J Physiol Volume 512, Number 2, 395-406, October 15, 1998
The Journal of Physiology (1998), 512.2, pp. 395-406
© Copyright 1998 The Physiological Society
Cytoplasmic terminus domains of Kir6.x confer different nucleotide-dependent gating on the ATP-sensitive K+ channel
Makoto Takano, Lai-Hua Xie, Hideo Otani * and Minoru Horie *
Department of Physiology and Biophysics and * Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan
Received 16 March 1998; accepted after revision 3 July 1998.
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ABSTRACT |
- In order to investigate the structural basis for the nucleotide-dependent gating of ATP-sensitive K+ channels (KATP), Kir6.1 (uKATP-1), Kir6.2 (BIR1) and chimeric channels were co-expressed with a common subtype of sulphonylurea receptor, SUR1, in COS7 cells. Representing the amino terminal domain-transmembrane domain-carboxyl-terminal domain of Kir6.1 as 1-1-1 and of Kir6.2 as 2-2-2, chimeric Kir6.x channels were constructed by swapping the amino and/or carboxyl terminal domains between Kir6.1 and Kir6.2 to give the chimeric x-1-x channels 1-1-2, 2-1-1 and 2-1-2, and the chimeric x-2-x channels 2-2-1, 1-2-2 and 1-2-1.
- Inside-out patch clamp experiments revealed that both wild-type Kir6.1 and Kir6.2 formed inwardly rectifying K+ channels. Single-channel conductances were 36·3 and 66·1 pS, respectively. Chimeric x-1-x channels, whose transmembrane domain was that of Kir6.1, showed similar ion-pore properties to wild-type Kir6.1. Likewise, chimeric x-2-x channels had similar ion-pore properties to wild-type Kir6.2.
- Wild-type Kir6.1 and Kir6.2 possessed distinct gating properties towards intracellular nucleotides. The activity of Kir6.1 was entirely dependent on Mg2+ and nucleotide diphosphates (NDPs) such as UDP. In contrast, Kir6.2 was activated upon excision of patch membrane. When Kir6.2 underwent rundown, UDP reactivated the channel.
- In order to eliminate UDP dependence from Kir6.1, it was necessary to replace both N- and C-termini; chimera 2-1-2 opened in UDP-free conditions. With Kir6.2, substitution of the N-terminus with that of Kir6.1 conferred UDP dependence on chimeras 1-2-2 and 1-2-1. Chimera 2-2-1 opened in UDP-free conditions, but UDP potentiated the channel activity by > 20-fold.
- The kinetics of UDP-dependent activation were significantly different between Kir6.1 and Kir6.2. Kir6.1 maximally activated by UDP was sensitive to intracellular ATP, although its ATP sensitivity was significantly lower than that of Kir6.2 measured in identical conditions. The kinetics of UDP-dependent activation and ATP sensitivity could be transferred between Kir6.1 and Kir6.2 only when both N- and C-termini were replaced. We therefore concluded that nucleotide-dependent gating was regulated by the N- and C-terminal domains irrespective of the transmembrane domains.
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INTRODUCTION |
The ATP-sensitive K+ channels (KATP channels) are distributed in many organs, including brain, heart, vascular smooth muscle, skeletal muscle and pancreatic 
-cells. They play a pivotal role in coupling membrane excitation to cellular metabolism. The KATP channel is a heteromultimer composed of ion-pore and regulatory subunits (Inagaki et al. 1995a; Sakura et al. 1995). The ion-pore subunits are members of the inwardly rectifying potassium channel family (Kir), which have two membrane-spanning domains (M1 and M2) flanking the K+-selective ion-pore region (H5) and cytoplasmic amino (N-) and carboxyl (C-) termini (Isomoto et al. 1997). The regulatory subunits are sulphonylurea receptors (SURx), members of the ATP-binding cassette superfamily which have two nucleotide binding folds, NBF1 and NBF2 (Aguilar-Bryan et al. 1995). It was recently shown that SUR specifically associates with Kir6.x among the family of Kir channels (Clement et al. 1997). Because nucleotide binding motifs are found in SUR molecules, and neither Kir6.1 nor Kir6.2 possesses putative nucleotide-binding motifs, it has been reported that SUR subtypes primarily determine the ATP sensitivities and pharmacological properties of reconstituted KATP channels. However, Tucker et al. (1997) recently reported that neutralization of a positively charged amino acid residue on the C-terminus of Kir6.2 (K185Q) led to a significant change in the ATP sensitivity. Yamada et al. (1997) reported that Kir6.1-SUR2B closely resembles the nucleotide diphosphate (NDP)-activated K+ channel found in smooth muscle cells, rather than the classical KATP channel reported in pancreatic -cells or cardiac myocytes. These reports strongly suggested that Kir6.x could play a significant role in the regulation of gating mechanisms towards intracellular nucleotides.
In the present study, we firstly aimed to clarify the role of different Kir6.x subunits in determining the gating properties of KATP channels. For this purpose, we co-expressed Kir6.1 and Kir6.2 with SUR1, and compared the properties of the reconstituted K+ channels. We in fact found that Kir6.1 and Kir6.2 conferred distinct gating properties: Kir6.1-SUR1 required MgNDPs to open, whereas Kir6.2-SUR1 opened spontaneously in NDP-free conditions. Secondly, we tried to identify the domains of Kir6.x subunits responsible for the different gating properties. We constructed chimeric channels between Kir6.1 and Kir6.2 by swapping the amino (N-) terminus and carboxyl (C-) terminus immediately before or after the transmembrane core domains. Our results demonstrate that nucleotide-dependent gating properties are determined by the combination of N- and C-termini, irrespective of the transmembrane domains.
In the following text, Kir6.x-SUR1 channels are abridged to Kir6.x for brevity unless mentioned otherwise, because Kir6.x and chimeric channels were always co-expressed with SUR1.
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METHODS |
Molecular biological experiments
SUR1 cDNA (Aguilar-Bryan et al. 1995), Kir6.1 cDNA (Inagaki et al. 1995b) and Kir6.2 cDNA (Takano et al. 1996) were subcloned into pCI vector which had a cytomegalovirus (CMV) promoter/enhancer (Promega, Madison, WI, USA). Mutant green fluorescent protein (GFP A65T; Moriyoshi et al. 1996) cDNA was subcloned into pCA vector which had a CAG promoter (Niwa et al. 1991). In order to construct chimeric channels with Kir6.1 and Kir6.2, silent restriction sites were introduced by site-directed mutagenesis using overlap PCR; a SphI site was introduced into Kir6.1 cDNA, and an AccI site into Kir6.2 cDNA. They were digested at shared AccI and SphI restriction sites. Appropriate restriction fragments were isolated from agarose gels and purified using QiaEx II gel purification kits (Qiagen, Hilden, Germany). They were ligated into pCI vector and the nucleotide sequences were verified on both strands. Schematic structures of chimeric channels are illustrated in Fig. 5.
