Diazoxide

ABSTRACT The mechanism of ATP-sensitive potassium (K^sub ATP^) channel closure by ATP is unclear, and various kinetic models in which ATP binds to open or to closed states have previously been presented. Effects of phosphatidylinositol bisphosphate (PIP^sub 2^,) and multiple Kir6.2 mutations on ATP inhibition and open probability in the absence of ATP are explainable in kinetic models where ATP stabilizes a closed state and interaction with an open state is not required. Evidence that ATP can in fact interact with the open state of the channel is presented here. The mutant Kir6.2[L164C] is very sensitive to Cd^sup 2+^ block, but very insensitive to ATP, with no significant inhibition in 1 mM ATP. However, 1 mM ATP fully protects the channel from Cd^sup 2+^ block. Allosteric kinetic models in which the channel can be in either open or closed states with or without ATP bound are considered. Such models predict a pedestal in the ATP inhibition, i.e., a maximal amount of inhibition at saturating ATP concentrations. This pedestal is predicted to occur at >50 mM ATP in the L164C mutant, but at >1 mM in the double mutant L164C/R176A. As predicted, ATP inhibits Kir6.2[L164C/R176A] to a maximum of -40%, with a clear plateau beyond 2 mM. These results indicate that ATP acts as an allosteric ligand, interacting with both open and closed states of the channel.

INTRODUCTION

First described in 1983 by Akinori Noma, ATP-sensitive potassium (K^sub ATP^) channels are reversibly inhibited by the nonhydrolytic binding of intracellular ATP (Noma, 1983; Ashcroft, 1988; Nichols and Lederer, 1991). The KA.tp channel is a hetero-octamer formed by an inward-rectifying K+-channel subunit (Kir6.x) and a sulfonylurea receptor (SURx) (Aguilar-Bryan et al., 1995; Inagaki et al., 1995, 1996) in a 4:4 stoichiometry (Clement et al., 1997; Inagaki et al., 1997; Shyng and Nichols, 1997). ATP inhibition occurs through interaction with the Kir6.2 subunit (Shyng et al., 1997a; Tucker et al., 1997, 1998; Tanabe et al., 1999), whereas the SURx subunit confers high-affinity block by sulfonylureas and stimulation by K+ channel openers and MgADP (Aguilar-Bryan et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996; Nichols et al., 1996; Gribble et al., 1997a,b; Shyng et al., 1997b; Schwanstecher et al., 1998).

Although there are a few mutations that alter ATP sensitivity without affecting the channel gating in the absence of ATP (Tucker et al., 1997; Li et al., 2000), most mutations in the Kir6.2 subunit alter sensitivity to ATP inhibition (K^ sub 1/2,ATP^) and open probability in the absence of ATP (P^sub Ozero^) in a strongly correlated manner. This correlation can be quantitatively explained by models that assume ATP binds to the closed state of the channel (Shyng et al., 1997a; Enkvetchakul et al., 2000). In accord with this notion, open-time distributions are generally not altered in the presence of ATP (Alekseev et al., 1998; Drain et al., 1998; Trapp et al., 1998; Enkvetchakul et al., 2000; Li et al., 2000), and diverse steady-state and kinetic nucleotide sensitivity data of wild-type and mutant K^sub ATP^ are reproducible by such a gating scheme (Enkvetchakul et al., 2000).

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D. Enkvetchakul,* G. Loussouarn,^ E. Makhina,^ and C. G. Nichols^

*Division of Renal Medicine and ^Department of Cell Biology and Physiology, and Washington University School of Medicine, St. Louis, Missouri 63110 USA

Received for publication 23 August 2000 and in final form 8 November 2000.

Address reprint requests to Dr. C. G. Nichols, Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Tel.: 314-362-6630; Fax: 314-362-- 7463, E-mail: cnichols@cellbio.wustl.edu.

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