Introduction

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ATP-sensitive K⁺ (K_ATP) channels are inhibited by intracellular ATP and activated by ADP and thus provide a link between the cellular metabolic state and excitability (Ashcroft, 1988; Terzic et al., 1995). These channels are associated with such cellular functions as insulin secretion, cardiac preconditioning, vasodilatation, and neuroprotection (Ashcroft, 1988; Terzic et al., 1995; Nichols et al., 1996; Quayle et al., 1997; Yamada et al., 2001; Miki et al., 2002). K_ATP channels are composed of an ATP-binding cassette protein, sulfonylurea receptor (SUR), and an inwardly rectifying K⁺ channel (Kir) subunit, Kir6.0 (Aguilar-Bryan et al., 1995; Inagaki et al., 1995, 1996, 1997; Sakura et al., 1995; Clement et al., 1997; Shyng and Nichols, 1997). Detailed functional analyses of K_ATP channels composed of Kir6.0 and either of three types of SUR (SUR1, SUR2A, and SUR2B) indicate that SUR1, SUR2A, and SUR2B represent pancreatic, cardiac, and vascular smooth muscle types of SUR, respectively (Aguilar-Bryan et al., 1995; Inagaki et al., 1995, 1996; Isomoto et al., 1996; Yamada et al., 1997). SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels exhibit the same single-channel conductance but different responses to K⁺ channel openers and MgADP because SUR regulates the channel's gating in response to these substances (Inagaki et al., 1995, 1996; Isomoto et al., 1996; Matsuoka et al., 2000).

SUR types also determine the response of K_ATP channels to sulfonylurea and benzamido derivatives (Gribble et al., 1998b). Sulfonylurea derivatives tolbutamide and gliclazide provoke selective high-affinity inhibition of SUR1/Kir6.2 channels (Gribble et al., 1998b; Gribble and Ashcroft, 1999). However, glibenclamide containing both sulfonylurea and benzamido moieties, and meglitinide possessing a benzamido but not a sulfonylurea moiety, provoke high-affinity inhibition of all SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels (Gribble et al., 1998b; Ashfield et al., 1999). Therefore, some of the compounds used as hypoglycemic agents may cause an adverse cardiovascular effect by cross-reacting with cardiovascular K_ATP channels (Bernauer, 1997; Cleveland et al., 1997; UK Prospective Diabetes Study Group, 1998).

The phenylalanine derivative nateglinide (N-[(trans-4-isopropylcyclohexyl)-carbonyl]-D-phenylalanine; A-4166) is a novel oral hypoglycemic agent. Although nateglinide lacks either a sulfonylurea or a benzamido moiety (Akiyoshi et al., 1995; Ikenoue et al., 1997; Gribble et al., 2001), it stimulates insulin secretion by inhibiting -cell K_ATP channels. However, its effects on distinct types of K_ATP channels have not been examined in detail. In this study, we compared the effects of nateglinide on SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels with the patch-clamp method. We show that nateglinide provokes selective, reversible, high-affinity inhibition of the SUR1/Kir6.2 channel through the same site on SUR1 as tolbutamide. Intracellular MgADP apparently enhances the inhibitory effect of nateglinide because nateglinide suppresses the stimulatory but not inhibitory effect of the nucleotide on the channel. This is the first report to show that a compound lacking a sulfonylurea moiety exhibits the same mode of action as sulfonylurea derivatives. A part of this study has been published in an abstract form (Chachin et al., 2002).

	Materials and Methods

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Molecular Biology. The cDNAs of mouse Kir6.2 and different SURs were used (Isomoto et al., 1996; Matsuoka et al., 2000). The coding region of each cDNA was individually subcloned into expression vector pcDNA3 (Invitrogen, Carlsbad, CA). SUR1 whose serine at position 1237 was substituted with tyrosine [SUR1(S1237Y)], SUR1 whose lysine at position 1384 was substituted with alanine [SUR1(K1384A)], and Kir6.2 whose lysine at position 185 was substituted with glutamine [Kir6.2(K185Q)] were constructed using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI). Kir6.2 whose C-terminal 26 amino acids were truncated (Kir6.2C26) was made by introducing a stop codon at the appropriate position by site-directed mutagenesis (Tucker et al., 1997). The nucleotide sequence of all mutated SUR1, Kir6.2, and Kir6.2C26 genes was confirmed by DNA sequencing.