Transfection
The amounts of vector were as follows (µg per 35 mm culture dish): pCI vector containing Kir6.x or chimeric channel, 0·8; pCI vector containing SUR1, 0·8; pCA vector containing GFP, 0·4. A cocktail of the above vectors was co-transfected into COS7 cells using lipofectAMINE and optiMEM (Gibco BRL). COS7 cells were plated on coverslips and cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10 % v/v). We could identify transfected cells by green fluorescence 
24-48 h after the transfection.
Patch clamp experiment
Coverslips were transferred to a recording chamber and were perfused with physiological saline containing (mM): NaCl, 140; KCl, 5·4; Na2HPO4, 1; CaCl2, 1·8; MgCl2, 0·5; glucose, 5; and Hepes, 5 (pH 7·4 with NaOH). A gigaohm seal was established in physiological saline using a patch pipette filled with K+ external solution containing (mM): KCl, 140; CaCl2, 1·8; MgCl2, 0·5; and Hepes, 5 (pH 7·4 with KOH). The electrode resistance was 3-6 M
. The patch membrane was then excised in control internal solution containing (mM): KCl, 140; EGTA (Sigma), 2; MgCl2, 2; and Hepes, 5 (pH 7·2 with KOH). All experiments were carried out in the inside-out mode of the patch clamp technique (Hamill et al. 1981) using an Axopatch 200B amplifier (Axon Instruments). In test solutions, various amounts of MgATP (Sigma or Boehringer Mannheim), Na2GDP (Sigma), Na2UDP (Wako) and adenylyl imidodiphosphate tetralithium salt (AMP-PNP; Boehringer Mannheim) were dissolved in control internal solution, and pH was re-adjusted to 7·2 with KOH. In some experiments, MgCl2 was omitted from control internal solution. Free Mg2+ concentrations of all test solutions were > 0·2 mM. Test solutions were applied either by bath perfusion or by Y-tube apparatus which can exchange solutions within 50 ms (Ogata & Tatebayashi, 1991). All experiments were carried out at 22-25°C.
Data analysis
Current signals were stored on DAT tape (with a TEAC recorder). For computer analysis, the signal was played back and digitized through a Digipack 1200A interface (Axon Instruments). Data analysis was carried out using commercial software (pCLAMP 6, Axon Instruments) on an IBM PC-compatible computer. The integral of open channel current was divided by time for integration (mean patch current; MPC). MPC was measured for 5-10 s unless stated otherwise. Statistical data are means ± S.E.M. Statistical difference was estimated by Student's unpaired t test.
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RESULTS |
We first investigated the single-channel conductances of Kir6.1 and Kir6.2 co-expressed with SUR1. In Fig. 1A, inward-going unitary current of Kir6.1 recorded in the presence of 2 mM UDP showed flickering channel activity, whereas for the outward direction, the channel showed longer open times. Its current-voltage (I-V) relationship is shown in Fig. 1C (open circles). The single-channel conductance of inward unitary current was 36·3 ± 0·6 pS (n = 7). Outward current showed slight inward rectification, presumably due to blocking by intracellular Mg2+. Kir6.2 also showed flickering channel activity for the inward direction, and the open time of outward current was clearly longer (Fig. 1B). Single-channel conductance of Kir6.2 was 66·1 ± 1·8 pS (n = 8) for the inward direction (Fig. 1C, filled circles).
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Figure 1. Single-channel conductance of Kir6.x
Unitary current of Kir6.1-SUR1 (A) and Kir6.2-SUR1 (B). The holding potentials are shown to the left of the traces. The dotted lines indicate the closed channel level. C, mean I-V relationships of unitary Kir6.1 ( ) and Kir6.2 ( ). The continuous line (Kir6.1) and dashed line (Kir6.2) were drawn using the mean values of inward single-channel conductances measured in each set of experiments, and were extrapolated to the positive membrane potentials. Single-channel conductance of Kir6.1 was 36·3 ± 0·6 pS (n = 7) and that of Kir6.2 was 66·1 ± 1·8 pS (n = 8).
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Since nucleotide binding folds (NBF1 and NBF2) are only found on SUR1, and neither Kir6.1 nor Kir6.2 has putative nucleotide binding motifs, it seems reasonable to expect that Kir6.1 and Kir6.2 should have an identical gating property towards intracellular nucleotides. However, as shown in Fig. 2A and B, Kir6.1 and Kir6.2 had distinct gating properties. In Fig. 2A, no channel activity was found after patch excision from cells transfected with Kir6.1 and SUR1. UDP (10 mM), which was used to potentiate native ATP-sensitive K+ channels, induced channel activity in a reversible manner (Tung & Kurachi, 1991). This reaction, namely NDP-dependent activation, required intracellular Mg2+ (data not shown). Activation of Kir6.1 was observed in 20 % of GFP-positive cells. In contrast, we could observe an abrupt increase of Kir6.2 activity when the patch membrane was excised into control internal solution in 80 % of GFP-positive cells. As shown Fig. 2B, Kir6.2 remained open in ATP-free solution, and underwent rundown with various time courses. During the course of rundown, application of 1 mM GDP, which is the most potent compound to potentiate pancreatic KATP channels (Bokvist et al. 1991), reactivated the channel. NDP-dependent reactivation of Kir6.2 also required Mg2+ (data not shown).
