Received for publication May 14, 2004.
Revised July 9, 2004.
Accepted for publication July 13, 2004.

Mutation in Nucleotide-Binding Domains of Sulfonylurea Receptor 2 Evokes Na-ATP-Dependent Activation of ATP-Sensitive K⁺ Channels*: Implication for Dimerization of Nucleotide-Binding Domains to Induce Channel Opening

Abstract

The ATP-sensitive K⁺ (K_ATP) channel is composed of a sulfonylurea receptor (SUR) and a pore-forming subunit, Kir6.2. SUR is an ABC protein with two nucleotide-binding domains (NBD1 and NBD2). Intracellular ATP inhibits K_ATP channels through Kir6.2 and activates them through NBDs. However, it is still unknown how ATP-bound NBDs activate K_ATP channels. A prokaryotic ABC protein, MJ0796, which is entirely NBD, forms a dimer in the presence of Na-ATP when its glutamate at position 171 is substituted with glutamine. Mg²⁺ or K⁺ destabilizes the dimer. We made the corresponding mutation in the NBD1 (D834N) and/or NBD2 (E1471Q) of SUR2A and SUR2B. As measured in the inside-out configuration of the patch-clamp method, SUR2x(D834N, E1471)/Kir6.2 channels mediated significantly larger currents in the presence of internal 1 mM Na-ATP than K-ATP alone or Mg-ATP. The response to Na-ATP resulted from an increase in the open probability but not single-channel amplitude of the channels and was completely abolished by glibenclamide (10^-5 M). In the presence of 1 mM Mg²⁺-free ATP, Na⁺ increased the activity of the channels in a concentration-dependent manner. The Na-ATP-dependent activation was never observed with K_ATP channels including either the wild-type SUR2x, SUR2x(D834N) or SUR2x(E1471). Nicorandil activated SUR2x(D834N, E1471Q)/Kir6.2 channels more strongly in the presence of Na-ATP than K-ATP alone, whereas the reverse was true for wild-type SUR2x/Kir6.2 channels. Therefore, it is likely that NBDs of SUR2x dimerize in response to ATP and nicorandil. The dimerization induces the opening of the K_ATP channel probably by causing a conformational change of SUR2x.

Key words: Ion channel regulation, Potassium, Nucleoside/Nucleotide, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Single channel kinetics

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