A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K+ channels by G-protein betagamma-subunits
2000; Springer Nature; Volume: 19; Issue: 18 Linguagem: Inglês
10.1093/emboj/19.18.4915
ISSN1460-2075
AutoresYoshiyuki Wada, Toshikazu Yamashita, Koubun Imai, Reiko Miura, Kyoichi Takao, Miyuki Nishi, Hiroshi Takeshima, Tomiko Asano, Rika Morishita, Kazuhisa Nishizawa, Shinichiro Kokubun, Toshihide Nukada,
Tópico(s)Mechanical Circulatory Support Devices
ResumoArticle15 September 2000free access A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K+ channels by G-protein βγ-subunits Yoshiyuki Wada Yoshiyuki Wada Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Toshikazu Yamashita Toshikazu Yamashita Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Kohbun Imai Kohbun Imai Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Reiko Miura Reiko Miura Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Kyoichi Takao Kyoichi Takao Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Miyuki Nishi Miyuki Nishi Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Hiroshi Takeshima Hiroshi Takeshima Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Tomiko Asano Tomiko Asano Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi, 480-0392 Japan Search for more papers by this author Rika Morishita Rika Morishita Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi, 480-0392 Japan Search for more papers by this author Kazuhisa Nishizawa Kazuhisa Nishizawa Department of Neurobiology, Institute for Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8654 Japan Search for more papers by this author Shinichiro Kokubun Shinichiro Kokubun Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Toshihide Nukada Corresponding Author Toshihide Nukada Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Yoshiyuki Wada Yoshiyuki Wada Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Toshikazu Yamashita Toshikazu Yamashita Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Kohbun Imai Kohbun Imai Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Reiko Miura Reiko Miura Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Kyoichi Takao Kyoichi Takao Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Miyuki Nishi Miyuki Nishi Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Hiroshi Takeshima Hiroshi Takeshima Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Tomiko Asano Tomiko Asano Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi, 480-0392 Japan Search for more papers by this author Rika Morishita Rika Morishita Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi, 480-0392 Japan Search for more papers by this author Kazuhisa Nishizawa Kazuhisa Nishizawa Department of Neurobiology, Institute for Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8654 Japan Search for more papers by this author Shinichiro Kokubun Shinichiro Kokubun Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan Search for more papers by this author Toshihide Nukada Corresponding Author Toshihide Nukada Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan Search for more papers by this author Author Information Yoshiyuki Wada1,2, Toshikazu Yamashita1,2, Kohbun Imai1, Reiko Miura1, Kyoichi Takao1,2, Miyuki Nishi1, Hiroshi Takeshima1, Tomiko Asano3, Rika Morishita3, Kazuhisa Nishizawa4, Shinichiro Kokubun2 and Toshihide Nukada 1 1Department of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo, 156-8585 Japan 2Advanced Medical Research Center, Nihon University School of Medicine, 30-1 Ohyaguchi-kamimachi, Itabashi-ku, Tokyo, 173-8610 Japan 3Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya-cho, Kasugai, Aichi, 480-0392 Japan 4Department of Neurobiology, Institute for Brain Research, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8654 