Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25.
Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.
School of Public Health and Family Medicine, Capital University of Medical Sciences, Beijing, China.
Yan
Y
He
Youhou
Y
Kang
Yuk-Man
YM
Leung
Fuzhen
F
Xia
Xiaodong
X
Gao
Huanli
H
Xie
Herbert Y
HY
Gaisano
Robert G
RG
Tsushima
D000494:Allosteric Regulation; D000818:Animals; D018726:Anti-Dyskinesia Agents / Q000378:metabolism / Q000494:pharmacology; D001905:Botulinum Toxins / Q000378:metabolism / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D051669:Shab Potassium Channels / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019789:Tetraethylammonium / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D018726:Anti-Dyskinesia Agents; C496991:Kcnb1 protein, rat; D010455:Peptides; D011993:Recombinant Fusion Proteins; D051669:Shab Potassium Channels; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D019789:Tetraethylammonium; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096326:botulinum toxin type E