Functional Coexpression of SURs and Kir6.2 cDNAs. Using LipofectAMINE (Invitrogen), human embryonic kidney (HEK) 293T cells were transfected simultaneously with the plasmid containing Kir6.2, a plasmid containing either SUR1, SUR2A, or SUR2B, and pCA-GFP (S65A) bearing a gene for green fluorescence protein (GFP). The plasmid containing Kir6.2C26 was transfected into HEK293T cells alone with pCA-GFP (S65A). The cells expressing GFP were identified by fluorescence microscopy and used for electrophysiology.

Electrophysiology. The currents through K_ATP channels expressed in the transfected HEK293T cells were recorded with patch-clamp techniques using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Patch pipettes were fabricated from borosilicate capillaries, and their tips were coated with Sylgard (Dow Corning, Midland, MI) and heat-polished. Pipettes were filled with solution containing 145 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 5 mM HEPES-KOH (pH 7.4). The bath was perfused with modified Tyrode's solution containing 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.5 mM HEPES-NaOH (pH 7.4). After formation of the cell-attached configuration, the bath was perfused with "internal" solution containing 145 mM KCl, 5 mM EGTA, 2 mM MgCl₂, and 5 mM HEPES-KOH (pH 7.3). The concentration of free Mg²⁺ in this solution was 1.4 mM. After patch excision, the internal side of the patch membrane was perfused with the internal solution containing nucleotides and/or drugs. When nucleotides were added to the internal solution, the free Mg²⁺ concentration was adjusted to 1.4 mM by supplementing MgCl₂. K_ATP channel currents in inside-out patch membranes were recorded at 60 mV at room temperature (25°C). The data were recorded on videocassette tapes through a PCM converter (VR-10B; InstruTECH, Port Washington, NY). For analysis, the stored data were reproduced through the same PCM converter, low pass-filtered at 1 kHz (3 dB) with an eight-pore Bassel filter (Frequency Devices, Haverhill, MA), digitized at 5 kHz with an AD converter (ITC16; InstruTECH), and analyzed on a personal computer (Power Macintosh G3; Apple, Cupertino, CA) using commercially available software (Patch Analyst Pro; MT Corporation, Hyogo, Japan).

Data Analysis. The channel activity was estimated by measuring a mean current amplitude or NP_o (N is the number of functional channels and P_o is the open probability of each channel). The channel activity in the presence of nateglinide was expressed as a fraction of the channel activity recorded in the absence of the drug (relative channel activity). Nateglinide concentration-response curves were fit with the following equations according to Gribble et al. (1998b).

(1)

x is a term describing the high-affinity site, and y is that describing the low-affinity site.

(2)

(3)

where [Nateglinide] is the nateglinide concentration in moles per liter; K_i1 and K_i2 are the nateglinide concentrations at which the magnitude of inhibition is half the maximum at the high- and low-affinity sites, respectively; h₁ and h₂ are the Hill coefficients (slope factors) for the high- and low-affinity sites, respectively; and L is the fraction remaining when the high-affinity inhibitory sites are maximally occupied. When only a single inhibitory site is present, the equation reduces to the following:

(4)

All statistical values are indicated as mean ± S.E.M. The statistical difference was evaluated by Student's t test. Statistical probability of p < 0.05 was taken as a significant difference.

Drugs. A 100 mM stock solution of nateglinide was prepared in dimethyl sulfoxide. Drugs were diluted to the desired concentrations in the internal solution. The final concentration of dimethyl sulfoxide was 0.3%, at which the vehicle by itself did not affect the K_ATP channel currents measured in the inside-out patch membranes (n = 5). Nateglinide was a kind gift from Ajinomoto Co. Ltd. (Tokyo, Japan). ATP and ADP were purchased from Sigma-Aldrich (St. Louis, MO). UDP was obtained from Roche Diagnostics (Mannheim, Germany). Other chemicals and materials were purchased from commercial sources.

