>> WELCOME, EVERYBODY. I'M PLEASED TO ACTUALLY LOOK OUT AND SEE A PRETTY DECENT TURNOUT CONSIDERING IT'S THE MONDAY BEFORE CHRISTMAS, SO THANK YOU ALL FOR COMING AND STAYING AT NIH. IT'S A PLEASURE TO INTRODUCE DEJIAN REN. WE GO QUITE A WAYS BACK. BUT I'LL GET TO THAT IN A MINUTE. HE GOT HIS PH.D. IN BUFFALO WITH LINDA HALL, WHO TAUGHT MY FIRST ION CHANNEL CLASS THAT I TOOK, BEFORE OF MOVING TO BUFFALO, AND THEN MOVED TO DAVID CLATHAM'S LAB AT CHILDREN'S HOSPITAL IN BOSTON, WHERE HE WORKED IN ONE -- ONE OF THE PROJECTS HE WORKED ON WAS TO ISOLATE NACHBACK, A BACTERIAL SODIUM CHANNEL HOMOLOGUE, AND WE ACTUALLY TOGETHER FOR A WHILE THEN, TRYING TO MAKE 2D OF THAT PROTEIN, UNSUCCESSFULLY OBVIOUSLY IN RETROSPECT, BUT THAT WAS KIND OF FUN, SO WE'VE KIND OF BEEN TRACKING EACH OTHER SINCE BECAUSE WE BOTH GOT INTERESTED IN LYSOSOMES. HE MOVED AROUND THE SAME TIME I MOVE OFFED HERE, HE MOVED TO PENN IN THE BIOLOGY DEPARTMENT AND HAS BEEN THERE NOW THROUGH THE RANKS AND WORKING ON REALLY, YOU KNOW, VERY MUCH CHANNEL PHYSIOLOGY, BIOLOGICAL FUNCTION, BOTH SOME PLASMA MEMBRANE CHANNELS AND WHAT WE'LL HEAR ABOUT TODAY, ORGANELLE CHANNELS. WON'T GO FURTHER THAN THAT, JUST LET YOU TAKE IT OVER. >> THANK YOU FOR THE INVITATION AND THANK YOU FOR THE INTRODUCTION. I WANT TO THANK YOU, WHENEVER I COME FROM THE BUILDINGS ARE BASICALLY QUIET AND CLOSED NOW. OKAY. SO BEFORE I START MY TALK, I JUST WANT TO THANK THE PEOPLE WHO DID THE JOB FOR US BECAUSE I MAY NOT HAVE ENOUGH TIME IN THE END. SO THE WORK I'M GOING TO TALK ABOUT IS MAINLY DONE BY -- HE CURRENTLY HAS HIS OWN LAB IN THE UNIVERSITY OF SCIENCE AND TECHNOLOGY IN CHINA, AND A FEW OTHER POSTDOCS ALSO CONTRIBUTED. -- NIH AND UNIVERSITY OF PENNSYLVANIA. I STARTED AS AN ELECTRICAL ENGINEER, NEVER KNEW ANYTHING PHYSIOLOGY OR BIOLOGY AT ALL, BUT I GOT SORT OF ATTRACTED TO THE BIOLOGY SIMPLY BECAUSE OF THE PROPERTIES OF THOSE CELLS, WHAT YOU SEE -- NEURON, AND WHAT WAS STRIKING TO ME AT THAT POINT -- YOU SEE SPIKES, YOU HAVE ACTION POTENTIAL. THAT'S WHAT I LEARNED IN GRADUATE SCHOOL. BUT RECENTLY OR IN THE PAST SEVERAL YEARS, I STARTED TO REALIZE ALL THE -- WERE ACTUALLY FROM PLASMA MEMBRANE. LIKE MANY FROM CELL BODIES. IF YOU'RE TECHNICAL ENOUGH, YOU PROBABLY CAN -- SOME OF THE PROCESSES BUT THEY'RE ALL, AGAIN, PLASMA MEMBRANE PROPERTIES. NOW FOR MANY CELLS, FOR EXAMPLE, THIS NEURON, IT ALSO HAS INTERSECTING ORGANELLES. WHAT YOU SEE HERE IS A NEURON LABELED RFP-RAB7 ENDOSOME, YOU CAN SEE THOSE ORGANELLES ACTUALLY TRANSPORT. THIS IS TRUE FOR MOST OF CELLS. THE CELL BIOLOGY IT -- ACTUALLY PLASMA MEMBRANE SURFACE, LESS THAN 10% OF -- CELL MEMBRANE SURFACE. MY POINT IS THAT IF YOU'RE AN ELECTROPHYSIOLOGIST, ALL THAT YOU HAVE DONE IN THE PAST 40 OR 50 YEARS BASICALLY IS LESS THAN 10% OF THE TOTAL CELL MEMBRANE. THAT'S EXCITING BUT IT'S ALSO -- EXCITING IS THAT YOU JUST START YOUR GRADUATE SCHOOL, YOU'RE IN A -- YOU THINK EVERYTHING HAS BEEN DONE BY PEOPLE, YOU KNOW, OLDER THAN YOU, THIS IS NOT TRUE. THERE ARE A LOT -- IN THE CELL MEMBRANE. THIS IS BASICALLY WHAT I'M GOING TO TALK ABOUT. SO THE POINT IS THAT IN A CELL, YOU HAVE ALL THE INTERSECTING ORGANELLES, YOU HAVE MEMBRANES. A FEW QUESTIONS WE HAVE BEEN ASKING, LITERALLY WE STARTED PROJECT ABOUT THREE YEARS AGO, WE SAID WELL, IF WE -- IF WE SATURDAY OF PUT ELECTRODES ON THOSE ORGANELLES, WHAT DO WE SEE? A FEW QUESTIONS WE ASKED, WHAT IS MEMBRANE POTENTIAL? WHAT ARE THE ELECTRICAL -- WHAT PROTEINS FORM THOSE CONDUCTORS, WHAT ION CHANNELS, AND WHAT MECHANISM, AND AFTER ALL, WHAT'S THE FUNCTION OF THOSE EYE ON CHANNELS, RIGHT? WE KNOW ION CHANNELS ARE IMPORTANT FOR MANY THINGS, BUT WHAT'S A FUNCTION -- ION CHANNELS? SO THE ORGANELLES I'M GOING TO FOCUS ON MOSTLY ARE ENDOSOMES OR LYSOSOMES. LYSOSOME IS A GARBAGE CAN, IT'S AN IMPORTANT GARBAGE CAN BECAUSE IT RECYCLES MATERIALS. MORE LIKE YOUR STOMACH, IT HELPS TO HAVE VERY LOW -- IN THIS