10 6 0:30-0:40 Session 0:40-1:10 Rac Michelle Lennartz 1:10-1:40 nnos 1:40-2:10 2:10-2:20 Session 2:20-2:50 2:50-3:20 NMDA NR1 3:20-3:50 GABA 3:50-4:00 Session 4:00-4:30
4:30-5:00 Vacuolar-type H + -ATPase 5:00-5:30 ph 5:30-6:00 6:30-10 7 Session 8:45-9:15 KCNQ/M PIP2 PKC 9:15-9:45 HERG 9:45-10:15 Ca2+ TRPM2 10:15-10:25 Session 10:25-10:55 TRPV2 10:55-11:25 PLC-zeta Ca2+ 11:25-11:55 Ca2+ 11:55-12:25
Rac Michelle Lennartz Albany Medical College NADPH Rac1: 6 ; Rac3: 4 ; Rac2: 3 oxidase (Nox2, p22 phox ) PB GFP (p47 phox, p67 phox, p40 phox, Rac) PB small GTPase Rac protein lipid overlay assay Rac1 PB PI(3)P PI(4)P PI(5)P p47 phox -p67 phox -p40 phox PI(3,4,5)P3PA Rac2 PB Rac2 Rac (Rac(Q61L)) GFP Rac (1, 2, 3) FcγR PB Rac PB Rac C 10 polybasic region (PB) GFP Rac Rac1 GFP Rac Rac PB Rac2 PB Rac3Rac2 C PB PB
nnos 1) 1) 2) 2) 3) 3) 1) 1 2 3 MD L-Arginine (1 mm) (NO) NO TGF MD NaCl ([NaCl]l) TGF NO NO BAPTA-AM (Ca 2+ chelator) 50 µm 7-nitroindazole (nnos ) [NaCl][Na + ] (nnos) [Cl - ] [Na + ] NO NO MD (NE-MD) [NaCl]furosemide (Na + -K + -2Cl - Na + -H + MD nnos )amiloride (Na + -H + furosemide [C l - ]l ) nnos (Western blotting)no (NO ) Ca 2+ ph nnos furosemide
( ) UV EPSC (VGLUT) Trypan bluevglut EPSC 5.1 H + -10 calyx of H + -ATPase (V-ATPase) bafilomycin A1 Held UV VGLUT 2
1,2,3 2 1 2 JST ICORP/SORST (PREGS) PREGS PREGS LTP 2 NMDA LTD LTP 1Hz LTD, 100Hz LTD EPSP S PREGS PREGS
NMDA NR1 NMDA NR1 3 ACPCACBC cycloleucine ACPCACBC NR1NR2B NMDA 80% 42% NR1 cycloleucine NR1 Non-NMDA GluR2 cycloleucine NR1 GluR2 2
GABA mglur G GPCR mglur Ca 2+ o mglur1 (5) GABABR Ca 2+ o GABABR Gi/o (1) GABABRmGluR1 Ca 2+ o GABABR 1 mglurmglur1 mglur1 mglur1 (2, 3) Ca 2+ o mglur1 GPCR GPCR co-factor 1.Kubo Y, Miyashita T, Murata Y (1998) Science 279, 1722-1725., mglur1 B GABA GABABR Ca 2+ o GPCR (4) Ca 2+ ogababr 2. Tabata T, Aiba A, Kano M (2002) Mol Cell Neurosci 20, 56-68., 3. Tabata T, Kano M (2004) Mol Neurobiol 29, 261-270., 4. Kulik A, Nakadate K, Nyiri G, Notomi T, Malitschek B, Bettler B, Shigemoto R (2002) Eur J Neurosci 15, 291-307., 5. Tabata T, Araishi K, GABABR Hashimoto K, Hashimotodani Y, Van der Putten H, Bettler B, Kano Ca 2+ o M (2004) PNAS 101, 16952-16957.
pairing NMDA F-actin PA-GFP-actin F-actin
Vacuolar-type H + -ATPase ATP V-ATPase DCCD ruffled border H + V1 actin vacuolar-type H + -ATPase (V-ATPase) H + ATP V-ATPase H + actin filament membrane sectorvoatpase catalytic sector (V1) V-ATPase Vacuole V-ATPase H + ATP 0 mv DCCD H + V-ATPase holoenzyme ATP Vo/V 1 diassembly Vo H +
ph 1) 2) 1) 1 2) ph Na + /HCO3-4 (NBC4g) NBC4g NBC4c camp RNA NBC4 NBC4
1 1, 2, 3 1 2 3 (1) lipid raft lipid raft Transgenic lipid raft 1 m LAT lipid raft CP /GFP lipid raft LAT GFP raft LAT CP LAT CP /GFP MCD cholesterol Transgenic LAT CP /GFP lipid raft lipid raft lipid raft lipid tyrosine raft lipid raft (2) - - RanGTP 30 Ran 8 / 100 125 Mda 10nM100nM
KCNQ/M PIP2PKC 1,2 1,2 1..SORST,JST KCNQ/M Gq KCNQ/M KCNQ/M M 10 M oxo-m M1 KCNQ - (G-V ) PKC PMA KCNQ2 G-V 17.5mV 10 M PIP2 wortmannin PIP 2 PKC G-V PKC PIP 2 M PIP 2 PIP2 KCNQ/M PKC KCNQ M1 5 G-V PKC
HERG K 1) 3) 2 2) 3) HEK293 HERG 10 mm [K + ] HERG 500 mosm1500 mosm n [K + ]100 mm n 1 3 1) 1 2) -0.7 mv/? Osm n K + 1.6 [K + ]100 mm n 0.9 KcsA HERG
Ca 2+ TRPM2 TRPM2 (H2O2) Ca 2+ TRPM2 Ca 2+ TRPM2 TRPM2 TRPM2 extracellular signal-regulated kinase (ERK) NAD + ADP-ribose TRPM2 TRPM2 U937 C Nudix motif H2O2 IL-8 TRPM2 TRPM2 KO TRPM2 KO TRPM2 TRPM2 TRPM2 H2O2 TRPM2 Ca 2+
TRPV2 TRPV2 TRPV TRPV2 sirna Ca 2+ TRPV2 TRPV2 fmlp fmlp PI- fmlp Cs + TRPV ruthenium red TRPV2siRNA Cs + fmlp fmlp Ca 2+ ruthenium red TRPV2 TRPV2 TRPV2 Ca 2+ Ca 2+ ruthenium red
PLC-zeta Ca 2+ phospholipase C-zeta (PLCζ ) Ca 2+ portion) PLCζ Venus RNA 30 Ca 2+ Ca 2+ PLCζRNA PLCζ PLCζ N EF-hand domain X Y catalytic domain C C2 domain X Y PLC (hinge point mutation EF-hand domain N 10 Ca 2+ Ca 2+ PLCζ RNA EF-hand domain Ca 2+ -binding site
Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ IP 3 Ca 2+ IP 3 Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+ Ca 2+
Total Internal Reflection Fluorescence Microscopy: TIRFM 2 nm SN TIRFM TIRFM TIRF