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4915-4925https://doi.org/10.1093/emboj/19.18.4915 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To determine the interaction site(s) of ATP-sensitive K+ (KATP) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein βγ2-subunits (Gβγ2) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. Gβγ2 bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the Gβγ2-dependent alteration of the channel activities. These findings provide evidence that Gβγ modulates KATP channels through a direct interaction with the loop-NBD of SUR. Introduction ATP-sensitive K+ (KATP) channels (Noma, 1983), which are composed of the inwardly rectifying K+ channel (Kir6.0) and the sulfonylurea receptor (SUR) subunit (Inagaki et al., 1995), play an important role in various cellular responses by linking the metabolic status of the cell to its membrane potential (Ashcroft, 1988). KATP channels modulate action potential duration and excitability in heart, and control insulin secretion in pancreatic β cells. Furthermore, KATP channels mediate hypoxic vasodilatation in coronary and cerebral vascular smooth muscles. A direct effect of hypoxia on Ca2+ channels may also be critical for the decrease in smooth muscle force (Taggart and Wray, 1998). Whereas neuronal Ca2+ channels are regulated negatively by guanine nucleotide binding regulatory proteins (G-proteins) (Clapham and Neer, 1993; Hille, 1994), KATP channels are regulated positively by G-protein-coupled receptors such as galanin (Dunne et al., 1989), adenosine A1 (Kirsch et al., 1990) and somatostatin (Ribalet and Eddlestone, 1995) receptors. This regulation of KATP channels is thought to be mediated by the α-subunits (Gαs) of Gi family G-proteins (Kirsch et al., 1990; Terzic et al., 1994; Ribalet and Eddlestone, 1995; Sánchez et al., 1998). It remains to be elucidated, however, which subunit(s) of the G-protein complex (Gα or the βγ-subunits, Gβγ) preferentially mediates this modulation of KATP, which subunit of the KATP channel (SUR and/or Kir) interacts with G-proteins, and whether the interaction between KATP and G-protein is direct or indirect. To address these issues at the molecular level, KATP channels were functionally expressed in mammalian COS-7 cells by introducing Kir6.2 and SUR (SUR2A or SUR1) cDNAs, and the effects of Gβγ2 proteins on the channel activities were studied using the inside-out configuration of the patch–clamp technique. In addition, a direct association of Gβγ with KATP channels was determined by in vitro binding using glutathione S-transferase (GST) proteins fused with intracellular loop and C-terminal segments of SUR2A, and N- and C-terminal segments of Kir6.2. Finally, an amino acid residue critical for the Gβγ-dependent modulation of KATP channels was identified within the Gβγ-binding site of SUR by site-directed mutagenesis. Results Cloning of rat Kir6.2 and SUR1 Determination of the nucleotide and predicted amino acid sequences of the inserts of clones pVGK1 and pBGK1 (see Materials and methods) revealed that GK15 is a rat version of Kir6.2 (BIR; Inagaki et al., 1995). Three nucleotide changes in the sequences were shown not to alter the coding amino acid residues: A (15), T (40) and T (327) of rat BIR (Isomoto et al., 1996) were G, C and C in our clones, respectively. In addition, determination of the nucleotide and predicted amino acid sequences of the inserts of clone pRSUR1 revealed 15 nucleotide changes from the sequence of rat SUR1 (Aguilar-Bryan et al., 1995), which resulted in nine amino acid substitutions and one amino acid insertion: Ala116, Thr487, Pro626, Ala630, Ile699, Pro835, Gly836, Gly1313 and Pro1562 were determined as Gly, Ser, Ser, Thr, Thr, Gln, Arg, Arg and Ser, encoded by GGT, AGC, TCC, ACA, ACC, CAG, CGA, CGT and