MgATP is known to 'refresh' the activity of classical KATP channels after rundown (Ashcroft & Rorsman, 1989; Takano et al. 1990). Application of MgATP readily activated Kir6.1, although MgATP was much less effective than UDP; the amplitude of mean patch current (MPC) during the application of 10 mM MgATP was only 15·0 ± 3·6 % of that activated by 10 mM UDP (n = 9). Upon removal of MgATP, a transient increase of channel activity was observed (Fig. 2A). This finding suggested that MgATP had dual effects on Kir6.1: activation and inhibition. The transient increase was found in three out of nine experiments. This variety was presumably due to a variable delay in the removal of MgATP from the dead space of the pipette tip (Qin et al. 1991). In Mg2+-free conditions, the application of 10 mM ATP4- failed to activate Kir6.1. A non-hydrolysable analogue of ATP, 10 mM AMP-PNP, also failed to activate Kir6.1 (data not shown). The effect of MgATP on Kir6.2 was clearly different from that on Kir6.1. In Fig. 2B, we started the application of 10 mM MgATP after the rundown of Kir6.2 reached a steady level. MgATP completely inhibited the residual channel activity. When the application of MgATP was repetitively interrupted for 2-5 s, Kir6.2 was promptly activated, and the channel activity in control internal solution became gradually larger as the preceding duration of the MgATP application became longer. It took 60 s before MgATP-dependent recovery reached a steady level. In the steady state, the channel activity recovered to 80 ± 8·6 % of the initial level (n = 8). This effect, namely MgATP-dependent recovery, was obstructed either by omitting Mg2+ or replacing ATP with AMP-PNP (data not shown). These properties closely resembled MgATP-dependent recovery reported in native KATP channels.
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Figure 2. Nucleotide-dependent gating of Kir6.x
A, channel activity of Kir6.1-SUR1 recorded ~3 min after the excision of a patch membrane. Bathing solution was control ATP-free solution. UDP (10 mM) or MgATP (1 or 10 mM) was applied as indicated by bars above or below the current trace, respectively. The amplitude of the mean patch current (MPC) measured during initial application of UDP was -30·3 pA. MPC during MgATP application was -3·04 pA. Note that a transient increase of Kir6.1 activity was induced upon washing off of MgATP. At the peak, MPC was -17·5 pA. MPC during the second application of UDP was -32·0 pA. B, Kir6.2-SUR1. As A, but instead of 10 mM UDP, 1 mM GDP was applied. MPC measured at the peak activation upon the patch excision (arrow) was -335 pA. Before starting the application of 10 mM MgATP, MPC was -26 pA. MPC was restored to -274 pA after the application of MgATP. It should be noted that the time course of rundown became slower after the application of MgATP. Both in A and in B, the holding potential was -40 mV, and the dotted lines indicate closed channel levels.
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Although NDPs could activate both Kir6.1 and Kir6.2, we found that the kinetics of concentration dependence of activation were completely different. Figure 3A shows an original current trace of Kir6.1 activated by various concentrations of GDP or UDP. Neither [UDP] nor [GDP] lower than 1 mM activated the channel. The magnitude of maximal activation by GDP was smaller than that by UDP; MPC in 10 mM GDP was 54 ± 5·2 % of that in 10 mM UDP (n = 10). A current trace of Kir6.2 is shown in Fig. 3B. Clearly, NDPs could activate Kir6.2, which underwent rundown at lower concentrations such as 0·1 or 0·3 mM. Kir6.2 was maximally activated by 3 mM UDP. UDP at 10 mM did not produce further activation (n = 2, data not shown). In order to measure the dose-response curves for NDP-dependent activation of Kir6.2, we measured MPC before and during the application of NDPs and the difference between these two values was defined as NDP-induced current. Figure 3C summarizes the dose-response relationships. Data points were normalized by the maximal values in each category of experiment. In Kir6.1, the half-maximal concentration for the activation (EC50) was 4·02 ± 0·12 mM for UDP (open circles) and the Hill coefficient (nH) was 4·20 ± 0·38 (n = 5). Kir6.2 (filled circles) showed significantly higher sensitivity: EC50 was 0·30 ± 0·10 mM (n = 5, P < 0·01). The Hill coefficient was also significantly different in Kir6.2 (nH = 1·11 ± 0·09, n = 5, P < 0·01).
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Figure 3. NDP-dependent activation of Kir6.x
The bars above the current traces indicate the applications of UDP or GDP. The dotted lines are closed channel levels. Holding potential was -40 mV. A, Kir6.1. MPC was -5·67 pA in 10 mM GDP and -12·5 pA in 10 mM UDP. B, Kir6.2. The current trace was recorded ~15 min after the excision of the patch membrane. C, dose-response curves of UDP for the activation of Kir6.1 () and Kir6.2 (). The ordinate indicates the relative MPC normalized by the maximal values in each category of experiment. The lines were fitted using the Hill equation: Relative amplitude (%) = max/(1 + (UDP/EC50)nH),where max is maximal response, EC50 is concentration for the half-maximal activation and nH is the Hill coefficient. Kir6.1: EC50 was 4·02 ± 0·12 mM and nH was 4·2 ± 0·38 (n = 5). Kir6.2: EC50 was 0·30 ± 0·10 mM and nH was 1·11 ± 0·09 (n = 5).
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As shown in Fig. 2A, MgATP had dual effects on Kir6.1: activation and inhibition. In order to separate these effects, we first maximally activated Kir6.1 by the application of 10 mM UDP and then started to apply ATP. Since UDP-dependent activation of the Kir6.1 channel required Mg2+, we applied MgATP in order to minimize the change of free Mg2+ concentration. As shown in Fig. 4A, MgATP inhibited Kir6.1 in a dose-dependent manner. However, sizeable channel activity remained even in the presence of 10 mM MgATP. The maximal inhibition was 78·5 ± 4·9 % (n = 6). This was presumably due to the fact that MgATP possessed an activating effect as well as an inhibitory effect on Kir6.1.
For comparison, ATP sensitivity of Kir6.2 was examined in identical conditions. As shown in Fig. 4B, Kir6.2 which was potentiated by 10 mM UDP showed significantly higher ATP sensitivity: 10 mM MgATP completely inhibited Kir6.2. Figure 4C shows dose-inhibition relationships. The dose-response curves for Kir6.2 were obtained both in the presence (filled triangles) and in the absence (filled diamonds) of 10 mM UDP. UDP at 10 mM shifted the dose-response curve to the right, to higher ATP concentrations, as previously reported (Kakei et al. 1986; Tung & Kurachi, 1991). The half-maximal concentration of MgATP for the inhibition (IC50) was 16·5 ± 1·2 µM (n = 4) in the absence of UDP, and 66·1 ± 18·3 µM (n = 5) in the presence of 10 mM UDP (P < 0·05). IC50 for Kir6.1 was significantly higher: in the presence of 10 mM UDP, the IC50 value was 337·8 ± 86·9 µM (n = 4, P < 0·01).
