Palmitoylation Controls BK Channel Regulation By Phosphorylation

Abstract

Large conductance calcium- and voltage- gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C-terminus of the pore-forming α-subunits. However, how PKA phosphorylation of the α-subunit effects changes in channel activity are unknown. The STREX variant of BK channels is inhibited by PKA as a result of phosphorylation of a serine residue within the evolutionary conserved STREX insert. As this inhibition is dependent upon phosphorylation of only a single α-subunit in the channel tetramer we hypothesised that phosphorylation results in major conformational rearrangements of the C-terminus. Using a combined imaging, biochemical and electrophysiological strategy we have defined the mechanism of PKA-inhibition of BK channels. We demonstrate that the cytosolic C-terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionary conserved cysteine residues. PKA-phosphorylation of STREX dissociates the C-terminus from the plasma membrane resulting in channel inhibition. Abolition of channel palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl-transferases prevents PKA-mediated inhibition. Thus PKA inhibition of BK channels is conditional upon the palmitoylation status of the channel. Palmitoylation and phosphorylation are both dynamically regulated thus cross-talk between these two major post-translational signalling cascades provides a novel mechanism for conditional regulation of BK channels. Interplay of these distinct signalling cascades has important implications for the dynamic regulation of BK channels and the control of physiological homeostasis.