	Results

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Comparison of Effects of Nateglinide on SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 Channel Currents. To determine the tissue selectivity of nateglinide, we first compared the effects of nateglinide on SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels expressed in HEK293T cells. These constructs represent, respectively, the -cell, cardiac, and smooth muscle types of K_ATP channel. Figure 1, A to C (left column), shows the effects of nateglinide on spontaneous openings of each type of K_ATP channel in inside-out patch membranes. The application of 0.1 µM nateglinide to the intracellular side of patch membranes reversibly inhibited SUR1/Kir6.2 channel currents by 35.3 ± 3.3% (n = 8). However, nateglinide inhibited SUR2A/Kir6.2 (n = 5) and SUR2B/Kir6.2 channel currents (n = 4) in a concentration-dependent manner only at concentrations higher than 10 µM. The relationship between the nateglinide concentration and the normalized amplitude of SUR1/Kir6.2 channel currents was best fit with the two-site model (eqs. 1-3 under Materials and Methods) with K_i1 of 75 nM and K_i2 of 114 µM (Fig. 1A, right column). In contrast, the drug inhibited SUR2A/Kir6.2 and SUR2B/Kir6.2 channel currents only with low affinity with K_i2 of 105 and 111 µM, respectively (eqs. 3 and 4 under Materials and Methods) (Fig. 1, B and C, right column). Thus, nateglinide inhibited the pancreatic type of K_ATP channel (SUR1/Kir6.2) with high affinity, and it caused low-affinity inhibition of all three types of K_ATP channel.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Inhibitory effects of nateglinide on SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channel currents. Inhibitory effects of nateglinide on SUR1/Kir6.2 (A), SUR2A/Kir6.2 (B) and SUR2B/Kir6.2 (C) channel currents were measured at 60 mV in inside-out patches. The pipette contained 145 mM K+. Left column, inhibitory effects of nateglinide on spontaneously opening SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels. MgATP and nateglinide were added to the bath solution as indicated by bars. Right column, concentration-response relationships for inhibition of each type of KATP current by nateglinide. The relative channel activity represents the channel activity normalized to that seen in the absence of the drug. The symbols and bars represent the mean and S.E.M., respectively. The number of observations for each point was eight, five, and four in A, B, and C, respectively. A, concentration-response curve was fit with the two-site model (eqs. 1-3 in text) with Ki1 = 75 nM, h1 = 0.8, Ki2 = 114 µM, h2 = 1.3, and L = 0.37. B, concentration-response curve was fit with the single-site model (eqs. 3 and 4) with Ki2 = 105 µM and h2 = 1.2. C, concentration-response curve was fit with the single-site model with Ki2 = 111 µM and h2 = 1.1.

Inhibitory Effect of Nateglinide on Kir6.2C26 and SUR1(S1237Y)/Kir6.2 Channels. Sulfonylurea and benzamido derivatives are thought to cause low-affinity inhibition of K_ATP channels through their action upon the Kir6.2 subunit and not through the SUR (Gribble et al., 1998b; Gribble and Ashcroft, 1999; Reimann et al., 2001). Figure 2A shows the effect of nateglinide on channel currents mediated by a Kir6.2 subunit whose C-terminal 26 amino acids had been removed (Kir6.2C26) and which forms active K_ATP channels in the absence of SUR (Tucker et al., 1997). Nateglinide inhibited Kir6.2C26 channel currents that were recorded in the absence of a SUR only at high concentrations (>10 µM). The complete concentration-response relationship for this reaction could not be obtained because >300 µM of the drug could not be dissolved in water. The fitting of eq. 4 to the available data suggested a K_i2 value of 290 µM. This value was of the same order as those obtained for the low-affinity inhibition of SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels by nateglinide, suggesting that the low-affinity site resides on the Kir6.2 subunit.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Inhibitory effect of nateglinide on Kir6.2C26 and SUR1(S1237Y)/Kir6.2 channel currents. Left column, inhibitory effects of nateglinide on spontaneously opening Kir6.2C26 (A) and SUR1(S1237Y)/Kir6.2 (B) channels were measured at 60 mV in inside-out patches. ATP and nateglinide were added to the bath solution as indicated by bars. Right column, concentration-response relationships for inhibition of Kir6.2C26 (A) and SUR1(S1237Y)/Kir6.2 (B) channel currents by nateglinide. Symbols and bars indicate the mean and S.E.M., respectively. The number of observation at each point was five in each of the graphs. The lines were fit with the single-site model with Ki2 = 290 µM and h2 = 0.9 for Kir6.2C26 channels, and Ki2 = 72 µM and h2 = 0.7 for SUR1(S1237Y)/Kir6.2 channels. The dashed line in the graph in B depicts the concentration-response curve for the inhibition of SUR1/Kir6.2 channel currents by nateglinide shown in Fig. 1A.