CASE LYSOSOME PH IS ABOUT 4.5. OFTEN FOR THE CYCLING PROCESS, YOU ALSO HAVE TO EXPORT MATERIALS YOU HAVE DIGESTED. FOR EXAMPLE, AMINO ACIDS OF. MOST TRANSPORTERS ARE -- I'M NOT GOING TO TALK ABOUT ANY OF THEM, OKAY, BUT LYSOSOMES DO A LOT OF THINGS. FOR EXAMPLE, SOME LYSOSOMES ALSO UNDERGO EXOCYTOSIS, CAN FUSE WITH PLASMA MEMBRANE -- AT IT P, FOR EXAMPLE. LYSOSOMES CAN ALSO REPAIR PLASMA MEMBRANE DAMAGE. MANY CELLS IT -- SOME LYSOSOMES CAN MIGRATE HERE TO HELP REPAIR THE CELL MEMBRANE. -- CALCIUM CONCENTRATION ABOUT 5 MINI MOLAR -- SIGNALING KINETICS IN THE CELL. LYSOSOMES, WHILE THIS IS SORT OF MORE RECENT FINDING FROM SEVERAL LABS, WHAT'S SURPRISING IS THAT LYSOSOMES ACTUALLY ACCUMULATE MANY SIGNALING MOLECULES ON THE SURFACE. THE CLASSIC ONE IS THE MTOR, MTOR IS A KINASE, I'LL TALK BRIEFLY LATER. FOR EXAMPLE, MTOR TRANSLOCATED ON THE SURFACE OF LYSOSOME. ALL RIGHT? SO MANY OTHER SIGNALING MOLECULES, KINASE, FOR EXAMPLE, AKT. SO THE POINT IS WHY IS THAT, WHY DO YOU HAVE THOSE SIGNALING MOLECULES ACCUMULATE ON THE LYSOSOMES IF LYSOSOMES ARE BASICALLY GARBAGE CAN? THE SUGGESTION MAY BE THOSE ARE ALSO FOR SOMETHING ELSE. LYSOSOME, I WILL TELL YOU LAYER, LYSOSOME CAN ALSO OF ATP IN THE CYTOSOME. SO THE POINT IS THAT LYSOSOMES DO A LOT OF THINGS OF. WE STARTED -- A FEW YEARS AGO, THE STUDY IS VERY LYSOSOMES OR ENDOSOMES -- MUTANTS, A FEW 3-MICRONS. ALSO LYSOSOMES WITH CHEMICAL IT -- SOMETIMES LYSOSOMES, ENDOSOMES CAN BE IT -- WITHOUT ANY TREATMENT WHEN YOU OVEREXPRESS SOME PROTEINS. IN LARGE, LYSOSOMES -- SOME CELLS WITH PARKINSON'S MUTATIONS. SO WE HAVE LARGE LYSOSOME -- ONE OF THE ORGANELLES, SO THIS IS WHAT YOU DO, I CANNOT GET IT TO GO FORWARD. SO YOU HAVE A -- HERE, WE -- IN THIS CASE -- LYSOSOME. URBAN SO HAD THIS LYSOSOME OR ENDOSOME IS RELEASED WE START A PATCH BECAUSE THERE'S ONLY ABOUT 3-MICRONS YOU CAN USE -- TO RECORD THEM. OKAY. SO THERE ARE A FEW THINGS WE NEED TO DEFINE, THE VOLTAGE IS DEFINED AS A DIFFERENCE BETWEEN CYTOSOL AND THE LUMEN. -- WHICH REALLY MEANS THE CYTOSOL IS MORE -- THAN THE LUMEN. THIS IS SORT OF BASICALLY SAYING INWARD CURRENT IS ACTUALLY CURRENT MOVING TOWARDS CYTOSOME, FOR THOSE THAT CLAMP EXPERIMENT, YOU FLIP THE NUMBERS OFF YOUR AMPLIFIER, SO WE'RE GOING TO USE THIS KNOWLEDGE LATER. A FEW THINGS I WANT TO SORT OF -- OF COURSE THIS IS GOING TO BE HARD BECAUSE THIS IT RECORDING IS WAY HARDER THAN THE WHOLE CELL RECORDING, NORMALLY IT TAKES A FEW WEEKS WITH TRAINED GRADUATE STUDENTS. MANY OF THESE THINGS WE DON'T REALLY KNOW FOR SURE. FOR EXAMPLE, WHAT'S THE INSIDE, THE IONIC CONCENTRATION INSIDE THE LYSOSOME, WHAT'S THE SODIUM CONCENTRATION, WHAT'S THE POTASSIUM CONCENTRATION? WE ACTUALLY DON'T KNOW FOR SURE, AND THERE'S A PROBLEM THIS BECAUSE NOW THE CURRENT PLAN, YOU ARE MAKING -- -- THE VALLEY OF THE VOLTAGE -- ARTIFACT. RIGHT? BUT WE ARE SEEING A CHANGE, THE CHANGE CAN STILL GIVE YOU INFORMATION. ANOTHER WOULD BE -- LARGE ENDOSOMES OR LYSOSOMES, SO THE POINT IS ARE THEY THE SAME AS WHEN THEY'RE IN THE NATIVE CONDITIONS. WE DON'T HAVE ANSWERS TO MOST OF THE QUESTIONS HERE. SO THIS IS WHAT YOU'LL SEE -- LYSOSOME. CHANGE THE CYTOSOLIC -- THIS IS MORE MEMBRANE POTENTIAL, POTASSIUM DEPENDENT. IT'S ALSO SODIUM DEPENDENT, IN THIS CASE I HAVE IT -- CHANGE OF MEMBRANE POTENTIAL OF. AGAIN I'M GOING TO COME BACK IS THAT THIS MEMBRANE POTENTIAL IS ALSO SENSITIVE TO CYTOSOLIC ATP CONCENTRATION. WITHOUT ATP, THIS WOULD BE -- A OF TP. SO THERE'S A MEMBRANE POTENTIAL AND MEMBRANE POE IT TENSION CHANGES OF. -- SINCE THE 1940S, VERY SAME PROPERTY. THEN WE SWITCH THE VOLTAGE CLAMPS TO LOOK FOR WHAT ARE THE CONDUCTANCES. ALL YOU NEED TO SEE IS THIS IS A VOLTAGE STEP, RIGHT, THIS IS A VOLTAGE CLAMP 150 MINIMUM POTASSIUM OUTSIDE AND INSIDE, YOU SEE THIS VOLTAGE CURRENT. SO THOSE LYSOSOMES ARE PERMEABLE IT TO POTASSIUM. THIS IS FROM GLIAL CELLS, FROM NEURON, BUT POTASSIUM CONDUCTANCE IS IN -- ALMOST