHER BUT THAT'S OUR 
WORKING HYPOTHESIS.
AS FAR AS THE NUMBER OF 
PROTEINS, THESE PUNK TA THAT 
WE'RE SHOWING HERE, IT'S 
IMPORTANT TO REMEMBER THAT THIS 
IS Aw3 HIGHLY-OR THE OFFICIAL 
SITUATION WHERE WE'RE 
OVEREXPRESSING STEM AND AWRY, 
AND, UM, ACTUALLY, UM, MIKE 
CAHALAN IN AN EARLIER PAPER USED
THE ANTIBODY TO LOOK AT WHAT THE
ENDOGENOUS FAUNG IN A VARIETY OF
CELLS WOULD LOOK LIKE.
IT'S NOT NEARLY AS BEAUTIFUL AND
CLEARLY LINEjOd LYNNUATED AS THESE 
PUNK TA THAT WE'RE SEEING.
I THINK IT MEANS THAT IT'S VERY 
LIKELY THAT THESE PUNK TA ARE 
TELLING US ABOUT THE 
COMMUNICATION BETWEEN THEmy 
PROTEINS, BUT IT'S HIGHLY 
UNLIKELY THAT THE NATIVE PUNK TA
WOULD BE AS LARGE AND INVOLVE AS
MANY PROTEINS AS WHAT WE'RE 
SEEING HERE.
THIS IS PROBABLY ONE íRHUNS TO 
MAYBE THOUSANDS OF PROTEINS IN A
PUNK TA AND I'D BE SURPRISED IF 
THAT WERE THE CASE.
IT'S POSSIBLE THAT THERE IS SOME
ADD COOPERATIVITY FROM 
CLUSTERING THESE IN LIMITED 
AREAS AND THERE'S REALLY NO 
EVIDENCE FOR OR AGAINST AT THIS 
POINT, BUT IT'S CERTAINLY A 
POSSIBILITY.ç
>> [LOW AUDIO].
>> YEAH.
WELL THAT'S INTERESTING.
THE CALCIUM CERTAINLY GETS TAKEN
BACK UP INTO THE STORES AND 
THERE ARE STUDIESLPÑ SHOWING THAT 
IT SEEMS TO BE MORE EFFECTIVE AT
ER NEAR THE PLASMA MEMBRANE 
JUNCTIONS YOU MIGHT EXPECT AS TO
WHETHER THAT'S GUIDED BY AN 
ACCUMULATION OF CALCIUM PUMPS AT
THOSE LOCATIONS, I THINK IT'S 
NOT AS CLEAR.
WE DID SOME EXPERIMENTS EARLY ON
USING A FLUORESCENTLY-LABELED 
[INDISCERNIBLE] AS A PROBE AND 
FOUND THAT BASICALLY IT DID NOT 
SEEM TO CONCENTRATE AT THESE 
PLACES.
IT WAS PRETTY MUCH THROUGHOUT 
THEç ER, BUT -- 
>> [LOW AUDIO].
>> THE FAST INACTIVATION.
THE FACT IS THAT IT'S NOT KNOWN,
SO THE ONLY WAY TO REALLY AT 
DRESS THAT WOULD BE, YOU KNOW, 
TO, FOR EXAMPLE, EXPRESS A 
MUTANT STEM OR MUTANT AWRY, A 
KNOCKIN' MOUSE OR SOMETHING LIKE
SOME SYSTEM LIKE THAT WHERE YOU 
COULD THEN ASSAY CELL FUNCTION.
>> [LOW AUDIO] ..
>> [LOW AUDIO] .
>> MM-HMM.
>> [LOW AUDIO] ..
>> [LOW AUDIO] .
>> WELL -- YEAH.
>> [LOW AUDIO].
>> WE HAVEN'T LOOKED AT THAT.
I MEAN, IT'S POSSIBLE THAT IT'S 
PLAYING A ROLE IN MODULATING THE
INTERACTION OF STEM AND AWRY6d
DURING, YOU KNOW T DEVELOPMENT 
OF THE CURRENT.
I THINK WHAT WE'RE DOING NOW IS 
TRYING TO EXPRESS THESE MUTANT 
FORMS AGAINST A NULL BACKGROUND 
OF STEM AND AWRY, AND TO LOOK AT
CELL FUNCTION IN THESE CELLS TO 
SEE IF THERE'S SOMETHING ABOUT 
FAST INACTIVATION, YOU KNOW, 
EITHER ENHANCED OR INHIBITS 
PARTICULAR FUNCTIONS.
BUT I THINK OTHER THAN THAT, UM,
YOU KNOW, IT'S USUALLY, I MEAN 
THIS IS TRUE FOR 
CALCIUM-DEPENDENT INACTIVATION 
OF MANY TYPES OF CHANNELS.
IT'S SOMETHING THAT IS VERY 
REPRODUCIBLE, IT'S VERY COMMON, 
BUT IN VERY FEW CASES, YOU KNOW,
CAN IT BE SHOWN THAT I*PVçÑ HAS A 
PARTICULAR FUNCTION 
PHYSIOLOGICALLY.
YOU REALLY WANT TO KNOW THAT IF 
YOU DON'T WANT IT TO BE JUST A 
BIOPHYSICAL CURIOSITY.
>> [LOW AUDIO].
[APPLAUSE]
>> THANK YOU.
[END OF PROGRAM.]çóON