TCG, respectively. Ser encoded by AGC was inserted at position 741. Twenty-one nucleotide changes in the sequences were shown not to alter the coding amino acid residues: C (21), C (24), G (27), G (1971), C (1977), T (1978), A (1986), G (1992), G (1995), C (2109), T (2310), T (2610), T (2634), C (3762), A (3765), A (3777), C (3780), T (4723), C (4724), C (4725) and T (4728) were G, G, A, T, G, C, G, A, A, T, C, A, C, T, T, G, G, A, G, T and C in our clones, respectively. Thus, the insert of this clone contained a cDNA sequence encoding SUR1. Functional expression of Kir/SUR channels in COS cells To study the modulation of KATP channels by G-proteins at the molecular level, KATP channels were expressed in mammalian COS-7 cells by introducing SUR (SUR2A or SUR1) and Kir6.2 cDNAs. Figure 1 illustrates unitary K+ currents recorded from excised inside-out membrane patches of COS-7 cells that have been transfected with Kir6.2/SUR2A (left) or Kir6.2/SUR1 (right) cDNAs. Although unitary K+ currents through Kir6.2/SUR2A and Kir6.2/SUR1 channels were scarcely observed in cell-attached patches (n = 30), the channels were spontaneously activated upon formation of inside-out patches (Figure 1a). The open probability without ATP, NPoi (see Materials and methods), was 2.39 ± 0.84 (n = 9) (Kir6.2/SUR2A) or 0.96 ± 0.37 (n = 9) (Kir6.2/SUR1). These channel activities were dramatically inhibited by application of 100 μM ATP to the cytoplasmic side of the excised patches (Figure 1b) and recovered after wash-out of ATP (Figure 1e). The channels reconstituted with Kir6.2/SUR2A or Kir6.2/SUR1 had a unitary conductance of 81.1 ± 3.4 (n = 6) or 80.1 ± 2.9 (n = 3) pS, respectively, at a membrane potential of −50 mV, the values that were similar to those reported (Inagaki et al., 1995, 1996). These findings indicate that KATP channels are functionally reconstituted in COS-7 cells by transfection with a combination of Kir6.2/SUR2A or Kir6.2/SUR1 cDNAs. Figure 1.Effects of Gβγ2 on Kir6.2/SUR2A and Kir6.2/SUR1 channels expressed in COS-7 cells. Single channel currents were recorded continuously from COS-7 cells expressing Kir6.2/SUR2A (left) or Kir6.2/SUR1 (right) channels, using the inside-out patch configuration. (a) Control (in the absence of ATP); (b) in the presence of 100 μM ATP; (c) in the presence of both 100 μM ATP and 50 pM Gβγ2; (d) after wash-out of Gβγ2; (e) after wash-out of both Gβγ2 and ATP. The membrane potential was −50 mV. Arrowheads, the zero current levels. Down indicates inward in the current traces. Download figure Download PowerPoint Modulation of Kir/SUR channels by Gβγ As shown in Figure 1c (left), bath (i.e. intracellular) application of 50 pM Gβγ2 (purified from bovine brain) caused an ‘enhancement’ of Kir6.2/SUR2A channel activities in the presence of 100 μM ATP (or a ‘reduction’ of ATP-induced inhibition of channel activities). The unitary conductance remained unchanged, but the relative NPo increased ∼2.5-fold (Figure 2). This enhancement of channel activities by Gβγ2 was abolished after wash-out of Gβγ2 (Figure 1d, left). Gβγ2 exerted similar effects on Kir6.2/SUR1 channels (Figure 1c and d, right). In contrast, Gβγ2 could not influence either Kir6.2/SUR2A or Kir6.2/SUR1 channels unless ATP was present (n = 5), indicating that Gβγ2 increases NPo only when the channel activities are suppressed by ATP. Figure 2.