Distinct stoichiometry of BKCa channel tetrarner phosphorylation specifies channel activation and inhibition by cAMP-dependent protein kinase

Large conductance voltage- and calcium-activated potassium (BKCa) channels are important signaling molecules that are regulated by multiple protein kinases and protein phosphatases at multiple sites. The pore-forming alpha-subunits, derived from a single gene that undergoes extensive alternative pre-mRNA splicing, assemble as tetramers. Although consensus phosphorylation sites have been identified within the C-terminal domain of a-subunits, it is not known whether phosphorylation of all or single a-subunits within the tetramer is required for functional regulation of the channel. Here, we have exploited a strategy to study single-ion channels in which both the a-subunit splice-variant composition is defined and the number of consensus phosphorylation sites available within each tetramer is known. We have used this approach to demonstrate that cAMP-dependent protein kinase (PKA) phosphorylation of the conserved C-terminal PKA consensus site (S899) in all four a-subunits is required for channel activation. In contrast, inhibition of BKCa channel activity requires phosphorylation of only a single a-subunit at a splice insert (STREX)-specific PKA consensus site (S4(STREX)). Thus, distinct modes of BKCa channel regulation by PKA phosphorylation exist: an all-or-nothing rule for activation and a single-subunit rule for inhibition. This essentially digital regulation has important implications for the combinatorial and conditional regulation of BKCa channels by reversible protein phosphorylation.