Effect of Intracellular MgADP on Nateglinide-Induced Inhibition of K_ATP Channels. It is known that intracellular MgADP enhances inhibition of SUR1/Kir6.2 channel currents by tolbutamide (Zünkler et al., 1988; Schwanstecher et al., 1992; Gribble et al., 1997a, 1998b). Because the same amino acid residue in SUR1 mediates the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide and tolbutamide, we examined whether the inhibitory effect of nateglinide was also augmented by MgADP (Fig. 3A). MgADP (100 µM) increased opening of SUR1/Kir6.2 channels in the absence of nateglinide but decreased them in the presence of 10 µM nateglinide (Fig. 3A). Whereas 10 µM nateglinide inhibited SUR1/Kir6.2 channel currents by 53.9 ± 4.0% in the absence of MgADP, in its presence the drug inhibited the currents by 86.1 ± 0.9% (Fig. 3D) (p = 0.0014). Thus, nateglinide also seemed to inhibit the channel currents more strongly in the presence of MgADP than in its absence.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of nateglinide on SUR1/Kir6.2, SUR1(S1237Y)/Kir6.2, and SUR2A/Kir6.2 channels in the presence and absence of 100 µM MgADP. The inhibitory effects of nateglinide on SUR1/Kir6.2 (A), SUR1(S1237Y)/Kir6.2 (B), and SUR2A/Kir6.2 (C) channels were measured in inside-out patches. ATP, ADP, and nateglinide were added to the bath solution as indicated by bars. D, relative channel activity in the presence of 10 or 100 µM nateglinide without (open columns) and with (closed columns) 100 µM MgADP. The channel activity in the presence of nateglinide is expressed as a fraction of that observed in the absence of the drug. The columns and bars indicate the mean and S.E.M, respectively. **, p < 0.01; NS, not significantly different.

Nateglinide Abolishes the Stimulatory Effect and Does Not Affect the Inhibitory Effect of MgADP on SUR1/Kir6.2 Channels. MgADP has both stimulatory and inhibitory effects on SUR1/Kir6.2 channels (Gribble et al., 1997b). The former effect is mediated by SUR1, whereas the latter is mediated by Kir6.2 (Gribble et al., 1997b). Figure 3A indicates that the stimulatory effect of the nucleotide dominated the inhibitory effect in the absence of nateglinide but vice versa in the presence of the drug. We therefore examined the effect of nateglinide on the separate stimulatory and inhibitory effects of the nucleotide.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effect of nateglinide on SUR1/Kir6.2(K185Q), SUR1/Kir6.2, and SUR1(K1384A)/Kir6.2 channels in the presence and absence of nucleotides. Effects of nateglinide on SUR1/Kir6.2(K185Q) (A), SUR1/Kir6.2 (B), and SUR1(K1384A)/Kir6.2 (C) channel currents were measured in inside-out patches. D, relative channel activity in the presence of 10 µM nateglinide without nucleotides (open columns), with MgADP (closed columns), and with MgUDP (hatched column). The channel activity in the presence of nateglinide is expressed as a fraction of that observed in the absence of the drug. The columns and bars indicate the mean and S.E.M., respectively. *, p < 0.05; **, p < 0.01; NS, not significantly different.

	Discussion

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Nateglinide is a novel type of oral hypoglycemic agent that does not contain either a sulfonylurea or benzamido moiety (Akiyoshi et al., 1995; Ikenoue et al., 1997; Gribble et al., 2001). It was recently licensed and launched for therapy of patients with type II diabetes. We examined the effect of nateglinide on SUR1/Kir6.2, SUR2A/Kir6.2, and SUR2B/Kir6.2 channels expressed in a mammalian cell line. We found that nateglinide specifically inhibited SUR1/Kir6.2 channels with high affinity. On the other hand, Sunaga et al. (2001) reported that less than 1 µM nateglinide partially inhibited all SUR1/Kir6.2, SUR2A/Kir6.2 and SUR2B/Kir6.2 channels. Although the reason for this discrepancy is not clear, Hu et al. (1999) reported that nateglinide inhibited native K_ATP channels in rat pancreatic -cells ~100 times more potently than those in the smooth muscle cells, consistent with our observation. Oral hypoglycemic agents cross-reacting with extrapancreatic K_ATP channels potentially cause undesirable side effects. Indeed, it is still under dispute whether sulfonylurea and benzamido derivatives increase the risk of cardiovascular mortality (Bernauer, 1997; Cleveland et al., 1997; UK Prospective Diabetes Study Group, 1998). Taking the therapeutic plasma concentration of nateglinide (~10 µM) into account, we conclude that nateglinide would not cause detrimental cardiovascular side effects (Hu et al., 1999; Gribble et al., 2001).