‘Antagonistic’ actions of Gβγ2 against ATP-induced inhibition of Kir6.2/SUR2A channels. (A) Concentration–inhibition relationships for ATP in the absence (open circles) or presence (filled circles) of 50 pM Gβγ2 in Kir6.2/SUR2A channels. (B) Dose-dependent response relationships for Gβγ2 observed in Kir6.2/SUR2A currents in the presence of 100 μM ATP. Current responses (NPo) were normalized to those in the absence of ATP (Relative NPo, see Materials and methods). The number of experiments is indicated in parentheses. *P <0.05. **P <0.001. Download figure Download PowerPoint The pattern of channel activities induced by Gβγ2 in the presence of ATP was different for Kir6.2/SUR2A and Kir6.2/SUR1 channels (Figure 1b and c). Gβγ2 increased the burst length rather than the opening frequency of Kir6.2/SUR2A channels, whereas Gβγ2 increased the opening frequency of Kir6.2/SUR1 channels without obvious changes in the burst duration. These suggest that there is a different mechanism for the Gβγ2-induced modulation of SUR2A- and SUR1-linked Kir6.2 channels. To characterize further the modulation of Kir6.2/SUR2A channels by Gβγ2, their responses to Gβγ2 were studied with various concentrations of ATP (Figure 2A). The activities of Kir6.2/SUR2A channels were inhibited by ATP in a concentration-dependent manner (Figure 2A, open circles). The inhibition constant (Ki) of ATP was 16.8 μM, and the Hill coefficient (nH) was 1.35. When Gβγ2 complex was added to the intracellular solution at a concentration of 50 pM (Figure 2A, filled circles), the Kir6.2/SUR2A channel activities were enhanced by 2.8- and 1.3-fold in the presence of 100 and 10 μM ATP, respectively. Moreover, an increase in the Ki value of ATP from 16.8 to 40.7 μM was observed after addition of Gβγ2. In contrast, an nH of 1.30 was comparable to that (1.35) before addition of Gβγ2. Thus, Gβγ2 produced a rightward shift of the concentration–inhibition curves for ATP without affecting the Hill coefficient. Similar effects of Gβγ2 on the concentration–inhibition curves for ATP were observed in Kir6.2/SUR1 channels (n = 3), in which the Ki value of ATP was markedly increased from 3.43 to 18.9 μM by application of Gβγ2, while nH was not significantly influenced (1.33 and 1.28) in the absence and presence of Gβγ2, respectively. The Ki values of ATP for Kir6.2/SUR1 (3.43 μM) and for Kir6.2/SUR2A (16.8 μM) indicate that these channels are 3- or 5-fold more sensitive to ATP than those reported, respectively (Inagaki et al., 1995, 1996). This may be accounted for, at least in part, by different levels of membrane phospholipids (Baukrowitz et al., 1998; Shyng and Nichols, 1998) in various cell lines used. However, a higher sensitivity of Kir6.2/SUR1 channels to ATP (Inagaki et al., 1996) was conserved in ATP-induced inhibition as compared with Kir6.2/SUR2A channels. On the other hand, the ATP-induced inhibition of Kir6.2/SUR2A channels was reduced by Gβγ2 in a concentration-dependent manner (Figure 2B). These results indicate that Gβγ2 ‘antagonizes’ ATP-induced inhibition of Kir6.2/SUR channels, as observed in Giα-mediated modulation (Kirsch et al., 1990; Terzic et al., 1994; Sánchez et al., 1998). On the other hand, Goα at 10 pM failed to facilitate Kir6.2/SUR2A channels in the presence of ATP (n = 3), whereas Gβγ2 at a lower concentration of 5 pM appreciably facilitated the channels (Figure 2B), indicating that Goα is not involved in the G-protein-mediated modulation of KATP channels (Kirsch et al., 1990). The activities of Goα were verified by examining its ability to couple to adenosine A1 receptors as reported previously (Asano et al., 1995). Recently, Gβγ−i1 and Gβγ−i2 have been reported not to affect Kir6.2/SUR1 channels (Sánchez et al., 1998). The discrepancy between our results and earlier reports may reflect different isoforms of Gβγ that were purified from bovine brain by using different procedures (Asano et al., 1993). Moreover, bath application of 100 μM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H7, n = 5), an inhibitor of cyclic nucleotide-dependent protein kinase and protein kinase C (PKC), bath application of 50 μM wortmannin (n = 4), an inhibitor of phosphatidylinositol (PI) 3-kinase, or pre-treatment