Focused issue on KATP channels
A functional role of the C-terminal 42 amino acids of SUR2A and SUR2B in the physiology and pharmacology of cardiovascular ATP-sensitive K+ channels

https://doi.org/10.1016/j.yjmcc.2004.11.022Get rights and content

Abstract

The ATP-sensitive K+ (KATP) channel is composed of four pore-forming Kir6.2 subunits and four sulfonylurea receptors (SUR). Intracellular ATP inhibits KATP channels through Kir6.2. SUR is an ABC protein bearing transmembrane domains and two nucleotide-binding domains (NBD1 and NBD2). SUR increases the open probability of KATP channels by interacting with ATP and ADP through NBDs and with K+ channel openers such as nicorandil through its transmembrane domain. Because NBDs and the drug receptor allosterically interact with each other, nucleotides and drugs probably activate KATP channels by causing the same conformational change of SUR. SUR2A and SUR2B have the identical drug receptor and NBDs and differ only in the C-terminal 42 amino acids (C42). Nonetheless, nicorandil ~100 times more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Based on our allosteric model, we have analyzed the interaction between NBDs and the drug receptor in SUR2A and SUR2B and found that both nucleotide-bound NBD1 and NBD2 more strongly induce the conformational change in SUR2B than SUR2A. Therefore, C42 modulates the function of not only NBD2 which is close to C42 in a primary structure but NBD1 which is more than 630 amino acid N-terminal to C42. This raises the possibility that in the presence of nucleotides, NBD1 and NBD2 dimerize to induce the conformational change and that the dimerization enables C42 to gain access to both NBDs. Modulation of the nucleotide-NBD1 and -NBD2 interactions by C42 would determine the stability of the nucleotide-dependent dimer and thus, the physiological and pharmacological properties of KATP channels.

Introduction

The ATP-sensitive K+ (KATP) channel is the inwardly rectifying K+ channel which is inhibited by intracellular ATP and activated by intracellular ADP [1]. Thus, it opens under metabolic stress to suppress the excitability of the cell membrane. This channel is a heterooctamer composed of four pore-forming Kir6.x subunits and four sulfonylurea receptors (SUR) (Fig. 1B) [2], [3]. Kir6.x is a K+ channel subunit with two transmembrane domains [4], [5]. There are two genes for Kir6.x which encode Kir6.1 and Kir6.2. SUR is a member of ABC proteins with 17 transmembrane domains and two cytoplasmic nucleotide-binding domains between the 11th and 12th transmembrane domains (NBD1) and at the C-terminus following 17th transmembrane domain (NBD2) (Fig. 1A) [2]. There are two genes for SUR which encode SUR1 and SUR2 [6], [7], and two splice variants of SUR2 (SUR2A and SUR2B) [8]. SUR2A and SUR2B differ only in the C-terminal 42 amino acids (C42) which are located 5 amino acid C-terminal to NBD2. Intracellular ATP inhibits KATP channels through Kir6.2 [9], [10]. SUR increases the open probability of KATP channels by interacting with ATP and ADP through its NBDs [11]. In this reaction, NBD1 and NBD2 seem to operate not independently but cooperatively [12], [13].

SUR2A and Kir6.2 form the KATP channel in cardiac myocytes [7] whereas SUR2B and Kir6.1 make up one in vascular smooth muscle cells [14], [15]. The SUR2B/Kir6.1 channel differs from the KATP channels composed of Kir6.2 and either of the SUR types in the single-channel conductance and spontaneous activity in the absence of intracellular nucleotides due to the different Kir6.x subunits [14]. However, SUR2A and SUR2B form with Kir6.2 KATP channels with identical single-channel conductance and characteristic spontaneous activity [7], [8].

A number of agents collectively termed K+ channel openers activate cardiovascular KATP channels [1]. These drugs cause their effect by interacting with the 17th transmembrane domain of SUR2A or SUR2B [16], [17]. Many K+ channel openers activate KATP channels in concert with intracellular nucleotides [18], indicating that there is an allosteric interaction between the drug receptor and NBDs [19]. SUR2A and SUR2B have the identical drug receptor and NBDs [8], [16]. Nonetheless, SUR2A/Kir6.2 and SUR2B/Kir6.2 channels exhibit strikingly different responses to some agents such as nicorandil. Because C42 is close to NBD2 in a primary structure [8], C42 may modulate the nucleotide-NBD interactions, thereby creating the different profiles of SUR2A/Kir6.2 and SUR2B/Kir6.2 channels [20], [21].