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Figure 4. ATP sensitivities of Kir6.x
A, ATP sensitivity of Kir6.1. Kir6.1 was activated by the application of 10 mM UDP, indicated by the bar above the trace. After the activation of Kir6.1 reached steady state, MgATP was applied as indicated by the bars below the trace. It should be noted that MPC in 10 mM UDP was increased from -41·2 pA to -51·0 pA by the application of 10 mM MgATP. B, ATP sensitivity of Kir6.2 examined in the presence of 10 mM UDP. MPC at the beginning of the current trace was -12·2 pA, and was increased to -37·0 pA by the application of 10 mM UDP. Note that additional 1 mM MgATP completely inhibited the channel. In the current traces shown in A and B, the holding potential was -40 mV, and the dotted lines are closed channel levels. C, dose-inhibition curves of Kir6.1 and Kir6.2. Each data point indicates relative amplitude (%); MPC measured during the application of MgATP was normalized by MPC measured in the preceding control conditions. Lines were fitted using the equation: 100 - relative amplitude (%) = maxI/(1 + (ATP/IC50)nH),where maxI was maximal inhibition, IC50 was the concentration for the half-maximal inhibition and nH was the Hill coefficient. : Kir6.1, IC50 = 337·8 ± 86·9 µM, nH = 1·16 ± 0·25, maximal inhibition was 78·5 ± 4·9 % (n = 4).  : Kir6.2 without UDP, IC50 = 16·5 ± 1·2 µM, nH = 1·16 ± 0·08 (n = 4).  : Kir6.2 in the presence of 10 mM UDP, IC50 = 66·1 ± 18·3 µM, nH = 1·17 ± 0·16 (n = 5). There was no significant difference in nH (P > 0·05).
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Kir6.1 is composed of 417 amino acids and Kir6.2 is composed of 390 amino acids. In spite of their amino acid sequences being 70 % homologous, we have demonstrated that Kir6.1 and Kir6.2 possess different concentration dependences for NDP-dependent activation and different ATP sensitivities. In order to get an insight into the structural basis for these differences, we constructed a series of chimeric Kir6.x channels and co-expressed them with SUR1. Cytoplasmic N- and C-termini were swapped before and after the transmembrane domains between Kir6.1 and Kir6.2. For example, chimera 1-1-2 was the channel whose N-terminus and transmembrane domain were those of Kir6.1, and C-terminus was that of Kir6.2. Chimera 1-2-1 had a transmembrane domain of Kir6.2, and N- and C-termini of Kir6.1. The schematic structures of the chimeric channels are illustrated in Fig. 5.
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Figure 5. Schematic structures of Kir6.x and chimeric channels
A, schematic diagrams of primary structures of Kir6.1 and Kir6.2. M1 and M2 are putative transmembrane domains and H5 the putative pore region. AccI restriction sites on cDNA sequences correspond to 65D of Kir6.1 and 64D of Kir6.2. SphI restriction sites correspond to 197A of Kir6.1 and 187A of Kir6.2. B, schematic structures of chimeric channels swapping N-terminus and C-terminus. The thin continuous line indicates domains derived from Kir6.1; the thick grey line, domains from Kir6.2.
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The single-channel conductance of each chimeric channel was not significantly modulated by swapping the N- and/or C-terminus. As shown in Fig. 6, single-channel I-V relationships of chimera x-1-x, whose transmembrane domains were that of Kir6.1, overlapped that of wild-type Kir6.1 (continuous line in A). Single-channel I-V relationships of chimera x-2-x were very close to that of wild-type Kir6.2 (dashed line in B). Values of single-channel conductances measured between -100 and -20 mV are summarized in Fig. 6C. No significant difference was found between the values of chimeras 1-1-2, 2-1-1, 2-1-2 and wild-type Kir6.1 (P > 0·05). The same was the case for chimeras 2-2-1, 1-2-2, 1-2-1 and wild-type Kir6.2 (P > 0·05).
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Figure 6. Single-channel conductances of chimeric channels
A, mean I-V relationships of chimera x-1-x. The continuous line was drawn using the mean value of the inward single-channel conductance of wild-type Kir6.1 (36·7 pS). , chimera 1-1-2 (35·5 ± 3·0 pS, n = 4);  , chimera 2-1-1 (36·1 ± 3·5 pS, n = 5);  , chimera 2-1-2 (37·2 ± 1·6 pS, n = 5). B, mean I-V relationships of chimera x-2-x. The dashed line (wild-type Kir6.2; 66·3 pS) was drawn in the same way as in A. , chimera 2-2-1 (63·3 ± 1·4 pS, n = 6);  , chimera 1-2-2 (66·3 ± 3·2 pS, n = 4); , chimera 1-2-1 (64·8 ± 4·3 pS, n = 4). C, summary of single-channel conductances of chimeric channels. Open bars indicate the single-channel conductances of chimera x-1-x, and the filled bars, chimera x-2-x. No significant difference was found in each category of chimeric channel (P > 0·05).
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In the amino acid sequences of Kir6.1 and Kir6.2, C-terminus regions show relatively lower homology. We therefore expected that chimera 1-1-2, a Kir6.1 channel whose C-terminus was substituted by that of Kir6.2, might open spontaneously in NDP-free conditions. However, no channel activity was induced after the formation of inside-out patches as shown in Fig. 7A. Following application, UDP activated chimera 1-1-2 in a reversible manner. The activity of chimera 2-1-1 also remained UDP dependent (Fig. 7B). However, when both C- and N-termini of Kir6.1 were substituted with those of Kir6.2, chimera 2-1-2 opened in NDP-free conditions (Fig. 7C). Chimera 2-1-2 underwent rundown, and was reactivated by UDP at lower concentrations than those used to activate Kir6.1. Figure 7D-E shows activities of a series of chimeric channels whose transmembrane domains were that of Kir6.2. As shown in Fig. 7D, chimera 2-2-1 opened spontaneously in UDP-free conditions, although the magnitude of UDP-dependent activation was clearly greater than that in wild-type Kir6.2; MPC measured in the presence of 10 mM UDP was increased 20·7 ± 4·5-fold over the control level (n = 5). In contrast, substitution of the N-terminus with that of Kir6.1 conferred UDP dependence on chimeras 1-2-2 and 1-2-1 (Fig. 7E and F).
