Distinct stoichiometry of BK _Ca channel tetramer phosphorylation specifies channel activation and inhibition by cAMP-dependent protein kinase

Abstract

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 ␣-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 ␣-subunits, it is not known whether phosphorylation of all or single ␣-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 ␣-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 ␣-subunits is required for channel activation. In contrast, inhibition of BK Ca channel activity requires phosphorylation of only a single ␣-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 BK Ca channels by reversible protein phosphorylation.L arge conductance voltage-and calcium-activated potassium (BK Ca ) channels are important regulators of cellular function in the endocrine, nervous, cardiovascular, and immune systems (1-6). BK Ca channels are assembled as tetramers (7, 8) of pore-forming ␣-subunits encoded by a single gene (9) that undergoes extensive alternative splicing (10, 11). Distinct ␣-subunit splice-variant mRNAs may be expressed in the same cell, differentially expressed between tissues, or even neighboring cells (12, 13), and dynamic modification of splice-variant mRNA expression (14, 15) may result in altered BK Ca channel phenotype and cellular regulation (5).Similar to other tetrameric potassium channels BK Ca channels are potently regulated by a variety of serine͞threonine protein kinases (16), including cAMP-dependent protein kinase (PKA) (10,(17)(18)(19)(20). The functional response of BK Ca channels to PKA phosphorylation depends on the splice-variant ␣-subunit composition of the tetramer (10, 19). For example, PKA activates homotetramers of mammalian ZERO splice variants (10,17,19), whereas PKA inhibits homotetramers of STREX variants (10). This differential regulation of BK Ca channels by PKA depends on functional consensus of PKA phosphorylation sites within the C terminus of the ␣-subunit. PKA activation of ZERO variants requires a functional conserved C-terminal PKA site (S899) (10,17,19), whereas PKA inhibition of STREX requires a functional PKA site (S4 STREX ) within the STREX insert (10).However, it is unknown how many ␣-subunits...

Control of alternative pre-mRNA splicing by Ca++ signals

“…4B), varying the stimulus strength will induce a gradient of the relative levels of the two variant proteins leading to the formation of heterotetrameric channels with varying ratios of variant subunits and the generation of electrical currents of higher diversity than with only two homotetramers. In fact, splicing changes can be KCl concentration-dependent [84,105], and different ratios of two BK channel subunits generate channel current kinetics in-between the two homotetramer channels [19,201]. This way, alternative splicing regulation by Ca ++ signals will greatly contribute to the fine-tuning of electrical properties of individual neurons.…”

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

“…PKA inhibition of STREX channels results from phosphorylation of serine residue 3 within the STREX insert (10,14) that is just upstream of the site of palmitoylation (C12:13). We reasoned that phosphorylation of S3, which would introduce a negative charge into an otherwise basic region immediately upstream of C12:13, might result in destabilization of the STREX domain with the plasma membrane leading to significant structural rearrangements and thus channel inhibition.…”

“…Inclusion of the stress regulated exon (STREX) (15) in the intracellular C terminus generates an additional PKA consensus motif (serine residue 3 of the STREX insert, S3) that results in channel inhibition by PKA (10,14). PKA inhibition of STREX follows a single-subunit rule, whereby only 1 ␣-subunit within the BK channel tetramer is required to be phosphorylated by PKA on S3 for inhibition to be conferred (14). Thus, phosphorylation of a single STREX ␣-subunit probably induces major conformational rearrangements in the BK channel C terminus to mediate channel inhibition.…”

“…RQPS 899 results in BK channel activation (9,10,14). Inclusion of the stress regulated exon (STREX) (15) in the intracellular C terminus generates an additional PKA consensus motif (serine residue 3 of the STREX insert, S3) that results in channel inhibition by PKA (10,14). PKA inhibition of STREX follows a single-subunit rule, whereby only 1 ␣-subunit within the BK channel tetramer is required to be phosphorylated by PKA on S3 for inhibition to be conferred (14).…”

“…Previous studies have demonstrated that PKA phosphorylation of a conserved C-terminal phosphorylation motif. RQPS 899 results in BK channel activation (9,10,14). Inclusion of the stress regulated exon (STREX) (15) in the intracellular C terminus generates an additional PKA consensus motif (serine residue 3 of the STREX insert, S3) that results in channel inhibition by PKA (10,14).…”