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J. Biol. Chem., Vol. 280, Issue 10, 8793-8799, March 11, 2005

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Two SUR1-specific Histidine Residues Mandatory for Zinc-induced Activation of the Rat K_ATP Channel^*

Victor Bancila, Thierry Cens, Dominique Monnier, Frédéric Chanson, Cécile Faure¶, Yves Dunant, and Alain Bloc||

From the Neurosciences Fondamentales, CMU, 1 rue Michel Servet, 1211 Genève 04, Switzerland, the CRBM, CNRS FRE 2593, 34293 Montpellier Cedex 05, France, and the ^¶Synthélabo Recherche, 10 rue des Carrières, B.P. 248, 92504 Rueil Malmaison Cedex, France

Received for publication, November 29, 2004 , and in revised form, December 14, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Zinc at micromolar concentrations hyperpolarizes rat pancreatic -cells and brain nerve terminals by activating ATP-sensitive potassium channels (K_ATP). The molecular determinants of this effect were analyzed using insulinoma cell lines and cells transfected with either wild type or mutated K_ATP subunits. Zinc activated K_ATP in cells co-expressing rat Kir6.2 and SUR1 subunits, as in insulinoma cell lines. In contrast, zinc exerted an inhibitory action on SUR2A-containing cells. Therefore, SUR1 expression is required for the activating action of zinc, which also depended on extracellular pH and was blocked by diethyl pyrocarbonate, suggesting histidine involvement. The five SUR1-specific extracellular histidine residues were submitted to site-directed mutagenesis. Of them, two histidines (His-326 and His-332) were found to be critical for the activation of K_ATP by zinc, as confirmed by the double mutation H326A/H332A. In conclusion, zinc activates K_ATP by binding itself to extracellular His-326 and His-332 of the SUR1 subunit. Thereby zinc could exert a negative control on cell excitability and secretion process of pancreatic -and -cells. In fact, we have recently shown that such a mechanism occurs in hippocampal mossy fibers, a brain region characterized, like the pancreas, by an important accumulation of zinc and a high density of SUR1-containing K_ATP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
K_ATP¹ channels are tetradimeric complexes of two structurally unrelated subunits (1–4): an inwardly rectifying K⁺ channel subunit (Kir6.x), which serves as an ATP-inhibitable pore (5), and a sulfonylurea receptor subunit (SUR), which belongs to the ATP-binding cassette transporter superfamily and endows the channel with sensitivity to magnesium nucleotides, channel openers, and sulfonylureas (6). To date, two Kir6.x genes have been described, Kir6.1 and Kir6.2 (7–9). As for the SUR subunit, two closely related genes, SUR1 and SUR2, have been cloned with different splice variants such as SUR2A and SUR2B (6, 10–12). Depending on tissues or organs, the different molecular forms of SUR and Kir6.x proteins co-assemble to form K_ATP channels with different functional and pharmacological properties (13, 14). Cardiac and skeletal muscle K_ATP channels are comprised of Kir6.2 and SUR2A, whereas vascular smooth muscle K_ATP channels combine the subunit SUR2B with either Kir6.1 or Kir6.2. The pancreatic -cell K_ATP channels involved in insulin secretion are comprised of Kir6.2 and SUR1. This pattern is also abundant in the mammalian central nervous system, especially in the hippocampal mossy fiber nerve terminals (15). In addition, several other subunit combinations have been described in the central nervous system (14).

Strikingly, several structures possessing K_ATP channels of the Kir6.2 and SUR1 type, such as pancreatic -cells and hippocampal mossy fibers, also contain substantial amounts of zinc (16–18). Puzzled by this co-localization, we found that micromolar concentrations of zinc hyperpolarize -cells from an insulin-secreting pancreatic line (RINm5F) by activating K_ATP channels (19). Moreover, this mechanism was also found using mossy fiber synaptosomes of the rat hippocampus (20). In both cases K_ATP activation by zinc resulted in a decreased secretion of insulin from -cells and of glutamate from mossy fiber terminals. This effect might be physiologically relevant, because it suggests that the transition metal could be involved in a paracrine or autocrine negative feedback regulation of release. However, in contrast to our results, Kwok and Kass (21) observed a block of cardiac K_ATP channels by low concentrations of extracellular divalent cations, including zinc. This discrepancy led us to hypothesis that zinc could induce opposite effects on different types of K_ATP, depending on the subunit composition of the channel. The cloning of cDNAs encoding K_ATP channel subunits afforded us the opportunity to determine the molecular basis of zinc-induced activation of recombinant K_ATP channels.

