Palmitoylation of the K _ATP channel Kir6.2 subunit promotes channel opening by regulating PIP ₂ sensitivity

Abstract

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2. Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.

Regulatory effects of protein S-acylation on insulin secretion and insulin action

“…Similar experiments performed in rat cardiomyocytes confirmed the S-acylation of Kir6.2 and showed that this modification was enhanced following incubation of the cells with either palmitate or a combination of ML348 and ML349, selective inhibitors of the thioesterases APT1 and APT2, respectively [ 31 ]. Electrophysiological recordings of inside-out membrane patches showed that treatment with palmitate or ML348/ML349 promoted an increase in mean patch current and open time and decreased sensitivity to ATP; these effects were seen for endogenous channels in rat cardiomyocytes and INS1 pancreatic β-cells, and in HEK293 cells transfected with Kir6.2 together with SUR1 or SUR2A [ 31 ]. Assuming that the effect of these treatments reflects changes in the S-acylation of Kir6.2, it is likely that S-acylation regulates the gating or the modulation of K ATP activity as cell surface levels of the channel were unaffected [ 31 ] ( figure 2 ).…”

“…Functional K ATP channels consists of four Kir6.x subunits, which assemble together with 4 SUR subunits, and in pancreatic β-cells these channels are composed of Kir6.2 and SUR1 subunits. A recent study using ABE assays on co-transfected HEK293 cells suggested that Kir6.2 (but not SUR1) is modified by S-acylation [ 31 ]. Similar experiments performed in rat cardiomyocytes confirmed the S-acylation of Kir6.2 and showed that this modification was enhanced following incubation of the cells with either palmitate or a combination of ML348 and ML349, selective inhibitors of the thioesterases APT1 and APT2, respectively [ 31 ].…”

“…Electrophysiological recordings of inside-out membrane patches showed that treatment with palmitate or ML348/ML349 promoted an increase in mean patch current and open time and decreased sensitivity to ATP; these effects were seen for endogenous channels in rat cardiomyocytes and INS1 pancreatic β-cells, and in HEK293 cells transfected with Kir6.2 together with SUR1 or SUR2A [ 31 ]. Assuming that the effect of these treatments reflects changes in the S-acylation of Kir6.2, it is likely that S-acylation regulates the gating or the modulation of K ATP activity as cell surface levels of the channel were unaffected [ 31 ] ( figure 2 ). Indeed, further analysis suggested that palmitate and ML348/ML349 destabilized the closed state of the channel and increased sensitivity to phosphatidylinositol 4,5-bisphosphate (PIP2), which promotes the channel transition from closed to open state [ 31 ].…”

The Pancreatic β-Cell: The Perfect Redox System

“…ATP binding to one of four ATP binding sites has already been reported to close the channel [ 136 ]. Moreover, the palmitoylation of Cys166 of Kir6.2 was found to enhance its sensitivity to PIP 2 [ 137 ].…”

The fine‐tuning of cell membrane lipid bilayers accentuates their compositional complexity

“…Phosphatidylinositol 4,5‐bisphosphate (PIP2) can activate membrane protein channels (i.e., inward‐rectifying potassium channels) [ 105 ] by direct binding but the membrane environment or allosteric presence of other lipids also contributes to channel activity. [ 106 ] The inward rectifier K + channel Kir6.2 becomes sensitized to PIP2 when palmitoylated but an additional lipid acyl chain also contributes hydrophobic contacts to the PI binding site, [ 107 ] and the closely related Kir2.2 exhibits allosteric binding by PS at an additional, anionic lipid binding site. [ 106 ] PIP2 itself can act by different modes on different membrane proteins.…”