Syntaxin 1A co-associates with native rat brain and cloned large conductance, calcium-activated potassium channels in situ.
Large conductance, calcium-activated potassium channels (BKCa channels) are regulated by several distinct mechanisms, including phosphorylation/dephosphorylation events and protein-protein interactions. In this study, we have examined the interaction between BKCa channels and syntaxin 1A, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is reported to modulate the activity and/or localization of different classes of ion channels. Using a reciprocal co-immunoprecipitation strategy, we observed that native BKCa channels in rat hippocampus co-associate with syntaxin 1A, but not the closely related homologue syntaxin 3. This BKCa channel-syntaxin 1A interaction could be further demonstrated in a non-neuronal cell line (human embryonic kidney (HEK) 293 cells) following co-expression of rat syntaxin 1A and BKCa channels cloned from either mouse brain or bovine aorta. However, co-expression of these same channels with syntaxin 3 did not lead to a detectable protein-protein interaction. Immunofluorescent co-staining of HEK 293 cells expressing BKCa channels and syntaxin 1A demonstrated overlapping distribution of these two proteins in situ. Functionally, co-expression of BKCa channels with syntaxin 1A, but not syntaxin 3, was observed to enhance channel gating and kinetics at low concentrations (1-4 microM) of free cytosolic calcium, but not at higher concentrations (< or = 10 microM), as judged by macroscopic current recordings in excised membrane patches. Interactions between BKCa channels and neighbouring membrane proteins may thus play important roles in regulating the activity and/or distribution of these channels within specialized cellular compartments.
Ling
Shizhang
S
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Alberta, Canada.
Sheng
Jian-Zhong
JZ
Braun
Janice E A
JE
Braun
Andrew P
AP
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D001013:Aorta, Thoracic / Q000378:metabolism; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D015221:Potassium Channels / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D011233:Precipitin Tests; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D051381:Rats; D050827:Syntaxin 1; D014162:Transfection
D000954:Antigens, Surface; D018076:DNA, Complementary; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D024642:Potassium Channels, Voltage-Gated; D050765:Qa-SNARE Proteins; C494212:STX1A protein, human; C494214:Stx1a protein, mouse; C494213:Stx1a protein, rat; D050827:Syntaxin 1; C059983:potassium channel protein I(sk)