It is reported that physiological concentrations of MgADP enhance the inhibitory effect of sulfonylurea derivatives on native -cell K_ATP channels and reconstituted SUR1/Kir6.2 channels but not SUR2A/Kir6.2 channels (Zünkler et al., 1988; Schwanstecher et al., 1992; Gribble et al., 1997a, 1998b). This was also the case for nateglinide (Fig. 3, A and C). This characteristic would further enhance the selectivity of nateglinide for pancreatic K_ATP channels. The cumulative effects of nateglinide and MgADP upon the channels can be explained as follows. MgADP has dualistic effects upon K_ATP channels, evoking both stimulation and/or inhibition (Gribble et al., 1997b). Nateglinide inhibits SUR1/Kir6.2 channels and also suppresses the stimulatory effect of MgADP (Fig. 4A). The inhibitory effect of MgADP is not affected by nateglinide (Fig. 4C). Both compounds then have inhibitory effects and as a result, the channel activity becomes even smaller in the presence of nateglinide and MgADP than in the presence of nateglinide alone (Fig. 4C).

The high-affinity inhibition of the channels by nateglinide was eliminated by the S1237Y mutation of SUR1 (Fig. 2A). This mutation also abolishes the high-affinity inhibition of SUR1/Kir6.2 channels by the sulfonylurea derivative tolbutamide, but not inhibition evoked by the benzamido derivative meglitinide (Ashfield et al., 1999). This mutation also abolishes the binding of [³H]glibenclamide to membranes expressing SUR1 (Ashfield et al., 1999). Because glibenclamide bears both sulfonylurea and benzamido moieties, it is likely that S1237 forms an essential part of the binding site for sulfonylurea derivatives. It would therefore seem that nateglinide inhibits SUR1/Kir6.2 channels through the same site as sulfonylurea derivatives, even though it lacks a sulfonylurea moiety. Interestingly, nateglinide was a much more potent inhibitor of SUR1/Kir6.2 channels than tolbutamide and as potent as another sulfonylurea derivative, gliclazide (Gribble et al., 1998b; Gribble and Ashcroft, 1999). Mitiglinide is another antidiabetic agent that lacks either a sulfonylurea or benzamido moiety but causes high-affinity inhibition of SUR1/Kir6.2 channels that is abolished by the S1237Y mutation (Reimann et al., 2001). Mitiglinide therefore resembles nateglinide in this regard. But mitiglinide also inhibits SUR2A/Kir6.2 and SUR2B/Kir6.2 channels with high affinity, which must be mediated by a site on SUR2 that is different from the "sulfonylurea site" (Reimann et al., 2001).

It is not clear at present how nateglinide interacts with the sulfonylurea site on SUR1. Conformation analysis revealed that hypoglycemic agents such as nateglinide, glibenclamide, glimepiride, meglitinide, mitiglinide, repaglinide, and S3075 with diverse molecular structures nevertheless display a comparable U shape with hydrophobic cycles placed at the extremity of each hand and a peptidic bond placed at the bottom of U (Lins et al., 1995). Thus, molecular modeling of the agents is unable to define the particular molecular structure of the interface between nateglinide and SUR1. The molecular structure of SUR1 needs to be identified to further understand the molecular mechanism underlying the interaction between nateglinide and SUR1.

In conclusion, we have shown that nateglinide specifically inhibits SUR1/Kir6.2 channels with high affinity. Although nateglinide lacks a sulfonylurea moiety, its effect on SUR1/Kir6.2 channels resembles that of tolbutamide with respect to the S1237Y mutation and the interaction with intracellular MgADP. Nateglinide was, however, much more potent than tolbutamide. Because nateglinide has a clinically useful pharmacokinetic profile to compensate for the impaired first phase insulin response and thus suppress postprandial hyperglycemia, this compound seems to be an effective antidiabetic agent with less undesirable extrapancreatic side effects.