with 2 mM neomycin sulfate (n = 4), an inhibitor of phospholipase C (PLC), exerted no effect on the Gβγ2-induced facilitation of Kir6.2/SUR2A channels in the presence of 100 μM ATP. These results are consistent with the view that protein kinase A, PKC, PI 3-kinase and PLC are not primarily involved in this channel modulation by Gβγ, although adenylyl cyclase, PI 3-kinase and PLC are known to be stimulated by Gβγ. Effects of mutant Kir6.2 subunits on Gβγ-dependent modulation of KATP channels To determine which subunit of KATP channels (Kir6.2 and/or SUR) interacts with Gβγ, we studied the effect of Gβγ2 on deletion mutant Kir6.2 channels when co-expressed with wild-type SUR2A (Figure 3). The mutant Kir6.2ΔC, where the C-terminal region (amino acid residues 366–390) of Kir6.2 had been removed, showed channel activities without the SUR2A subunit (Kir6.2ΔC, open box; Tucker et al., 1997), in which the sensitivity to ATP-induced inhibition was lower than that of wild-type Kir6.2/SUR2A channels (Kir6.2/SUR2A, open box). Moreover, Gβγ2 did not further enhance the channel activities of Kir6.2ΔC in the presence of ATP (Kir6.2ΔC, filled box). In contrast, co-expression of SUR2A with Kir6.2ΔC gained a high sensitivity to ATP (Kir6.2ΔC/SUR2A, open box; Tucker et al., 1997) and also a Gβγ2-mediated reduction of ATP-induced inhibition (Kir6.2ΔC/SUR2A, filled box), as observed in the wild-type Kir6.2/SUR2A channels. Thus, Gβγ reversed the high sensitivity of Kir6.2ΔC to ATP that was induced by SUR2A. Figure 3.Mutational effects of the Kir6.2 subunit on the Gβγ2-dependent modulation of KATP channels. The mutant Kir6.2 (Kir6.2ΔC or Kir6.2ΔN) was co-expressed with the wild-type SUR2A in COS-7 cells (Kir6.2ΔC/SUR2A or Kir6.2ΔN/SUR2A) or expressed alone (Kir6.2ΔC). For comparison, co-expression of the wild-type Kir6.2 and the SUR2A was also carried out (Kir6.2/SUR2A). Current responses (NPo) to 100 μM ATP were measured in the absence (open boxes) or presence (filled boxes) of 50 pM Gβγ2. Some Gβγ2 was heat treated at 90°C for 3 min (hatched box). NPo are expressed as a ratio to those in the absence of ATP. The number of experiments is indicated in parentheses. *P <0.05. **P <0.001. NS, not significant. Download figure Download PowerPoint On the other hand, another mutant Kir6.2 (Kir6.2ΔN) channels with a deletion of the N-terminal region (amino acid residues 2–14 of Kir6.2) were less sensitive to ATP when compared with wild-type or Kir6.2ΔC channels, even though wild-type SUR2A was co-expressed (Kir6.2ΔN/SUR2A, open box; Koster et al., 1999; Reimann et al., 1999). The Kir6.2ΔN/SUR2A channels showed no reduction of the ATP-induced inhibition in the presence of Gβγ2 (Kir6.2ΔN/SUR2A, filled box), standing in contrast to Kir6.2/SUR2A and Kir6.2ΔC/SUR2A channels. This ‘antagonistic’ action of Gβγ2 against ATP-induced inhibition as typically observed in the wild-type Kir6.2/SUR2A channels was abolished by heat denaturation of Gβγ2 proteins added (Kir6.2/SUR2A, hatched box). These findings indicate that SUR is necessary for KATP channels to allow Gβγ to ‘antagonize’ the ATP-induced channel inhibition. It is further suggested that Gβγ might interfere with the interaction between SUR and Kir6.2 by acting directly on SUR, but not through diffusible second messengers, and that Gβγ-mediated modulation of KATP channels might take place preferentially in a high ATP-sensitive channel state that is closely associated with SUR. Gβγ2 binds to the intracellular loop and C-terminus of SUR SUR, a member of the ATP-binding cassette (ABC) superfamily, has two domains (NBD-1 and NBD-2) for binding and hydrolysis of nucleotides (Aguilar-Bryan et al., 1995). ATP/ADP binds to NBDs of SUR (Ueda et al., 1997), and NBDs in turn function as ATP/ADP sensors (Nichols et al., 