Two SUR1-specific Histidine Residues Mandatory for Zinc-induced Activation of the Rat KATP Channel

Dominique Monnier,Cécile Faure,Yves Dunant,Frédéric Chanson,Victor Bancila,Thierry Cens,Alain Bloc

doi:10.1074/jbc.m413426200

Abstract

Zinc at micromolar concentrations hyperpolarizes rat pancreatic beta-cells and brain nerve terminals by activating ATP-sensitive potassium channels (KATP). The molecular determinants of this effect were analyzed using insulinoma cell lines and cells transfected with either wild type or mutated KATP subunits. Zinc activated KATP in cells co-expressing rat Kir6.2 and SUR1 subunits, as in insulinoma cell lines. In contrast, zinc exerted an inhibitory action on SUR2A-containing cells. Therefore, SUR1 expression is required for the activating action of zinc, which also depended on extracellular pH and was blocked by diethyl pyrocarbonate, suggesting histidine involvement. The five SUR1-specific extracellular histidine residues were submitted to site-directed mutagenesis. Of them, two histidines (His-326 and His-332) were found to be critical for the activation of KATP by zinc, as confirmed by the double mutation H326A/H332A. In conclusion, zinc activates KATP by binding itself to extracellular His-326 and His-332 of the SUR1 subunit. Thereby zinc could exert a negative control on cell excitability and secretion process of pancreatic beta-and alpha-cells. In fact, we have recently shown that such a mechanism occurs in hippocampal mossy fibers, a brain region characterized, like the pancreas, by an important accumulation of zinc and a high density of SUR1-containing KATP.

Highlights

KATP1 channels are tetradimeric complexes of two structurally unrelated subunits [1,2,3,4]: an inwardly rectifying Kϩ channel subunit (Kir6.x), which serves as an ATP-inhibitable pore [5], and a sulfonylurea receptor subunit (SUR), which belongs to the ATP-binding cassette transporter superfamily and endows the channel with sensitivity to magnesium nucleotides, channel openers, and sulfonylureas [6]
Several structures possessing KATP channels of the Kir6.2 and SUR1 type, such as pancreatic ␤-cells and hippocampal mossy fibers, contain substantial amounts of zinc (16 –18). Puzzled by this co-localization, we found that micromolar concentrations of zinc hyperpolarize ␤-cells from an insulin-secreting pancreatic line (RINm5F) by activating KATP channels [19]
The rat Kir6.2/SUR1 KATP was reconstituted in HEK293T cells, a human embryonic kidney cell line, which does not express endogenous KATP channels [23]

Summary

Introduction

KATP1 channels are tetradimeric complexes of two structurally unrelated subunits [1,2,3,4]: an inwardly rectifying Kϩ channel subunit (Kir6.x), which serves as an ATP-inhibitable pore [5], and a sulfonylurea receptor subunit (SUR), which belongs to the ATP-binding cassette transporter superfamily and endows the channel with sensitivity to magnesium nucleotides, channel openers, and sulfonylureas [6]. The pancreatic ␤-cell KATP channels involved in insulin secretion are comprised of Kir6.2 and SUR1 This pattern is abundant in the mammalian central nervous system, especially in the hippocampal mossy fiber nerve terminals [15]. Several structures possessing KATP channels of the Kir6.2 and SUR1 type, such as pancreatic ␤-cells and hippocampal mossy fibers, contain substantial amounts of zinc (16 –18) Puzzled by this co-localization, we found that micromolar concentrations of zinc hyperpolarize ␤-cells from an insulin-secreting pancreatic line (RINm5F) by activating KATP channels [19]. In contrast to our results, Kwok and Kass [21] observed a block of cardiac KATP channels by low concentrations of extracellular divalent cations, including zinc This discrepancy led us to hypothesis that zinc could induce opposite effects on different types of KATP, depending on the subunit composition of the channel. By using pH modifications, pharmacological tools, and site-directed mutagenesis, we identified two histidine residues present in the extracellular side of SUR1 as mandatory for activation of KATP channels by zinc

Results

Discussion

Conclusion