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Figure 7. UDP dependence of chimeric channels
A-C, chimera x-1-x; D-F, x-2-x. In all panels, applications of 10 mM UDP are indicated by the bars above the traces and applications of 10 mM MgATP are indicated by the bars below the traces. The dotted lines indicate the closed channel levels. Holding potential was -40 mV. A, chimera 1-1-2. MPC during the application of UDP was -41·2 pA before the application of MgATP. After the application of MgATP, it was increased to -162·6 pA. No channel activity was induced during the application of MgATP. B, chimera 2-1-1. MPC in the first and second UDP application was -22·2 pA and -35·8 pA, respectively. C, chimera 2-1-2. In the left panel, the patch membrane was excised at the time indicated by the arrow. At the peak activation, MPC measured for 0·5 s was -121·0 pA. The current trace in the right panel was recorded ~6 min after patch excision. Before starting the application of MgATP, MPC was -35·3 pA; after the application of MgATP, -151·5 pA. D, chimera 2-2-1. The patch membrane was excised at the beginning of the current trace, and MPC was -12·7 pA. During the first application of UDP, MPC was -297 pA. The inset shows the expanded trace of spontaneous activity of chimera 2-2-1. Before starting the application of MgATP, MPC was -10·5 pA. The spontaneous activity increased after the application of MgATP to -98 pA. During the second application of UDP, MPC was -804 pA. E, chimera 1-2-2. MPC in the first and second UDP applications was -8·40 pA and -13·0 pA, respectively. Note that no channel activity was found in the absence of UDP. F, chimera 1-2-1. MgATP also directly activated chimera 1-2-1. Mean patch current was -1·37 pA in 10 mM ATP. Note that transient increase of channel activity was induced upon the washing out of MgATP. At the peak, MPC was -6·18 pA. MPC in the first UDP application was -8·34 pA and in the second UDP application, -10·3 pA.
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MgATP-dependent recovery found in wild-type Kir6.2 was only observed in chimeras 2-2-1 and 2-1-2, which opened spontaneously in UDP-free conditions. MgATP at 10 mM directly activated chimeras 1-2-1 and 1-2-2 in the same way as Kir6·1. In other types of chimeric channels whose activities were dependent on UDP, application of MgATP increased the amplitudes of UDP-activated currents, as typically shown in chimera 1-1-2 (Fig. 7A).
Kir6.1 could be recorded in 20 % of GFP-positive cells, whereas Kir6.2 was recorded in 80 % of GFP-positive cells. The mechanisms underlying the variety in functional expression level remain unclear. Kir6.1 may be expressed on the plasma membrane only at low levels, trapped in cytoplasmic compartments such as mitochondria (Suzuki et al. 1997). However, when both N- and C-termini of Kir6.1 were replaced with those of Kir6.2, chimera 2-1-2 could be recorded in 70 % of GFP-positive cells. Vice versa, chimera 1-2-1 was recorded in 30 % of GFP-positive cells. Therefore, cytoplasmic termini of Kir6.x may regulate the trafficking of channel molecules to the plasma membrane, as has been reported for Kir3.1 and Kir3.2 (Stevens et al. 1997).
The kinetics of NDP-dependent activation were completely different between Kir6.1 and Kir6.2 as shown in Fig. 3. In the chimeric channels, kinetics of UDP-dependent activation also changed in accordance with the observation in Fig. 7. When either the C- or N-terminus of Kir6.1 alone was replaced with that of Kir6.2, the activities of chimeras 1-1-2 and 2-1-1 remained dependent on intracellular UDP in the same way as Kir6.1. In Fig. 8A, dose-response relationships for the UDP-dependent activation of chimera 1-1-2 (open circles) and 2-1-1 (open squares) were close to that of Kir6.1 (continuous line). In contrast, chimera 2-1-2, which opened in UDP-free conditions in the same way as Kir6.2, was activated by UDP at much lower concentrations (open triangles; EC50 = 0·74 ± 0·12 mM, n = 4, P < 0·01). Furthermore, the dose-response curve was well fitted with nH = 1·13 ± 0·13 (n = 4, P < 0·01).
Figure 8B shows the dose-response relationship for UDP-dependent activation of chimera x-2-x. The EC50 of chimera 2-2-1 (filled circles; 1·07 ± 0·17 mM, n = 4) was significantly larger than that of Kir6.2 (P < 0·01), whereas nH (1·31 ± 0·35) remained similar to the value for Kir6.2 (P > 0·05). Replacement of the N-terminus alone produced a larger change in the dose-response curve of chimera 1-2-2 (filled squares; EC50 = 2·62 ± 0·27 mM, nH = 2·01 ± 0·20, n = 4, P < 0·01). When both the N- and C-termini were replaced, the kinetics of chimera 1-2-1 became similar to that of wild-type Kir6.1, rather than that of Kir6.2 (filled triangles; EC50 = 3·71 ± 0·12 mM, nH = 3·24 ± 0·17, n = 4, P < 0·01).
As summarized in Fig. 8C and D, EC50 and nH of wild-type Kir6.1 (chimera 1-1-1) and chimera 1-2-1 were not significantly different (P > 0·05). Also, the same was the case for wild-type Kir6.2 (chimera 2-2-2) and chimera 2-1-2 (P > 0·05). Thus, it seemed likely that the combination of N- and C-terminus domains, i.e. 1-x-1 and 2-x-2 structures, determined the kinetics of UDP-dependent activation, irrespective of the transmembrane domains.
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Figure 8. Dose-response relationships for UDP-dependent activation
The continuous and dashed lines in A and B are dose-response curves for wild-type Kir6.1 and Kir6.2, respectively. The dotted lines were drawn using the average values of nH and EC50 of each chimeric channel. A, chimera x-1-x. Different symbols indicate different chimeric channels. , chimera 1-1-2 (n = 4); , chimera 2-1-1 (n = 4); , chimera 2-1-2 (n = 4). B, chimera x-2-x. , chimera 2-2-1 (n = 4); , chimera 1-2-2 (n = 4); , chimera 1-2-1 (n = 4). C, summary of EC50. Open and filled bars indicate the values of chimera x-1-x and x-2-x, respectively. No significant difference was present between wild-type Kir6.1 (i.e. 1-1-1) and chimera 1-2-1, or between wild-type Kir6.2 (i.e. 2-2-2) and chimera 2-1-2. D, summary of Hill coefficients. Labelling as in C. Values of EC50 and nH are indicated in the text.