1996; Gribble et al., 1997), and thus, SUR regulates the ATP inhibition of KATP channels (Babenko et al., 1999). On the other hand, Kir6.2, a pore-forming subunit of KATP channels, is also defined as the primary site of the ATP-induced channel inhibition (Tucker et al., 1997). The C- and N-termini of Kir6.2 are both involved in this ATP inhibition (Drain et al., 1998; Tucker et al., 1998). Therefore, Gβγ might interact with these ATP-binding sites, thereby reducing the inhibitory action of ATP. To examine this hypothesis, two fragments (SL1 and ST1) from the intracellular loop containing NBD-1 (referred to as ‘loop-NBD’ in the present study) and the C-terminus containing NBD-2 of SUR2A, were expressed in Escherichia coli as GST fusion proteins, respectively (Figure 7A, SL1 and ST1). In addition, the N-terminus (corresponding to amino acid residues 2–73) and the C-terminus (corresponding to amino acid residues 169–390) of the Kir6.2 subunit (KN1 and KT1) were also fused with GST, respectively. Then, as shown in Figure 4, their ability to bind Gβγ was tested by immunoblotting analysis using the antibody M-14 against Gβ1. As described previously (Furukawa et al., 1998), Gβγ2 did not bind to GST proteins alone. Figure 4.Direct binding of Gβγ2 complex to the SUR2A subunit of KATP channels. Bovine brain Gβγ2 released from glutathione beads was detected by the anti-Gβ1 antibody M-14. Gβγ2 was incubated with the beads that had been immobilized with GST proteins fused with one of the following SUR2A fragments: SL1 (lane 2), ST1 (lane 3), KT1 (lane 4), SL2 (lane 6), SL3 (lane 7), SL4 (lane 9), ST2 (lanes 12 and 15), ST3 (lanes 13 and 14), ST4 (lane 18) and ST5 (lane 19) (see Figure 7A). For immunoblot analysis, proteins bound to the beads were separated by 12% (lanes 1–7 and 10–16) or 11% (lanes 8, 9 and 17–19) SDS–PAGE, together with 25 ng (lanes 10 and 16) or 12.5 ng (lanes 1, 5, 8, 11 and 17) of purified Gβγ2. The antibody was pre-incubated with the peptide antigen sc-261P (lanes 14–16). Arrowheads, positions of purified Gβ (Furukawa et al., 1998). Download figure Download PowerPoint The antibody M-14 reacted with a 36 kDa Gβ purified from brain (Figure 4, lanes 1, 5, 8, 10, 11 and 17), as well as a 36 kDa polypeptide released from both SL1- and ST1-bound beads (Figure 4, lanes 2 and 3) that had been incubated with the purified Gβγ2. These reactivities of M-14 with the 36 kDa polypeptides were inhibited by pre-incubation of M-14 with the peptide antigen sc-261P for M-14 (lane 16; n = 3), indicating that the 36 kDa immunoreactive polypeptide was recognized specifically by the antibody against Gβ when released from the loop-NBD and C-terminus of SUR2A. In contrast, this antibody scarcely detected a 36 kDa polypeptide released from KT1- (lane 4) or KN1-bound (n = 3) beads, the beads that had been incubated with the purified Gβγ2. These results support the idea obtained from the electrophysiological experiments that the Kir6.2/SUR channel is under the regulation of Gβγ interacting directly with the SUR subunit. To confirm further the binding sites of Gβγ on SUR, various fragments derived from the loop-NBD (Figure 7A, SL2 to SL4) and the C-terminus (Figure 7A, ST2 to ST5) of SUR2A were also fused with GST and tested for Gβγ binding in vitro. The antibody M-14 reacted with a 36 kDa polypeptide released from both SL2- and SL3-bound beads (Figure 4, lanes 6 and 7), but not from SL4-bound beads (lane 9), when these beads had been incubated with the purified Gβγ2. Moreover, M-14 reacted with a 36 kDa polypeptide released from both ST2- and ST3-bound beads (lanes 12 and 13), but not from both ST4- and ST5-bound beads (lanes 18 and 19). These reactivities of M-14 with the 36 kDa polypeptide were inhibited by pre-incubation with the peptide antigen