We recently proposed an allosteric model of SUR2A and SUR2B [19]. In this article, we quantitatively analyze the responses of SUR2A/Kir6.2 and SUR2B/Kir6.2 channels to nicorandil with this model and thereby delineate the functional role of C42 in the nucleotide- and drug-induced activation of cardiovascular KATP channels.

Section snippets

Allosteric model of SUR2x

Fig. 1B shows our allosteric model of SUR2A or SUR2B (SUR2x) [19]. It is plausible that intracellular nucleotides and drugs induce the opening the Kir6.2 channel pore by causing a conformational change of SUR [11], [18], [22], [23], [24], [25]. For the sake of simplicity, we assume that SUR2x can adopt two distinct conformations which are able (R conformation) and unable (T conformation) to induce the pore opening, and that these two conformations are intrinsically in equilibrium determined by

Analysis of the concentration-dependent effect of nicorandil on SUR2x/Kir6.2 channels with the allosteric model

Fig. 2 shows the concentration–response relationships of the nicorandil-induced activation of SUR2x/Kir6.2 channels [19]. In this analysis, we used the SUR2x with or without the mutations in the Walker A motif in NBD1 (K708A) and/or NBD2 (K1349A) which disrupt the nucleotide-NBD interactions. The currents through wild-type SUR2x/Kir6.2 (SUR2x(Wt)/Kir6.2), SUR2x(K708A)/Kir6.2, SUR2x(K1349A)/Kir6.2, and SUR2x(K708A, K1349A)/Kir6.2 channels were measured at –60 mV with the symmetrical ~140 mM K+

Implication for the structure–function relationship of SUR2x

The above results indicate that C42 affects the function of both NBD1 and NBD2. C42 is only 5 amino acid C-terminal to NBD2 but more than 630 amino acid C-terminal to NBD1 [8]. Therefore, C42 seems to exist in the vicinity of both NBD1 and NBD2 in a tertiary structure. This raises the possibility that NBD1 and NBD2 of SUR2x dimerize as is the case for the NBD of some ABC proteins [24], [25], [26], [27], and that in the dimer, C42 can gain access to both NBDs. Fig. 4 shows the structure of the

Limitation of our allosteric model and remaining issues

The fitting of the concentration–response relationships of the nicorandil-induced activation of SUR2x/Kir6.2 channels with our model was not perfect (for instance, see the difference between the measured and calculated channel activities of SUR2A(Wt)/Kir6.2 channel in the presence of 1 mM ATP and 100 μM nicorandil). Therefore, the system may be more complex than our model describes.

In future studies, it is necessary to show the physical association of NBDs with biochemical and/or bio-imaging

Acknowledgments

This work was supported by a grant-in-aid for scientific research on priority areas (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Yoshihisa Kurachi and a grant-in-aid for scientific research C from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a research grant for cardiovascular diseases (12C-7) from the Ministry of Health, Labor and Welfare of Japan to Mitsuhiko Yamada.

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      This action is mediated by the binding of the sulfonylurea to the SUR (Ashford et al., 1986; Gribble et al., 1998; Schwanstecher et al., 1992). The SUR subunits of KATP channels are encoded by SUR1 and SUR2, and alternative splicing of the last exon of SUR2 gives rise to the isoforms of SUR2A and SUR2B; they are of high homology with each other (Chutkow et al., 1996), and their molecules differ only in the last 42 amino acids (Isomoto et al., 1996; Yamada and Kurachi, 2005). Northern blot analysis of SUR2 (Inagaki et al., 1996; Isomoto et al., 1996) and in situ hybridization were performed in various rodent tissues, including the brain (Hernandez-Sanchez et al., 1997).

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    Present address: Department of Molecular Pharmacology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.

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