ALL THE CELLS. THE POINT IS THAT MOST LYSOSOMES OR ENDOSOMES ARE PERMEABLE TO POTASSIUM. THEY'RE ALSO PERMEABLE TO SODIUM. OKAY? AGAIN, HAD IS RECORDING 150 MINIMUM OF SODIUM INSIDE 150 -- OUTSIDE, YOU SEE THIS CURRENT. THIS IS A LINEAR CURRENT, WHICH MEANS IF YOU IT -- THE VOLTAGE WITH THE CURRENT VERSUS THE VOLTAGE, THE RELATIONSHIP IS LINEAR, SO WE JUST CALL THIS LEAK-LIKE SODIUM CONDUCTORS. SO THE POINT IS THAT LYSOSOMES, ENDOSOMES ARE ALSO TO SODIUM. SO ONE STRIKING THING IT WE FOUND IS SOME OF THE -- LYSOSOMES -- CORE SODIUM CONDUCTORS, THE SAME VOLTAGE HERE AS YOU WILL SEE HERE, YOU SEE HERE IN THIS YOU SEE CURRENTS, INWARD CURRENTS AND OUTWARD CURRENTS, SO INWARD CURRENTS, POSITIVE VOLTAGE, SO THE SODIUM CAN IT GO BOTH WAYS, INTO LYSOSOMES AND OUT OF LYSOSOMES. BUT IN THIS CASE, YOU SEE IN THIS ONE, THERE'S NO INWARD NOW, BUT WHEN YOU DEPOLARIZE THE LYSOSOME, YOU CHANGE THE VOLTAGE TO A POSITIVE, NOW YOU START TO SEE CURRENT, OKAY, THIS IS A VOLTAGE WITH SODIUM CONDUCTORS. IF YOU PLUG THE CURRENTS VERSUS VOLTAGE, THE RELATIONSHIP IS LIKE THIS. SO THIS IS MORE LIKE A CLASSIC VOLTAGE -- SODIUM CHANNELS IN THE NEURON, IN THE PLASMA MEMBRANE. I'M GOING TO COME BACK, WHY WOULD THE LYSOSOME, ENDOSOME HAVE VOLTAGE ACTIVE SODIUM CHANNEL? POTASSIUM CONDUCTANCE IS TWO SODIUM CONDUCTANCE, PROTON CONDUCTANTS. PRESUMABLY THERE'S A CALCIUM CONDUCTANT. SO THE NEXT MAJOR QUESTION WE ASKED WAS WHAT ARE THE PROTEINS, WHAT ARE THE ION CHANNEL PROTEINS FORMS THOUGH CONDUCTANTS. SO THE APPROACH WE USE WITH BASICALLY COMBINATION -- PROTEOMICS ASKING THE QUESTION, WHAT OTHER PROTEINS ON LYSOSOME MEMBRANE OF COURSE -- NOT SURPRISINGLY, OF COURSE, IF YOU'RE A PHYSIOLOGIST, SOME MAY LOOK LIKE ION CHANNELS. THOSE ARE MAJOR APPROACHES WE USE. I'M GOING TO START THE POTASSIUM CONDUCTANCE. WE'RE LUCKY WE HAVE A COLLABORATOR IT -- IN FRANCE, WHAT THEY DID WAS THE PROTEOMICS -- THE LIVER, FRACTIONATION, LYSOSOME DEFICIENCY FRACTION, PROTEOMICS, YOU ASK -- PROTEINS ARE MORE OR LESS LYSOSOME SPECIFIC, 734 LYSOSOME SPECIFIC, 400 TRANSMEMBRANE PROTEINS, 46 NOVEL. NOW, HYPOTHETICALLY YOU CAN SAY, WELL, DOES ANY ONE OF THE POTASSIUM CHANNEL? RIGHT? -- 403 TRANSMEMBRANE PROTEIN BUT THAT'S GOING TO BE A LOT OF WORK. NO IT ONE WOULD DO THAT. SO AGAIN, WE WERE LUCKY, SO THEY GAVE US 12 -- CLONES, HERE ARE THE 12 CLONES I HAVE, MAYBE -- WHO KNOWS, WELL THEY ARE, MAYBE YOU SHOULD TRY, WHICH IS 1 BY 1, I LABEL THIS -- BECAUSE I HAVE NO IDEA WHAT ARE THE GIVEN NAME, TRANSPORTER XYZ, I LABEL 1, 2, 3, 4, 5 TO 12. SO WE TRIED ONE BY ONE OF. THE FIRST 11 CLONES DID NOT GIVE US ANY CURRENT. BUT THE LAST ONE, NUMBER 12, GAVE US REALLY NICE POTASSIUM CURRENT. OKAY, YOU TRANSFECT -- LYSOSOME POTASSIUM CHANNEL CURRENT. ONE OF THE CLONES IS CALLED TMEM175 SO THAT CLONE GAVE US CONDUCT POTASSIUM CHANNEL. AFTER THAT, WE SAY LET'S TRY A FEW MORE, ANOTHER FOUR CURRENTS. IT TMEM175 GAVE US CURRENTS, YOU PLOT THE -- PREDICT THE STRUCTURE, IT'S 6 TRANSMEMBRANE BY -- WELL, 2 BY 6, RIGHT, SO IT'S A 2 BY 6 TRANSMEMBRANE STRUCTURE, SO MANY CHANNELS, POTASSIUM CHANNELS, SODIUM CHANNELS, THEY ALL HAVE WHAT'S GOT A P-LOOP WHERE THE IT -- BACK IN THE CELL MEMBRANE AND THAT FORMS THE ION CHANNEL FILTER IT AND MOST PROTEINS DON'T HAVE THAT. THE -- SIX TRANSMEMBRANE HERE, FOR EXAMPLE, IT -- IS ONLY ACIDS. ALSO POTASSIUM CHANNELS ON A PLASMA MEMBRANE HAVE -- GYG OR GFG SIGNATURE THAT'S HOMOGENEOUS, ALSO -- ALL THE LIFE, BACTERIA, SO IT'S A NOVEL PROTEIN, WE KNOW THIS FORMS THE CHANNEL, WE OVEREXPRESS ENDOSOMES, FOR EXAMPLE, THIS IS A VOLTAGE STEP, COMES FROM MINUS 100, PLUS 100 MILLIVOLT, OF COURSE YOU REMOVE THE POTASSIUM FROM -- REPLACE WITH A LARGER ION AND YOU SEE CURRENT, THERE'S NO OUTWARD CURRENT. -- WHEN YOU OVEREXPRESS TMEM175. THIS IS A CURRENT VOLTAGE RELATIONSHIP WITH POTASSIUM INSIDE, POTASSIUM OUTSIDE. THESE ARE LINEAR CURRENTS, WHEN YOU REPLACE POTASSIUM WITH -- YOU