sc-261P (lanes 14 and 15; n = 3). Thus, the results indicate that Gβγ interacts with both intracellular (loop-NBD) and C-terminal segments of SUR2A corresponding to SL3 (amino acid residues 616–691) and ST3 (amino acid residues 1461–1545), respectively (see Figure 7A). Molecular determination of the critical amino acid residue(s) for Gβγ-dependent modulation of Kir/SUR channels As shown in Figure 7B (SUR2A Loop, SUR2A Tail), a homology search has revealed that the regions SL3 and ST3 of SUR2A contain similar amino acid sequences. Aiming at identifying the critical amino acid residues on the SUR subunit interacting with Gβγ2, conserved basic amino acid residues within these amino acid sequences, Arg656 and Arg657 in SL3, and His1501 and Arg1502 in ST3, were substituted by Ala to generate four mutant SUR2As: SLM1 (Arg656,657Ala), SLM2 (Arg657Ala), SLM3 (Arg656Ala) and STM1 (His1501Ala and Arg1502Ala). These three kinds of mutant SUR2As with substitution of amino acids in loop-NBD (SLM1, SLM2 or SLM3) were co-expressed for each with wild-type Kir6.2, and ATP-dependent channel activities were observed (Figure 5, Kir6.2/SLM1, Kir6.2/SLM2 and Kir6.2/SLM3; A, a, b, e; B, open boxes), as shown previously for wild-type SUR2A (Figures 1 and 5B, Kir6.2/SUR2A, open box). Again, Gβγ2 reduced ATP-induced inhibition of Kir6.2/SLM2 channels (Figure 5, Kir6.2/SLM2; A, c, d; B, filled box), as observed in Kir6.2/SUR2A channels (Figure 5B, Kir6.2/SUR2A, filled box). In contrast, Gβγ2 failed to enhance the channel activities that were inhibited by ATP in either Kir6.2/SLM1 (Figure 5, Kir6.2/SLM1; A, c, d; B, filled box) or Kir6.2/SLM3 channels (Figure 5, Kir6.2/SLM3; A, c, d; B, filled box). When three kinds of segment in SL3 of SUR2A, namely amino acid residues 616–689 (SLΔ1, n = 10), 616–658 (SLΔ2, n = 5) or 616–640 (SLΔ3, n = 5), were deleted, no channel activities were detectable in spite of the co-expression with wild-type Kir6.2. Figure 5.Mutational effects of the SUR2A subunit on the Gβγ2-dependent modulation of KATP channels. (A) Single channel currents were recorded continuously from COS-7 cells co-expressed with the wild-type Kir6.2 subunit together with four kinds of mutant SUR2A subunit (SLM1, SLM2, SLM3 or STM1). (a) Control (in the absence of ATP); (b) in the presence of 100 μM ATP or (c) 100 μM ATP plus 50 pM Gβγ2; and (d) after wash-out of Gβγ2 or (e) Gβγ2 plus ATP. Arrowheads, the zero current levels. (B) Current responses (NPo) to 100 μM ATP were measured in the absence (open boxes) or presence (filled boxes) of 50 pM Gβγ2 in the wild-type or mutant channels as indicated, and expressed as a ratio to those in the absence of ATP. The number of experiments is indicated in parentheses. *P <0.05. **P <0.001. NS, not significant. Download figure Download PowerPoint On the other hand, the mutant STM1, with substitution of two amino acid residues in the C-terminus, also produced ATP-sensitive channels when co-expressed with wild-type Kir6.2 (Figure 5, Kir6.2/STM1; A, a, b, e; B, open box). Moreover, Gβγ2 reversibly ‘antagonized’ the ATP-induced inhibition of Kir6.2/STM1 channels (Figure 5, Kir6.2/STM1; A, c, d; B, filled box), as observed in wild-type channels (Figures 1 and 5B, Kir6.2/SUR2A, filled box). These results clearly indicate that a single amino acid residue in the loop-NBD of SUR2A, i.e. Arg656, plays a critical role in the modulation of Kir6.2/SUR2A channels by Gβγ. Similarly, Kir6.2/SUR1 channels were investigated with respect to the importance of loop-NBD in the regulation of Gβγ-dependent modulation. Arg665 in SUR1, which is a basic amino acid residue located at a position homologous to Arg656 in SUR2A (Inagaki et al., 1996), was replaced with Ala by site-directed mutagenesis t
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