Syntaxin 1A co-associates with native rat brain and cloned large conductance, calcium-activated potassium channels in situ

Abstract

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 μM) of free cytosolic calcium, but not at higher concentrations (≤ 10 μM), 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.