SEE -- CURRENTS. YOU CAN ALSO REPLACE POTASSIUM WITH CALCIUM OR SODIUM, YOU ALSO SEE THIS OUTWARD CURRENT DONE. THAT'S HOW YOU -- THIS TMEM175 IS NOT PERMEABLE TO SODIUM, IS NOT PERMEABLE TO CALCIUM, SO -- FOR POTASSIUM. YOU CAN USE WHAT'S CALLED A REVERSE OF POTENTIAL -- PKP SODIUM, FOR EXAMPLE, 36, OKAY, IT'S SLIGHTLY SORT OF IT'S SLIGHT IT -- THAN THE CLASSIC POTASSIUM NEURONAL MEMBRANE, PLASMA MEMBRANE, BUT AGAIN -- IT'S THIS CHANNEL ACTUALLY -- PRETTY WELL. OKAY -- IN THE MACROPHAGE CELL WITH CRISPR -- CURRENTS, CURRENTS BASICALLY -- OF COURSE YOU CAN RESCUE THIS CURRENT, LOCK OUT CELLS -- I'M SORRY I'M USING -- IT TMEM175 -- ENDOSOME, LYSOSOME POTASSIUM CHANNELS. IN THE MOUSE SELL NOW YOU CAN -- HUMAN OF KO, CRISPR KNOCKOUT, COVER THIS CURVE. SO LYSOSOMES HAVE ONE MAJOR POTASSIUM CHANNEL, WHICH IS TMEM175. AGAIN, THIS IS A LITTLE SURPRISING -- DIFFERENT POTASSIUM CHANNELS ON A PLASMA MEMBRANE. DEPENDS ON WHICH CELLS YOU'RE LOOKING AT. THERE'S ONLY ONE MAJOR ONE -- IF YOU KNOCK OUT, NOW YOU CAN RECORD THE MEMBRANE POTENTIAL, 140 MINIMAL -- I SHOWED YOU THE WILD TYPE, MEMBRANE POTENTIAL -- YOU CHANGE POTASSIUM CONCENTRATION. IN THE KNOCKOUT, IT CHANGES VERY LITTLE. SO THIS CALCIUM TMEM175 IS A MAJOR CHANNEL PERMEABLE TO POTASSIUM ON THE LYSOSOME. WHAT ABOUT SODIUM? SODIUM CONDUCTORS. SODIUM CONDUCTORS ARE TWO PROTEINS, ONE IS CALLED TPC2. REMEMBER I TOLD YOU THERE'S TWO KINDS OF CONDUCTORS, ONE IS A LEAK CURRENT VOLTAGE RELATIONSHIP, ANOTHER IS A VOLTAGE GATED, TPC1. AGAIN, THOSE PROTEINS ARE MORE OR LESS LAVA PROTEINS OR IN A CLASSIC TEXTBOOK, YOU WILL HAVE LEARNED VOLTAGE -- SODIUM CHANNELS -- BUT IN THE GENOME, HUMAN GENOMES, YOU ANOTHER CLASS OF PROTEIN CALLED TPC PROTEINS THAT HALF THE SIZE. THEY HAVE SEQUENCING MINORITIES. CLEARLY THEY -- ORIGINATED FROM PROBABLY THE COMMON ANCESTOR. THOSE ARE 2 BY 6 TRANSMEMBRANES. SOME -- IT TPC3, OF COURSE YOU SEARCH BACK IN OUR GENOMES, WE ALSO HAVE 6 -- BACTERIA YOU HAVE -- SODIUM CHANNELS FORMED BY SIX TRANSMEMBRANE DOMAINS. YOU ALSO HAVE MORE THAN OF HALF OF THE POPULATION PEOPLE IN THIS AUDIENCE WHILE BASICALLY MALES, YOU ALSO HAVE ONLY EXPRESSED IN SPERM CELLS, THOSE ARE FOR PROTEINIZATION, CALCIUM CHANNELS. SO EVIDENCE FOR TPW IS HERE -- LYSOSOMES, PLOT -- CURVE, IT'S -- LINEAR. TPC1, HERE AGAIN -- BUT THERE'S NO INWARD CURRENT, INWARD CURRENT, OUTWARD CURRENTS, YOU HAVE DEPOLARIZATION, THIS WAS RELATIVELY SELECTIVE SODIUM CHANNELS, CLASSIC VOLTAGE SODIUM CHANNELS FOR NEURONS FOR THE ACTION POTENTIALS. SO THE VOLTAGE DEPENDENTS PRESUMABLY COMES FROM WHAT'S CALLED S4 DOMAINS, TPC1 FOR EXAMPLE, 1S4 AND 2S4. STRUCTURE WISE WE SUSPECT IT'S SIMILAR -- SODIUM CHANNELS, WE ALSO WORKED OUT WHERE WE'RE -- AT SOME POINT WE WORK OUT -- THIS -- IS ALSO IN THE PLANS. WE WERE WORKING ON THE PLANS -- NORMALLY IT'S LOCALIZED IN A VACUUM, YOU CAN GET -- CURRENTS ON A PLASMA MEMBRANE. SO -- RECENTLY -- STRUCTURE IS MORE LIKE A PREDICTIVE -- I THINK THIS PAPER IS ONLINE TODAY. SO -- THE PROTEINS -- CHANNEL IS FROM -- ONE -- I'M NOT GONNA SHOW HERE BUT RELATIVELY -- TO THE VOLTAGE -- SODIUM CHANNELS FROM THE BACTERIA, WHICH HAS A CRYSTAL YOU'RE. OF SO THE POINT IS LYSOSOMES -- SODIUM CHANNELS, THIS ONE IS KIND OF -- IT'S ALSO PERMEABLE CALCIUM -- THAT BINDS CALCIUM, SO ANIMAL -- TPC1 THAT IS NOT -- BY CALCIUM BUT CAN BE POTENTIATED BY PH. THIS IS WHAT YOU SEE HERE, TPC1, IF YOU MEASURE DIFFERENT PH, PH 4.6 AND PH 5.6, IT -- 4.5, 5.6, YOU CAN SEE THE SHAPE OF ACTIVATION TOWARD LEFT BY -- 60 -- THIS CHANNEL IS EXTREMELY SENSITIVE TO PH OF THE LYSOSOME. SO WHEN YOU HAVE A LITTLE PH INCREASE, THIS CHANNEL CAN ACTIVATED. SO -- >> [INAUDIBLE] IS PIPETTE PH INSIDE THE LYSOSOME. FOR THOSE OF YOU WHO ARE NOT USED TO -- CURVE, INWARD CURVE IT -- YOU SAY INWARD CURRENT HERE, RIGHT, THIS IS CURRENT VERSUS VOLTAGE, THIS IS MORE LIKE YOUR CLASSIC VOLTAGE SODIUM CHANNEL CURRENTS, YOU HAVE YOUR -- CURRENTS, BUT WHEN YOU INCREASE THE PH5.6, THIS -- CURVE 60MILLIVOLTS, SO THE POINT IS THAT WHY WOULD YOU WANT TO HAVE A VOLTAGE IT -- CHANNEL IN THE LYSOSOMES. WE ALL TALK ABOUT EXCITABILITY ACTION POTENTIAL, THOSE PROPERTIES OF THE NEURONS. SO WE CAN DID ONE THING, WE SAID WHAT IF YOU OVEREXPRESS THE CHANNEL HEC293 CELLS, THEY HAVE VERY LITTLE IT TPC1. THIS IS WHAT YOU HAVE. -- THE CURRENT CLAMP, YOU IT GIVE CURRENT INJECTION, WHEN YOUR INJECTION CAN -- THE VOLTAGE COMES BACK, BUT WHEN YOU INCREASE THE AMPLITUDE OF YOUR CURRENT INJECTION, WHEN YOU INCREASE YOUR POLAR SAY, AFTER YOU REACH A THRESHOLD, YOU SEE HERE THIS KIND OF SPIKE, SO THE MEMBRANE POTENTIAL STAYS THERE FOR LONG, IN FACT, IF DEPOLARIZE THIS LONG MUST HAVE OR STRONG ENOUGH, THIS MEMBRANE -- CAN STAY THERE FOR MANY, MANY LOT OF TIMES IT DOESN'T EVEN COME BACK, YOU CALL THIS -- MEMBRANE POTENTIAL. SO THIS IS NON-TRANSFECTED CELLS. THIS IS NOT JUST AN ARTIFACT OF OVEREXPRESSION BECAUSE YOU CAN ALSO SEE THIS -- FROM -- MYOCYTES, THIS IS A WILD DEPOLARIZATION CURRENT, ONCE YOU REACH A THRESHOLD, YOU SEE THE MEMBRANE POTENTIAL STAYS THERE FOR A LONG TIME. THIS IS A KNOCKOUT, OF TPC KNOCKOUT, YOU DON'T SEE THIS. SO WE THINK SOME LYSOSOMES MAY BE ABLE TO GENERATE DEPOLARIZATION SPIKE, THAT'S MORE LIKE ACTION POTENTIAL. WE THINK THEY CAN BE EXCITABLE. WE HAVE NO IDEA WHAT IT'S GOING TO IT -- EXCITABILITY WOULD DO TO LYSOSOMES, BUT THEY CAN DO THIS. NOW FOR THOSE OF WHO YOU ARE USED TO RECORDING ACTION POTENTIALS IN NEURONS, YOU WOULD SAY WELL, THIS -- THIS LASTS FOR SOME MANY, MANY, MANY SECONDS, SOMETIMES MANY SECONDS. I JUST WANT TO REMIND YOU, EVEN ON THE PLASMA MEMBRANE CELLS, THERE ARE ANIMALS THAT ACTUALLY GENERATE LONG ACTION POTENTIAL THAT LAST MANY MINUTES. THE LONGEST ONE I HAVE FOUND IS FROM -- X, YOU CAN SEE ACTION POE IT TENSION, THAT'S 30 MINUTES. YOU CAN -- CELL, THERE'S ACTION POTENTIAL, YOU CAN GO OUT, HAVE YOUR COFFEE AND THE ACTION POTENTIAL IS STILL THERE. SO MY POINT IS -- EVOLUTION OR EVEN NOW, SOME ANIMALS STILL USE THIS -- ACTION POE IT TENSION. SO -- BUT BACK IT TO LYSOSOMES, WE DON'T KNOW ANYTHING IF THIS IS USED AT ALL. SO NOW -- AGAIN TO SUMMARIZE, WE HAVE IDENTIFIED -- OF MAJOR ION CHANNELS CONDUCTANCES, I'M NOT TALKING TO YOU ABOUT IT -- MOST LIKELY IT'S THE CHANNEL -- WHAT -- STARTED SEVERAL YEARS AGO IN THE LYSOSOMES, CLC7, CALCIUM CONDUCTORS, IT -- BUT THERE ARE CHANNELS THAT ARE EXPRESSED ON A LYSOSOME CLEARLY WHEREVER YOU EXPRESS THEM, THEY ARE CALCIUM-PERMEABLE. BUT NOW I WANT TO SWITCH GEAR, I WANT TO LOOK AT THE FUNCTION OF THE ION CHANNELS, WHAT THEY DO. WHAT'S A MEMBRANE POTENTIAL, WHAT DOES A MEMBRANE POTENTIAL DO, AGAIN, PROBABLY FOR THOSE WHO HAVE TALKED TO JOE, YOU PROBABLY ALREADY KNOW THAT, WHEN YOU HAVE THE INSIDE THE LYSOSOME TO LOWER THE PH TO 4.5 OR 4.6, EXAMPLE. YOU ARE ALSO TAKING IT -- YOU'RE TAKING POSITIVE CHARGE INSIDE A CELL, RIGHT, MEMBRANE POTENTIAL CAN -- VERY POSITIVE INSIDE, THAT LIMITS YOUR ABILITY TO -- IT PROTEIN INSIDE A CELL. SO THERE'S A NEED FOR WHAT'S CALLED A COUNTER ION. COUNTER IONS COULD BE -- TO NEUTRALIZE THE CHARGE OR PRESUMABLY YOU CAN HAVE -- SODIUM BUT POTASSIUM WILL BE UP. SO THE POINT IS THAT IT -- SODIUM OR POTASSIUM CONDUCTANTS IS ALSO USED TO NEUTRALIZE THE POSITIVE CHARGE. THERE IS SOME EVIDENCE SUGGESTING MAYBE THEY ARE DOING THIS. ONE PIECE OF EVIDENCE IS THAT IN A KNOCKOUT, THE SODIUM CHANNEL KNOCKOUT IS. PC KNOCKOUT, IS FROM MACROPHAGE, THE PH LYSOSOME PH, THE HISTOGRAM OF LYSOSOME PH, WE HAVE TO STARVE THE CELLS, MACROPHAGE CELLS, BEFORE STARVATION, AFTER STARVATION. THERE'S A LITTLE CHAIN OF DISTRIBUTION, BUT YOU AVERAGE THEY'RE ROUGHLY SIMILAR IN THE WILD TYPE. SO WE THINK IN THE WILD TYPE, THE PH IS RELATIVELY STABLE. NOW IF YOU LOOK AT IT KNOCKOUT, OKAY, THIS IS BEFORE AFTER STARVATION, THERE'S A SHAPE OF THIS OF PH, SO IF YOU AVERAGE ALL THE LYSOSOME PH, THERE