Distinct stoichiometry of BKCa channel tetramer phosphorylation specifies channel activation and inhibition by cAMP-dependent protein kinase.
Large conductance voltage- and calcium-activated potassium (BK(Ca)) 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 alpha-subunits, it is not known whether phosphorylation of all or single alpha-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 alpha-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 alpha-subunits is required for channel activation. In contrast, inhibition of BK(Ca) channel activity requires phosphorylation of only a single alpha-subunit at a splice insert (STREX)-specific PKA consensus site (S4(STREX)). Thus, distinct modes of BK(Ca) 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.
Membrane Biology Group, Division of Biomedical Science, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Lijun
L
Tian
Lorraine S
LS
Coghill
Heather
H
McClafferty
Stephen H-F
SH
MacDonald
Ferenc A
FA
Antoni
Peter
P
Ruth
Hans-Guenther
HG
Knaus
Michael J
MJ
Shipston
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D016384:Consensus Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004594:Electrophysiology; D051379:Mice; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D020033:Protein Isoforms; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism
D015221:Potassium Channels; D020033:Protein Isoforms; D021122:Protein Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium