Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters: indications for a long-chain acyl-CoA ester binding motif

Abstract

Aims/hypothesis The pancreatic beta cell ATP-sensitive potassium (K ATP ) channel, composed of the pore-forming α subunit Kir6.2, a member of the inward rectifier K+ channel family, and the regulatory β subunit sulfonylurea receptor 1 (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate K ATP channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. Materials and methods Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif. The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1.Results Oocytes expressing Kir6.2ΔC26 K332A repeatedly generated K + currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 μmol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2ΔC26), Kir6.2ΔC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2ΔC26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K ATP channel and Kir6.2ΔC26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. Conclusions/interpretation These results verify that LCCoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.

Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells

“…LC-CoAs esters are also potent stimulators of K ATP channels activity in human beta cells and do not require the presence of Mg 21 or nucleotides (Bränström et al, 2004). The increase in the activity of K ATP channels occurs by interaction of LC-CoAs in the carboxyl-terminal domain of Kir6.2 subunit (Gribble et al, 1998b), where the Lys332 residue is a key structural determinant (Bränström et al, 2007). Moreover, the effects of LC-CoAs on the activity of K ATP channels depend on both the length and the saturation degree of the acyl chain.…”

The pyruvate carboxylase-pyruvate dehydrogenase axis in islet pyruvate metabolism: Going round in circles?

“…59,60 There is evidence that increased levels of cytosolic long-chain acyl-CoA may stimulate or potentiate GSIS through activation of atypical PKC 61 or via interaction at the pore-forming subunit Kir6.2. 62 There is also evidence that addition of palmitate, but not palmitoyl-CoA, to mouse islets increases insulin secretion by a K ATP channel-independent mechanism. 63 This effect of palmitate was exerted at the level of exocytosis and involved augmentation of L-type Ca 2+ currents and an increase in the readily releasable pool of secretory granules.…”

A beta cell-specific knockout of hormone-sensitive lipase in mice results in hyperglycaemia and disruption of exocytosis

“…Here, HSL may generate lipid coupling signals for insulin secretion via lipolysis, including long-chain acyl-CoAs (LC-CoAs) and diacylglycerol [1,[6][7][8][9]. To this end, LC-CoAs have been shown to stimulate insulin secretion, possibly via the activation of protein kinase C [10], direct interaction with the exocytotic machinery [11] or via activation of the ATP-sensitive K + channel [12]. The critical role of lipolysis has been further highlighted by studies using lipase inhibitors: thus orlistat, a pan-lipase inhibitor, blocks lipolysis and insulin secretion in rat islets [13], while a selective inhibitor of HSL has been shown to inhibit insulin secretion from rat islets [14].…”