IS AN INCREASE OF PH IN THE LYSOSOMES IN MUTANT LYSOSOMES. SO WE THINK THOSE CHANNELS CAN BE USED TO HELP STABILIZE LYSOSOMES. I'M NOT SHOWING YOU THE POTASSIUM CHANNEL KNOCKOUTS ALSO HAVE THIS PROBLEM. ANOTHER THING WE LOOK AT IS, IS IT POSSIBLE -- THE POINT IS WHAT DOES THE VOLTAGE OF A MEMBRANE -- WHAT DOES THE VOLTAGE ACROSS THE LYSOSOME MEMBRANE DO? ANOTHER THING WE LOOK AT IS THE LYSOSOME FUSED WITH OTHER ORGANELLES, LYSOSOME FUSED WITH ENDOSOMES, AUTO PHAGOSOMES, WE MONITOR THEM BY THIS CONSTRUCT, SO THIS MARKER, IF -- REMEMBER THE GFP IS NOT FLUORESCENT WHEN LOW PH, WHICH MEANS WHEN THE AUTO IS FUSED WITH -- CONTROL CELLS, KNOCKOUT CELLS, STARVE FOR A LITTLE WHILE, SO THE CONTROL CELLS, TFP SIGNAL HERE, YOU'LL SEE SOME -- THAT MEANS THE AUTO PHAGOSOMES -- AND THIS -- CHERRY SIGNAL AND TFP SIGNAL, BUT LOOK AT THIS CELL. YOU HAVE POSITIVE -- TEST, YOU HAVE AUTO PHAGOSOMES, IT APPEARS MOST ARE ALREADY FUSED WITH LYSOSOMES. WE THINK -- AND LYSOSOMES. -- TO COVER THIS. -- CONTROLS. SO TAKE HOME MESSAGE IS THAT IF A KNOCKOUT OF POTASSIUM CHANNEL, WE THINK THERE'S A CHANGE OF -- BETWEEN THE AUTO PHAGOSOMES AND LYSOSOMES, BUT -- CAN INDICATE THAT. ANOTHER FUNCTION OF THOSE CHANNELS ARE IT -- WE THINK THEY CAN BE USED TO SENSE NEUTRONS. WHEN I SAY NEUTRONS, THIS CAN BE ATP CONCENTRATION AND AMINO ACID CONCENTRATION OUTSIDE. ONE OF THE EARLY EXPERIMENTS AS I JUST SHOWED YOU ACTUALLY IS A MEMBRANE POTENTIAL CAN -- SENSITIVE ATP CONCENTRATION. OKAY. RIGHT? TURNED OUT THE SODIUM CHANNELS CAN ACTUALLY -- CONCENTRATION, THIS IS -- FOR THOSE THAT CARE, THIS CHANNEL ALSO REQUIRED -- MAXIMUM ACTIVATION ALSO REQUIRES IT -- THIS WAS FIRST FOUND IN -- LAB IN UNIVERSITY OF MICHIGAN ANN ARBOR. YOU GET OCCURRENCE, INWARD OCCURRENCE, NOW YOU ADD ATP. THE CURRENT GETS -- IS GONE. -- THE SAFETY OF ATP THIS OCCURRENCE THIS ATP CONCENTRATION. WHEN YOU INCREASE ATP CONCENTRATION, THE CURRENT IS OKAY, THIS IS IT -- EACH CURRENT, BUT THIS JUST TELLS YOU THAT ATP -- SODIUM CURRENT. WE KNOW THIS IS A TPC CURRENT BECAUSE WHEN YOU OVEREXPRESS TPC -- CELLS, THE ONE I JUST USED IS NATIVE CURRENT, THIS IS A LOW ATP, USE A LARGE CURRENT, ATP, MUCH SMALLER CURRENT, BUT ANOTHER CHANNEL OF THE LYSOSOME, CALCIUM-PERMEABLE CHANNEL -- ATP. THIS CHANNEL ALSO SERVES THE AVAILABILITY OF AMINO ACIDS OUTSIDE THE CELLS. THE EXPERIMENT WAS LIKE THIS. YOU STARVE THE CELL OF AMINO ACIDS FOR ONE HOUR, FOR EXAMPLE, NOW YOU DO THE SAME RECORDING. THIS IS CONTROL, THE CELLS THAT HAVE NEUTRONS, ONE IT -- ATP, IT -- A. IT P, RIGHT? BUT WHEN YOU START FOR HALF -- THIS SODIUM CONDUCTOR IS NO LONGER SENSITIVE TO ATP. REMEMBER WE ARE STARVING THE EXTRA CELLULAR AMINO ACIDS, YOU CAN FADE IT, THIS IS I THINK FIVE MINUTES OR IT 10 MINUTES, AND THIS IT -- COVERS. SO SOMEHOW REMEMBER WE ARE RECORDING LYSOSOMES, SOMEHOW THE LYSOSOME KNOWS THE AMOUNT OF AMINO ACID OUTSIDE THE CELL. AGAIN TO MAKE A LONG STORY SHORT, TO FIGURE OUT HOW IS YOUR CHANNEL -- LYSOSOME, AMINO ACID OUTSIDE THE HOW TO YOU SENSE THAT? TURNS OUT THIS REQUIRES A PROTEIN CALLED MTOR, IF YOU BLOCK MTOR WITH RAPAMYCIN -- IF YOU SHAPE THAT MTOR, NOW YOU ADD ATP, THERE'S NO -- THE CHANNEL IS NO LONGER SENSITIVE TO ATP. THAT'S A CONTROL, RIGHT? YOU CAN OF OVER -- OVEREXPRESS MTOR, NOW THE ATP SENSITIVITY IS MUCH HIGHER. CONCENTRATION DEPENDENCE. STANDARD CONTROL, OVEREXPRESS M IT TORE. SO MTOR IS REQUIRED FOR THE SENSITIVITY, ALSO -- ENHANCE SENSITIVITY. SO THE IDEA IS THAT AGAIN, THIS WAS WORKED ON OTHER SYSTEM, TURNED OUT SEVERAL LABS -- MTOR ON THE CELL, MTOR TRANSLOCATES ON THROUGH THE LYSOSOME SURFACE, WHEN YOU HAVE AMINO ACIDS OUTSIDE. BUT MTOR MOVES AWAY FROM LYSOSOME, YOU DON'T HAVE AMINO CONSISTENTLY THIS MODEL, WE SHOW -- IMMUNO -- WE TPC CAN PRECIPITATE WITH MTOR, AGAIN I'M NOT GOING TO SHOW YOU -- APPROACHES IS ALSO CO-LOCALIZED WITH MTOR WHEN YOU HAVE AMINO ACIDS OUTSIDE. NOW THE LAST PIECE