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As well as UDP-dependent activation, 1-x-1 and 2-x-2 structures of Kir6.x also determined the nature of ATP-dependent inhibition. Figure 9A shows the dose-inhibition curves of the chimera x-1-x. Chimeras 1-1-2 and 2-1-1 were activated by 10 mM UDP, and then MgATP was applied to inhibit the channels. Wild-type Kir6.1 was not inhibited completely even by the application of 10 mM MgATP; the maximal inhibition was 78·5 ± 4·9 %. However, chimeras 1-1-2 and 2-1-1 were almost completely inhibited by 10 mM MgATP. IC50 was 178 ± 32·4 µM (open circles; n = 4) in chimera 1-1-2 and 285·9 ± 53·4 µM (open squares; n = 4) in chimera 2-1-1, indicating that less significant changes were produced by swapping either the N-terminus or C-terminus alone. However, when both the C- and N-termini were swapped, a drastic change was found in ATP-dependent inhibition of chimera 2-1-2. Since chimera 2-1-2 spontaneously opened in UDP-free conditions, we measured dose-inhibition curves both in the presence and in the absence of 10 mM UDP. It is evident from Fig. 9A, that dose-inhibition curves of chimera 2-1-2 overlapped those of wild-type Kir6.2. IC50 values in the presence and absence of 10 mM UDP were 59·5 ± 9·8 µM (open triangles; n = 4) and 16·5 ± 1·2 µM (open diamonds; n = 4), respectively. These values were not significantly different from those of wild-type Kir6.2 measured in identical conditions (P > 0·05).
The dose-inhibition curves of chimera x-2-x are shown in Fig. 9B. Although the activity of chimera 1-2-2 was entirely dependent on UDP, its ATP sensitivity (filled squares; IC50 = 58·1 ± 16·3 µM, n = 4) was not significantly different from that of wild-type Kir6.2 measured in the presence of 10 mM UDP (P > 0·05). Chimera 2-2-1 was less sensitive to ATP. Furthermore, ATP sensitivity of chimera 2-2-1 measured in the presence of UDP (filled circles; 99·7 ± 12·9 µM, n = 5) and absence of 10 mM UDP (filled diamonds; 113·4 ± 29·1 µM, n = 5) were not significantly different (P > 0·05), suggesting that the C-terminus of Kir6.1 eliminated antagonistic action of NDPs upon ATP-dependent inhibition. Chimera 1-2-1 showed the lowest ATP sensitivity among the chimera x-2-x (filled triangles; 313·6 ± 16·9 µM, n = 4), which was not significantly different from that of Kir6.1 (P > 0·05). Furthermore, chimera 1-2-1 was not completely inhibited by 10 mM MgATP; the maximal inhibition was 88·9 ± 2·1 % (n = 4). Thus, kinetics of ATP-dependent inhibition could be almost completely exchanged between Kir6.1 and Kir6.2 by replacing both N- and C-termini.
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Figure 9. ATP-sensitivities of chimeric channels
In both A and B, the dashed line indicates the dose-inhibition curve of Kir6.2 in the presence of UDP and the dashed-dotted line indicates that in the absence of UDP, respectively. The continuous line is the dose-response curve of Kir6.1. A, dose-inhibition relationships of chimera 1-x-1. , chimera 1-1-2 (n = 4); , chimera 2-1-1 (n = 4);  , chimera 2-1-2 in the absence of UDP (n = 4); , chimera 2-1-2 in the presence of UDP (n = 4). The dotted lines were drawn using the average values of maximal inhibition, nH and IC50 of each chimeric channel. B, dose-inhibition curves of chimera x-2-x. , chimera 2-2-1 in the absence of UDP (n = 5); , chimera 2-2-1 in the presence of UDP (n = 5). , chimera 1-2-2 (n = 4); , chimera 1-2-1 (n = 4). C, summary of IC50 values. Open bars and filled bars indicate the values of chimera x-1-x and x-2-x, respectively. IC50 values are shown in the text. nH ranged from 1·00 ± 0·06 (chimera 1-1-2) to 1·29 ± 0·36 (chimera 2-2-1, in the presence of UDP). No significant differences were found in nH values (P > 0·05).
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DISCUSSION |
We have demonstrated that the pore-forming subunits Kir6.1 and Kir6.2 confer distinct nucleotide-dependent gating even when they are co-expressed with the same subtype of SUR molecule. The physiological properties of Kir6.2-SUR1 were very similar to those in the previous reports (Inagaki et al. 1995a; Sakura et al. 1995; Gribble et al. 1997a). In the present study, Kir6.1-SUR1 opened only in the presence of NDPs, and Kir6.1-SUR1 activated by UDP was partially inhibited by additional ATP. Yamada et al. (1997) also demonstrated that the activity of Kir6.1-SUR2B is dependent on NDPs. However, Kir6.1-SUR2B activated by UDP was 'ATP insensitive'. We do not have a ready explanation for this difference. It may be simply because different SUR subunits confer different ATP sensitivities. It should be noted that Kir6.1-SUR2B may be potentially ATP sensitive because the dose-response curve for ATP activation showed a 'bell shape' when the channel was activated by K+ channel openers plus ATP.
The subcellular distribution of Kir6.1 appears to be a matter of debate. Yamada et al. (1997) suggested that Kir6.1- SUR2B closely resembles the nucleotide-activated K+ channels found on the plasma membrane of smooth muscle cells (Kajioka et al. 1991). On the other hand, Suzuki et al. (1997) suggested that Kir6.1 may be a pore-forming subunit of mitochondrial KATP. In the latter case, Kir6.1-SUR1 may not correspond to the mitochondrial KATP channel, because the mitochondrial KATP channel opens in the absence of MgNDPs and is completely inhibited by ATP (Inoue et al. 1991).
The functional KATP channel is a (Kir6.2-SUR1)4 octamer (Clement et al. 1997; Inagaki et al. 1997; Shyng & Nichols, 1997). Although the stoichiometry of the Kir6.1-SUR1 heteromultimer remains unclear, it appears reasonable to assume that an octameric (Kir6.1-SUR1)4 structure is also the case. Therefore, both the (Kir6.1)4 and (Kir6.2)4 ion pores should be regulated by four surrounding SUR1 molecules. In the present study, the dose-response curve for UDP-dependent activation of Kir6.2 was fitted with nH = 1·10. This might imply that one of four SUR1 molecules activated by MgNDPs could exert regulatory effects on a (Kir6.2)4 ion pore. The kinetics of activation of Kir6.1 were completely different. Since nH was 4 in this reaction, four SUR1 molecules may be required to activate a (Kir6.1)4 ion pore.
