Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

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 poreforming ␣-subunits. However, the molecular mechanism(s) by which phosphorylation of the ␣-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single ␣-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.L arge conductance calcium-and voltage-gated potassium (BK) channels are potently regulated by protein phosphorylation (1) and are important determinants of neuronal, cardiovascular, endocrine, and epithelial function where channel dysfunction may lead to major disorders such as hypertension (2, 3), ataxia (4), epilepsy (5, 6), and incontinence (7). BK channels are potently regulated by phosphorylation, and several putative phosphorylation motifs on the pore-forming ␣-subunit have been identified (8-12). However, as for other potassium channels, the molecular basis through which phosphorylation of the ␣-subunit effects changes in BK channel activity is essentially unknown.BK channel pore-forming ␣-subunits are encoded by a single gene, KCNMA1 (13), and native BK channels show functional heterogeneity in their response to protein kinase A (PKA)-mediated phosphorylation. This diversity results, in large part, from the extensive alternative pre-mRNA splicing of the pore-forming ␣-subunits (10, 12). 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 ...

Unconventional myristoylation of large-conductance Ca 2+ -activated K + channel (Slo1) via serine/threonine residues regulates channel surface expression

“…This treatment ruptures labile thioester bonds such as those of Spalmitoylated proteins but leaves intact oxyester and amide linkages (12). Cells radiolabeled with [ 3 H]palmitic acid confirmed the incorporation of this fatty acid into hSlo1, as reported earlier for murine Slo1 (13). As expected, when [ 3 H]palmitatelabeled hSlo1 was treated with 1.4 M β-mercaptoethanol, the signal was almost completely removed (Fig.…”

“…Recent reports indicate that palmitoylation modulates Kv1.1 channel voltage sensitivity and channel kinetics (21), whereas it modulates Kv1.5 surface expression (22). Also, palmitoylation of the mSlo1-STREX variant allows efficient plasma membrane targeting of the intracellular C terminus, supporting channel inhibition by protein kinase A phosphorylation (13). We now show that myristoylation regulates hSlo1 surface expression but does not affect the channel steadystate voltage-dependent activation.…”

“…Although ion channels are integral parts of the cell signaling machinery, only a few studies have identified ion channel palmitoylation (13,(19)(20)(21)(22). Furthermore, no information on direct myristoylation of any ion channel has been reported yet.…”

Dual role of protein kinase C on BK channel regulation

“…To allow complete dephosphorylation of PKC sites, inside-out patches were first superfused for 5 min with ATP-free solution to which 5 μM PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] had been added. This solution, which did not significantly alter NP o , was then changed for another 5 min to a solution containing ATP, PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] , and either PKG or PKA. Similar to the results obtained with the mutant BK channel S 1151 A (Fig.…”

“…Reduced G max could not be reversed by increasing voltage or Ca 2+ . The inhibitory PKC effect was abolished when inside-out patches were concurrently superfused for at least 5 min with PKC c and 5 μM of the PKC pseudosubstrate inhibitor peptide PKC [19][20][21][22][23][24][25][26][27][28][29][30][31] (Fig. 1A).…”