EVIDENCE FOR THIS IS THAT WE KNOCK OUT THE TPC, THE SODIUM CHANNELS, OKAY, NOW RECORD MEMBRANE POTENTIAL, THE MEMBRANE POTENTIAL IS NO LONGER SENSITIVE TO ATP ANYMORE, NO LONGER SENSITIVE TO SODIUM CONCENTRATION. OKAY. SO WE THINK THE SODIUM CHANNELS CAN ALSO SENSE NEUTRONS OUTSIDE. WHAT ELSE DOES IT DO? ANOTHER POSITIVE BEN IT FIT -- MEMBRANE POE IT TENSION HERE OR CHANNELS PER SE, DO THEY AFFECT THE EXPORT OF AMINO ACIDS? RIGHT? THIS IS MEDIATED BY TRANSPORTERS, SO IS IT POSSIBLE SOME TRANSPORTERS ARE ALSO SENSITIVE TO VOLTAGE. IT TURNS OUT, AGAIN, THIS IS -- USING DID -- LYSOSOMES, YOU -- LABELED LYSINE, 14 LABELS, AND YOU MEASURE E FLUX RATES, SO THIS IS A WILD TYPE EFFLUX MUCH FASTER THAN THE KNOCKOUTS, SO THE CHANNELS OR MEMBRANE POTENTIAL ALSO AFFECT THE EFFLUX -- TRANSPORTER ACTIVITIES. AGAIN WE DON'T HAVE CONVINCING EVIDENCE BUT THOSE ARE SOME CLUES. NOW WHAT HAPPENS TO WHOLE ANIMALS? SO WE THINK OKAY, THIS AFFECTS AMINO ACID EXPORT, YOU MAY BE ABLE TO SEE SOME OF THE CHANGE OF AMINO ACID LEVEL. SO WHEN YOU -- ANIMALS, NOW IF YOU'RE NOT -- FOR MAYBE ONE -- YOU BURN YOUR GLUCOSE. IF YOU'RE NOT -- FOR ONE DAY OR TWO DAYS OR THREE DAYS AND YOU START TO BURN YOUR -- FAST, THOSE AB MALLS, THOSE ARE MICE THE SAME, YOU'RE FAST THIS, IS A FAST WILD TYPE, KNOCKOUT, YOU COMPARE THE AMINO ACIDS LEVEL IN THE -- SO YOU NORMALIZE THE -- TO THE ONE BEFORE FASTING, WHERE THIS RELATIONSHIP IS ONE ON ONE NOW, SO IN THE -- YOU DON'T SEE THAT. ESSENTIAL AMINO ACID, THIS INCREASES EVEN HIGHER OF. AGAIN, YOU DON'T SEE THAT IN THE WILD IT TYPE, IN THE MUTANTS, OR TMEM, YOU HAVE AN INCREASE AFTER FASTING BUT YOU SEE VERY LITTLE INCREASE IN THE MUTANT, WE HAVE DECREASE. -- YOU CAN -- THE IT TEST WAS WE ASKED MICE ON THE TREADMILLS, SO THIS IS A WILD TYPE, THIS IS BEFORE YOU ASK HOW MANY -- CAN THEY -- ON THE TREADMILLS, THIS IS BEFORE IT -- NOT SURE MY POINTER IS STILL WORKING. SO BEFORE FASTING AND AFTER FASTING, BASICALLY THE WILD FINE, AFTER FASTING. BUT LOOK AT THE MUTANTS. AFTER FASTING, REALLY CANNOT -- ANYMORE. SO WE THINK THIS IS AN INDICATION OF -- DURING FASTING. SO THE POINT IS WE THINK ANIMALS, INDEED THOSE CHANNELS STRESS IN TERMS OF LET'S SAY A LACK OF FOOD, FOR SO YOU HAVE IT -- IN THE WILD, YOU HAVE TO HAVE CHANNELS OH SURVIVE. ALL OUR MICE ARE IT IN THE VICINITY WHERE FOOD IS UPON US -- TO SUMMARIZE WHAT I HAVE TAUGHT TO YOU, I THINK WE HAVE -- MOST OF IT PERHAPS, IONIC CONDUCTANCE IN THE LYSOSOME, CHLORIDE CONDUCTANCE, THERE ARE SEVERAL OTHER CONDUCTORS WHICH OTHER LABS HAVE WORKED ON. WE THINK THE CHANNELS CAN SENSE THE PH INSIDE THE LYSOSOME. THE VOLTAGE ACROSS THE LYSOSOME MEMBRANE -- OUTSIDE. WE DON'T KNOW OTHER THINGS -- PHYSIOLOGICAL WISE, MEMBRANE POTENTIAL CLEAR -- PH PERHAPS, FUSION, AMINO ACID EXPORT AND -- THERE MUST BE OTHER FUNCTIONS. AGAIN, WE DON'T REALLY KNOW. THERE ARE REPORTS SHOWING THAT THE MUTANT MICE ARE ACTUALLY -- MUTANT MICE -- RESISTANT FOR EXAMPLE EBOLA INFECTION. NO IDEA HOW THAT WORKS. SO IT'S POSSIBLE THOSE LYSOSOMES ARE ACTUALLY -- SOME OTHER PROCESS WHICH WE DON'T KNOW. I WANT TO LEAVE WITH YOU WHAT WE STARTED WITH, MANY OF YOU PERHAPS ARE MORE INTERESTED IN THE NEURONS ON THE QUESTIONS THAT WHAT DO THE ORGANELLES DO TO THOSE NEURONS? MEMBRANES DUE TO THOSE NEURONS, AND WE HAVE NO IDEA. WE HAVE -- SEE IF THEY HAVE ANY DEFECTS IN -- YOU KNOW, IT'S POSSIBLE, REMEMBER SOME ORGANELLES, FOR EXAMPLE, THEY TRANSPORT, OF COURSE, BUT SOME OF THEM ALSO FUSE WITH THE PLASMA MEMBRANE, IS A POSSIBILITY, THEY ACTUALLY CAN BE USED TO CONTROL THE PLASMA MEMBRANE EXCITABILITY. SO MY POINT IS THAT THERE'S A LOT OF -- BUT WE THINK WE HAVE STARTED WITH SOMETHING THAT WE'RE, AGAIN, HOPEFULLY WILL GET IT IN SOME YEARS. I WANT TO THANK YOU FOR SITTING HERE AND I'D BE HAPPY TO ANSWER QUESTIONS. [APPLAUSE] >> [INAUDIBLE] >> YES, WE DON'T KNOW -- IT DEFECT -- PER SE. WE LOOK AT SOME AUTO PHAGE MARKERS, WE DON'T -- CERTAINLY STILL THERE -- ALL THE THINGS ARE STILL THERE. NOW, WHETHER THERE IS A LITTLE CHANGE OF LEVELS, WE'RE NOT SURE YET, SO WE HAVE NOT ANALYZED THEM. >> [INAUDIBLE] >> THEY CANNOT, WE THINK ONE POSSIBILITY IS THE EXPORT OF AMINO ACIDS IS DEFECTIVE. BUT IT CAN -- >> [INAUDIBLE] >> YES. >> [INAUDIBLE] >> YES -- BUT IF THEY DON'T EXPORT AMINO ACIDS, IT'S USELESS. SO YOU GENERATE SOMETHING BUT YOU CAN NOT EXPORT THEM. BUT ON THE OTHER HAND, THE POTASSIUM CHANNEL LOCKOUT -- CELL LINE, CLEARLY THERE SEEMS TO BE DIFFERENT -- THE LAST STEP, WE HAVE NOT ANALYZED THE KNOCKOUT MICE, THE KNOCKOUT MICE CERTAINLY ARE ALIVE, THEY ARE FERTILE, WE HAVE NOT ANALYZED IF THEY HAVE IT -- >> [INAUDIBLE] >> THAT'S RIGHT, WE HAVE -- WE ARE -- WE ARE ACTUALLY MAKING A TPC2 AND A OF TMEM -- I WAS GOING TO MAKE A -- BUT -- MY LACK OF GENETIC -- I FORGOT -- TPC -- AND THE TMEM175 ARE ON THE SAME CHROMOSOME. -- FOR LACK OF -- BECAUSE THEY'RE ON THE SAME CHROMOSOME. WE'RE DOING THOSE KIND OF EXPERIMENTS. >> A QUESTION, SO YOU DIDN'T SHOW US THE LOCALIZATION OF TPC. ARE THEY ALWAYS -- IF YOU LOOK ACROSS TISSUES, ARE THEY ALWAYS IN THE EXTRACELLULAR MEMBRANE OR IS THERE VARIATION FROM CELL-TO-CELL? >> SO WE DID NOT LOOK AT THEM. SEVERAL OTHER LABS HAVE LOOKED AT THEM. MANY -- MANY, THEY ARE ALL FROM EVERY EXPRESS CELLS. SO TPC2 IS -- WITH MARKERS ON THE LYSOSOMES, TPC1 IS IN THE ENDOSOMES. NOW ONE THING, IF YOU OVEREXPRESS THOSE CHANNELS, YOU RECORD -- OCCURRENCE WHICH REFLECTS THE PLASMA MEMBRANE CURRENTS, WHICH TELLS IT YOU EVEN WHEN THEY'RE ON THE MEMBRANE, THEY'RE FUNCTIONING. NOW PP OF C OF 1 -- REMEMBER THAT I SHOWED YOU SOME OF THE LYSOSOMES -- OCCURRED FROM CARDIAC -- YOU CAN SEE THIS ACTION POE IT TENSION LIKE -- BUT YOU DON'T SEE THIS IN MANY OTHER CELLS. NOW TPC1 IS NOT IN EVERY CELL. MY POINT IS THAT THERE IS NOT -- NOT ALL THE LYSOSOMES ARE THE SAME HE YOU MATE THINK ALL THE MITOCHONDRIA IT -- FROM CAN DIFFERENT CELLS ARE THE SAME BUT LYSOSOMES ARE NOT. >> SO YOU MENTIONED EACH CELL -- [INAUDIBLE] >> WE HAVE NOT DONE THAT AND THAT'S A VERY GOOD POINT. THERE IS ALSO COMPLICATION IT -- IDEALLY -- LYSOSOMES THAT'S NOT ENLARGED, MAYBE -- NOW YOU'RE -- WHOLE LYSOSOME RECORDING UNTIL DURING THE ISOLATION PROCESS -- THE SAME CONCENTRATION, ESPECIALLY WHEN YOU HAVE POTASSIUM LEAK THERE, FOR EXAMPLE. >> I'LL END WITH MY FAVORITE QUESTION TO ASK YOU ABOUT, WHICH IS -- AND YOU MENTIONED TO BEGIN WITH ABOUT WHAT MIGHT HAPPEN, HOW MUCH OF WHAT YOU LOOK AT, YOU'RE ALWAYS ENLARGED LYSOSOMES OR ENDOSOMES AND WHAT -- YOU KNOW, WE DON'T KNOW WHAT HAPPENED THOUGH TO THE CURRENT. DO YOU HAVE ANY INSIGHT IN THE MECHANISM OF THE ENLARGEMENT, WHETHER YOU'RE -- EVEN WHETHER YOU'RE RECRUITING MEMBRANE AND GETTING FUSION OF MULTIPLE ORGANELLES OH GET THIS, OR SWELLING BY OSMOTIC -- >> YEAH, SO THAT'S A FAIR QUESTION. SO WE USE SEVERAL DIFFERENT ENLARGEMENT METHODS. ONE, LYSOSOMES, FOR EXAMPLE -- AND -- MOLECULES. THAT SOMETIMES CAUSE ENLARGEMENTS. -- WILL HAVE ENLARGED LYSOSOMES. NOW IF YOU SEE A CURRENT THAT ALL THE -- IN THE LYSOSOME -- ALL THE DIFFERENT METHODS, IF THEY'RE ALL THEN WE THINK MAYBE -- OCCURRENCE UNLESS, ALL THE ENLARGEMENT USE THE SAME MECHANISM. NOW THE MECHANISM OF ENLARGEMENT IS NOT IS CLEAR -- FOR EXAMPLE WE USE, IT'S POSSIBLE THAT ACTUALLY IT -- IT'S SUFFICIENT. SO NOW YOU CAN ASK -- NOW THE CURRENT ARE -- RECORDED -- REALLY -- OCCURRENCE, BUT THERE ARE STILL CRITERIA WE USE. ONE, THE PROTEIN IS LOCALIZED ON LYSOSOME, TWO -- THE CURRENTS AND THE -- AND SODIUM CURRENTS, USUALLY YOU DON'T SEE ON THE PLASMA MEMBRANE, SO THE THAT PERHAPS THEY ARE ONLY FUNCTIONAL ON THE FLOOR -- LYSOSOME ENDOSOME MEMBRANE. NOW THE QUESTION WILL BE OF COURSE THIS IS TRUE FOR OTHER PATCH CLAMP EXPERIMENTS, IS WHAT YOU SEE IS THAT WHAT THE CELL USE. AGAIN I DON'T THINK WE HAVE A GOOD ANSWER FOR THAT. >> ANY OTHER QUESTIONS? THANKS AGAIN. [APPLAUSE]