The functional roles of the Kir and SUR subunits have been investigated using a truncated form of Kir6.2. Tucker et al. (1997) reported that Kir6.2
C26, which lacked the last twenty-six amino acid residues, was active without SUR1. Kir6.2C26 was inhibited by ATP without SUR1. Therefore, the ATP sensitivity seemed intrinsic to Kir6.2. SUR1 increased the ATP sensitivity of Kir6.2, although its mechanism remains unclear (Tucker et al. 1997; Shyng et al. 1997b). Tucker et al. (1997) also demonstrated that Kir6.2C26-K185Q showed substantially lower ATP sensitivity. In the C-terminus of Kir6.2, the region immediately behind the second transmembrane domain appeared, at least partially, to form an 'ATP-sensitive gate'. Functional roles of the N-terminus of Kir6.2 are not well understood. When the N-terminus of Kir6.2 was substituted by that of Kir6.1, the activity of chimera 1-2-2 was entirely dependent on NDPs whereas the ATP sensitivity of chimera 1-2-2 was identical to that of wild-type Kir6.2 measured in the same conditions. Therefore, the N-terminus of Kir6.2 seemed to play an essential role in stabilizing the channel activity rather than in the regulation of ATP sensitivity.
SUR1 conferred the sulphonylurea sensitivity on Kir6.2-SUR1. The same was the case for the potentiatory effects of MgNDPs and K+ channel openers (Tucker et al. 1997). Mutations in the Walker-A motifs of NBF1 and/or NBF2 abolished the potentiatory effect of MgADP on Kir6.2-SUR1 (Gribble et al. 1997b). Mutations in the Walker-B motif or linker region of NBF2 showed a similar effect (Nichols et al. 1996; Shyng et al. 1997b). Thus, it seems likely that the potentiatory effect of NDPs is intrinsic to the SUR subunit. However, as shown in the present study, 1-x-1 and 2-x-2 structures of Kir6.x subunits determined the kinetics of the potentiatory effect of UDP. At present, the mechanisms remain unclear; Kir6.x may modify the affinity of SUR1 for UDP, or Kir6.x may modify apparent concentration dependence by an allosteric mechanism (Shyng et al. 1997a).
The mutation study in the Walker-A motif also revealed that antagonistic action of MgADP upon the ATP sensitivity of Kir6.2-SUR1 was mediated by the same mechanism as the potentiatory effect (Gribble et al. 1997b). In the present study, the ATP sensitivity of chimera 2-2-1 was not significantly different in the presence and absence of UDP even though the activity of chimera 2-2-1 was potentiated by UDP. The cytoplasmic C-terminus of Kir6.1 seemed to dissociate the potentiatory effect from the antagonistic effect on the ATP sensitivity.
The activity of Kir6.2C26 was persistently 'refreshed' by MgATP without the coexistence of SUR1, suggesting that MgATP-dependent recovery was also intrinsic to Kir6.2 (Tucker et al. 1997). The effect of MgATP upon Kir6.1 appears to be a different entity, since the time course of activation was faster, and the effect was readily reversible. MgATP seemed to activate Kir6.1 in a similar way to MgUDP. In the present study, the activities of chimera 2-1-2 and chimera 2-2-1, both of which opened spontaneously in NDP-free conditions, were substantially increased after the application of MgATP. In other chimeric channels, MgATP up-regulated the amplitudes of UDP-activated current, although the magnitudes of upregulation varied widely. Therefore, MgATP may simply augment the operative state of Kir6.x and chimeric channels, irrespective of their UDP dependence. The mechanism of MgATP-dependent recovery remains unclear. Since Mg2+ was needed for this effect, and AMP-PNP could not mimic it, it has been speculated that hydrolysis of ATP and phosphorylation were involved (Findlay & Dunne, 1986; Ashcroft & Rorsman, 1989; Gribble et al. 1997b). The time course of MgATP-dependent recovery may also support this hypothesis (Ohno-Shosaku et al. 1987; Takano et al. 1990). However, sites of phosphorylation remain totally unclear. The target may be membrane lipids rather than Kir6.x or SURs (Hilgeman & Ball, 1996).
In conclusion, the quantitative, as well as the qualitative, nature of nucleotide-dependent gating was almost completely transferred between Kir6.1 and Kir6.2 when both the N- and C-termini were replaced. This finding suggests a new idea, that 1-x-1 and 2-x-2 structures of Kir6.x play a crucial role in the transmission of the conformational signals between SURs.
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REFERENCES |
| Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P. IV, Boyd, A. E. III, González, G., Herrera-Sosa, H., Nguy, K., Bryan, J. & Nelson, D. A. (1995). Cloning of the -cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268, 423-426 |
[Medline] |
| Ashcroft, F. M. & Rorsman, P. (1989). Electrophysiology of the pancreatic -cell. Progress in Biophysics and Molecular Biology 54, 87-143 |
[Medline] |
| Bokvist, K., Ämmälä, C., Ashcroft, F. M., Berggren, P. O., Larsson, O. & Rorsman, P. (1991). Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K+ channels in mouse pancreatic -cells. Proceedings of the Royal Society B 243, 139-144 |
[Medline] |
| Clement, J. P. IV, Kunjilwar, K., González, G., Schwanstecher, M., Panten, M., Aguilar-Bryan, L. & Bryan, J. (1997). Association and stoichiometry of KATP channel subunits. Neuron 18, 827-838 |
[Medline] |
| Findlay, I. & Dunne, M. J. (1986). ATP maintains ATP-inhibited K+ channels in an operational state. Pflügers Archiv 407, 238-240 |
[Medline] |
| Gribble, F. M., Ashfield, R., Ämmälä, C. & Ashcroft, F. M. (1997a). Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes. The Journal of Physiology 498, 87-98 |
[Abstract] |
| Gribble, F. M., Tucker, S. J. & Ashcroft, F. M. (1997b). The essential role of the Walker A motifs of SUR1 in KATP channel activation by Mg-ADP and diazoxide. EMBO Journal 16, 1145-1152 |
[Abstract/Full Text] |
| Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981). Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches. Pflügers Archiv 391, 85-100 |
[Medline] |
| Hilgemann, D. & Ball, R. (1996). Regulation of cardiac Na+, Ca2+ exchange and KATP potassium channels by PIP2. Science 273, 956-959 |
[Abstract] |
| Inagaki, N., Gonoi, T., Clement, J. P. IV, Namba, N., Inazawa, J., Gonzalez, G., Aguilar-Bryan, L., Seino, S. & Bryan, J. (1995a). Reconstitution of IKATP: An inward rectifier subunit plus the sulfonylurea receptor. Science 270, 1166-117 |
[Abstract] |
| Inagaki, N., Gonoi, T., Clement, J. P. IV, Wang, C. Z., Aguilar-Bryan, L., Bryan, J. & Seino, S. (1996). A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16, 1011-1017 |
[Medline] |
| Inagaki, N., Gonoi, T. & Seino, S. (1997). Subunit stoichiometry of the pancreatic -cell ATP-sensitive K+ channel. FEBS Letters 409, 232-236 |
[Medline] |
| Inagaki, N., Tsuura, Y., Namba, N., Masuda, K., Gonoi, T., Horie, M., Seino, Y., Mizuta, M. & Seino, S. (1995b). Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart. Journal of Biological Chemistry 270, 5691-5694 |
[Abstract/Full Text] |
| Inoue, I., Nagase, H., Kishi, K. & Higuti, T. (1991). ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature 352, 244-247 |
[Medline] |
| Isomoto, S., Kondo, C. & Kurachi, Y. (1997). Inwardly rectifying K+ channels: their molecular heterogeneity and function. Japanese The Journal of Physiology 47, 11-39 |
[Medline] |
| Kajioka, S., Kitamura, K. & Kuriyama, H. (1991). Guanosine diphosphate activates an adenosine 5'-triphosphate-sensitive K+ channel in the rabbit portal vein. The Journal of Physiology 444, 397-418 |
[Abstract] |
| Kakei, M., Kelly, R. P., Ashcroft, S. J. H. & Ashcroft, F. M. (1986). The ATP sensitivity of K+ channels in rat pancreatic -cells is modulated by ADP. FEBS Letters 208, 63-66 |
[Medline] |
| Moriyoshi, K., Richards, L. J., Akazawa, C., O'Leary, D. D. M. & Nakanishi, S. (1996). Labeling neural cells using adenoviral gene transfer of membrane-tagged GFP. Neuron 16, 255-260 |
[Medline] |
| Nichols, C. G., Shyng, S. L., Nestorowicz, A., Glaser, B., Clement, J. P. IV, Gonzalez, G., Aguilar-Bryan, L., Permutt, M. A. & Bryan, J. (1996). Adenosine diphosphate as an intracellular regulator of insulin secretion. Science 272, 1785-1787 |
[Abstract] |
| Niwa, H., Yamamura, K. & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193-200 |
[Medline] |
| Ogata, N. & Tatebayashi, H. (1991). A simple and multi-purpose 'concentration clamp' method for rapid superfusion. Journal of Neuroscience Methods 39, 175-183 |
[Medline] |
| Ohno-Shosaku, T., Zünkler, B. J. & Trube, G. (1987). Dual effects of ATP on K+ currents of mouse pancreatic -cells. Pflügers Archiv 408, 133-138 |
[Medline] |
| Qin, D., Yoshida, A. & Noma, A. (1991). Limitations due to unstirred layers in measuring channel response of excised membrane patch using rapid solution exchange methods. Japanese The Journal of Physiology 41, 333-339 |
[Medline] |
| Sakura, H., Ämmälä, C., Smith, P. A., Gribble, F. M. & Ashcroft, F. M. (1995). Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic -cells, brain, heart and skeletal muscle. FEBS Letters 377, 338-344 |
[Medline] |
| Shyng, S.-L., Ferrigni, T. & Nichols, C. G. (1997a). Control of rectification and gating of cloned KATP channels by Kir6.2 subunit. Journal of General Physiology 110, 141-153 |
[Abstract/Full Text] |
| Shyng, S.-L., Ferrigni, T. & Nichols, C. G. (1997b). Regulation of KATP channel activity by diazoxide and MgADP. Distinct functions of the two nucleotide binding folds of the sulfonylurea receptor. Journal of General Physiology 110, 643-654 |
[Abstract/Full Text] |
| Shyng, S.-L. & Nichols, C. G. (1997).Octameric stoichiometry of KATP channel complex. Journal of General Physiology 110, 655-664 |
[Abstract/Full Text] |
| Stevens, E., Woodward, R., Ho, I. H. M. & Murrel-Lagnado, R. (1997). Identification of regions that regulate the expression and activity of G protein-gated inward rectifier K+ channels in Xenopus oocytes. The Journal of Physiology 503, 547-562 |
[Abstract] |
| Suzuki, M., Kotabe, K., Fujikura, K., Inagaki, N., Suzuki, T., Goni, T., Seino, S. & Takata, K. (1997). Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria. Biochemical and Biophysical Research Communications 241, 693-697 |
[Medline] |
| Takano, M., Ishii, T. & Xie, L. H. (1996). Cloning and functional expression of the rat brain Kir6.2 channel. Japanese The Journal of Physiology 46, 491-495 |
[Medline] |
| Takano, M., Qin, D. & Noma, A. (1990). ATP-dependent decay and recovery of K+ channels in guinea pig cardiac myocytes. American Journal of Physiology 258, H45-50 |
[Medline] |
| Terzic, A., Findlay, I., Hosoya, Y. & Kurachi, Y. (1994). Dualistic behavior of ATP-sensitive K+ channels toward intracellular nucleoside diphosphates. Neuron 12, 1049-1058 |
[Medline] |
| Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S. & Ashcroft, F. M. (1997). Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 387, 179-183 |
[Medline] |
| Tung, R. T. & Kurachi, Y. (1991). On the mechanism of nucleotide diphosphate activation of the ATP-sensitive K+ channel in ventricular cell of guinea-pig. The Journal of Physiology 437, 239-256 |
[Abstract] |
| Yamada, Y., Isomoto, S., Matsumoto, S., Kondo, C., Shindo, T., Horio, Y. & Kurachi, Y. (1997). Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea-sensitive but ATP-insensitive K+ channel. The Journal of Physiology 499, 715-720 |
[Abstract] |
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Acknowledgements
We are grateful to Drs L. Aguilar-Bryan and J. Bryan for the SUR1 clone, Professor S. Seino for the Kir6.1 (uKATP-1) clone, Dr K. Moriyoshi for the GFP clone and Professor J. Miyazaki for the CAG promoter. We thank Mr M. Fukao and Ms K. Tuji for technical support. We are especially grateful to Professor A. Noma for helpful discussions. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and by the Japan Heart Foundation and an IBM Japan Research Grant.
Corresponding author
M. Takano: Department of Physiology and Biophysics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan.
Email: takano{at}med.kyoto-u.ac.jp
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Introduction
