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.
Tian
Lijun
L
Membrane Biology Group, Division of Biomedical Science, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Coghill
Lorraine S
LS
McClafferty
Heather
H
MacDonald
Stephen H-F
SH
Antoni
Ferenc A
FA
Ruth
Peter
P
Knaus
Hans-Guenther
HG
Shipston
Michael J
MJ
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