RACK1 is a BKCa channel binding protein

Abstract

The large conductance calcium-activated potassium channel, or BKCa channel, plays an important feedback role in a variety of physiological processes, including neurotransmitter release and smooth muscle contraction. Some reports have suggested that this channel forms a stable complex with regulators of its function, including several kinases and phosphatases. To further define such signaling complexes, we used the yeast two-hybrid system to screen a human aorta cDNA library for proteins that bind to the BKCa channel's intracellular, COOH-terminal “tail”. One of the interactors we identified is the protein receptor for activated C kinase 1 (RACK1). RACK1 is a member of the WD40 protein family, which also includes the G protein β-subunits. Consistent with an important role in BKCa-channel regulation, RACK1 has been shown to be a scaffolding protein that interacts with a wide variety of signaling molecules, including cSRC and PKC. We have confirmed the interaction between RACK1 and the BKCa channel biochemically with GST pull-down and coimmunoprecipitation experiments. We have observed some co-localization of RACK1 with the BKCa channel in vascular smooth muscle cells with immunocytochemical experiments, and we have found that RACK1 has effects on the BKCa channel's biophysical properties. Thus RACK1 binds to the BKCa channel and it may form part of a BKCa-channel regulatory complex in vascular smooth muscle.