Leucine zipper domain targets cAMP-dependent protein kinase to mammalian BK channels.
Large conductance, calcium- and voltage-activated potassium (BK) channels control excitability in many tissues and are regulated by several protein kinases and phosphatases that remain associated with the channels in cell-free patches of membrane. Here, we report the identification of a highly conserved, non-canonical, leucine zipper (LZ1) in the C terminus of mammalian BK channels that is required for cAMP-dependent protein kinase (PKA) to associate with the channel and regulate its activity. A synthetic polypeptide encompassing the central d position leucine residues in LZ1 blocks the regulation of recombinant mouse BK channels by endogenous PKA in HEK293 cells. In contrast, neither an alanine-substituted LZ1 peptide nor a peptide corresponding to another, more C-terminal putative leucine zipper, LZ2, had any effect on regulation of the channels by endogenous PKA. Mutagenesis of the central two LZ1 d position leucines to alanine in the BK channel also eliminated regulation by endogenous PKA in HEK293 cells without altering the channel sensitivity to activation by voltage or by exogenous purified PKA. Inclusion of the STREX splice insert in the BK channel protein, which switches channel regulation by PKA from stimulation to inhibition, did not alter the requirement for an intact LZ1. Although PKA does not bind directly to the channel protein in vitro, mutation of LZ1 abolished co-immunoprecipitation of PKA and the respective BK channel splice variant from HEK293 cells. Furthermore, a 127-amino acid fusion protein encompassing the functional LZ1 domain co-immunoprecipitates a PKA-signaling complex from rat brain. Thus LZ1 is required for the association and regulation of mammalian BK channels by PKA, and other putative leucine zippers in the BK channel protein may provide anchoring for other regulatory enzyme complexes.
Tian
Lijun
L
Membrane Biology Group, Division of Biomedical Sciences, University of Edinburgh Medical School, Scotland, United Kingdom.
Coghill
Lorraine S
LS
MacDonald
Stephen H-F
SH
Armstrong
David L
DL
Shipston
Michael J
MJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels; D016350:Leucine Zippers; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D024681:Potassium Channels, Calcium-Activated / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases