Isacson CK, Lu Q, Karas RH, Cox DH. RACK1 is a BKCa channel binding protein. Am J Physiol Cell Physiol 292: C1459 -C1466, 2007. First published January 31, 2006; doi:10.1152/ajpcell.00322.2006.-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 BK Ca 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 ␤-subunits. Consistent with an important role in BK Ca-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 BK Ca channel biochemically with GST pull-down and coimmunoprecipitation experiments. We have observed some co-localization of RACK1 with the BK Ca channel in vascular smooth muscle cells with immunocytochemical experiments, and we have found that RACK1 has effects on the BK Ca channel's biophysical properties. Thus RACK1 binds to the BK Ca channel and it may form part of a BK Ca-channel regulatory complex in vascular smooth muscle.calcium-activated potassium channel; protein kinase C; smooth muscle LARGE CONDUCTANCE Ca 2ϩ -activated potassium channels (BK Ca channels) are uniquely sensitive to both intracellular Ca 2ϩ concentration and membrane voltage. They are found in many tissues, and they are particularly abundant in nerve and smooth muscle, where they provide feedback control over the Ca 2ϩ and voltage-dependent processes of neurotransmitter release and smooth muscle contraction. In the nervous system, BK Ca channels are activated by Ca 2ϩ entering through nearby calcium channels (26,39,41), and their activity regulates the duration of the nerve-terminal action potential (20,40,47). In smooth muscle, they are activated by Ca 2ϩ released from internal stores (6,30). This causes the membrane potential to hyperpolarize and Ca 2ϩ entry through voltage-gated Ca 2ϩ channels to decrease (17,30,31). In both cases, BK Ca channels regulate tissue function indirectly by regulating Ca 2ϩ entry. Given the BK Ca channel's role as an important regulator of Ca 2ϩ -dependent processes, it is perhaps not surprising that the BK Ca channel itself is regulated by second messengers in addition to calcium, including protein phosphatases (8, 55, 56), heterotrimeric GTP binding proteins (18,19), and protein kinases (36 -38). The BK Ca channel's pore-forming ␣-subunit is phosphorylated by cAMP-dependent protein kinase (PKA) (50, 58), protein kinase C (PKC) (38...