Thus, the present study aimed to investigate the mechanisms by which zinc activates or inhibits K_ATP channels and more particularly whether definite molecular compositions of SUR and Kir subunits are critical for these effects. Preliminary results (22) suggested that the SUR1 subunit was involved in K_ATP activation by extracellular zinc. So far, however, systematic subunit-combination analysis and clear-cut identification of domain and/or amino acid residues involved in zinc binding to K_ATP have not been performed. To achieve this, we used electrophysiological techniques to investigate the effects of zinc on recombinant K_ATP reconstituted in human embryonic kidney (HEK293T) cells by transient transfection of various combinations of rat SURx and Kir6.x subunits. By using pH modifications, pharmacological tools, and site-directed mutagenesis, we identified two histidine residues present in the extracellular side of SUR1 as mandatory for activation of K_ATP channels by zinc.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture—HEK293T cells and insulin-producing cell lines (RINm5F and INS-1E) were obtained from the Laboratoire de Biochimie Clinique, CMU, Geneva. Cells were cultured at 37 °C and in a humidified 5% CO₂, 95% air atmosphere in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum, 11 mM glucose, 100 units/ml penicillin, and 100 µg/ml streptomycin. 1–5 days before experiments were carried out, RINm5F, INS-1E, and transfected HEK293T cells were seeded out onto Falcon 3001-type Petri dishes (BD Biosciences) at 100–200,000 cells/dish in RPMI 1640 medium.

Cloning—Rat Kir6.1 and Kir6.2 cDNAs (GenBank D42145 [GenBank] .1 and X97041 [GenBank] .1, respectively) were cloned from rat lung and the rat insulinoma cell line RINm5F, respectively, using a RT-PCR-based strategy with 5'- and 3'-untranslated region-specific primers based upon the published sequence (7, 8). The resulting PCR products (1290 and 1180 bp for Kir6.1 and Kir6.2, respectively) were subcloned into pRCII (Invitrogen), and the sequences were confirmed on six independently isolated clones. cDNA encoding the rat sulfonylurea receptor SUR1 (GenBank X97279 [GenBank] .1) was isolated by RT-PCR from RINm5F cells. Three overlapping PCR products were generated using specific oligonucleotides primer pairs based upon the known sequence (10). The amplified products were subcloned into pBluescript II KS+ (Invitrogen). The sequences were confirmed on several independently isolated clones before the reconstitution of the full-length cDNA.

Site-directed Mutagenesis—The sequences of the SUR1 and SUR2A subunits of K_ATP were obtained from GenBank (accession numbers X97279 [GenBank] .1 and D83598 [GenBank] .1, respectively). In vitro site-directed mutagenesis of selected histidine residues on the rat SUR1 gene were performed each separately on the pcDNA3/SUR1 vector using the QuikChange site-directed mutagenesis kit from Stratagene. Each mutation introduced a single amino acid, changing a histidine to an alanine residue in the corresponding coding region of the cDNA. The double mutation H326A/H332A was done on the single cDNA mutant histidine 332 by using mutagenic primers (F/326 and R/326) introducing the H326A mutation. Mutagenesis and amplification reactions were performed with a PCR Mastercycler (Eppendorf) in a 50-µl reaction volume, containing 1.5 mM MgCl₂, with 50 ng of DNA pcDNA3/SUR1, 125 ng of each primer, 1 ml of mix dNTPs 10 mM, 3 plaque-forming units and plaque-forming unit buffer. Incorporation and extension of the mutagenic primers were done with the following cycling program, 1 run at 95 °C during 30 s and 25 runs of 30 s at 95 °C, 1 min at 55 °C, and 20 min at 68 °C. The non-mutated parental DNA template was digested with DpnI during 1 h at 37 °C, and 5 µl of the PCR mix was used to transform DH5-competent bacteria by heat shock at 37 °C. The sequence of each mutated cDNA was confirmed by full sequencing.

Transfections—The coding regions of wild type rat Kir6.1, Kir6.2, SUR1, SUR2A, or mutated SUR1 cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen). The combinations of these plasmids were transfected into HEK293T cells grown to 70% confluence in Falcon 3004-type Petri dishes. Transfections were performed overnight by CaCl₂ precipitation with 4 µg/well of Kir6.x + SURx combinations (ratio 1:3), together with 0.5 µg of vector pEGFP (Invitrogen) in fresh Dulbecco's modified Eagle's medium plus 10% fetal calf serum medium. Twelve hours after transfection cells were seeded out onto Falcon 3001-type Petri dishes and cultured in RPMI 1640 medium. The cells expressing green fluorescent protein were identified by fluorescence microscopy and used for electrophysiological recordings.

Electrophysiology—Whole-cell recordings were performed using fire-polished electrodes pulled from borosilicate glass and showing an open resistance of 2–3 megohms. Signals were amplified using an Axopatch 200-B amplifier and filtered through a 4-pole low pass Bessel filter at 1 or 2 kHz, before digitization with a Digidata 1200 interface and analysis with pClamp 8 software (Axon Instruments, Inc., Foster City, CA). Capacitative transients and series resistance were compensated (70%), using the circuitry incorporated within the amplifier. The external solution contained 145 mM NaCl, 3 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 10 mM Hepes, and 10 mM D-glucose. The pH was adjusted (with NaOH) to 7.2 in standard conditions and from 5.6 to 8.0 for pH dependence studies. The patch pipette solution contained 10 mM NaCl, 140 mM KCl, 1 mM MgCl₂, 10 mM Hepes, 1 mM EGTA, 1 mM MgATP, pH 7.2 (KOH). All experiments were performed at room temperature (20–22 °C), and the equilibrium potential for K⁺ ions (E_K+), calculated after correction of the liquid junction potential, was about –82 mV.

Data Expression—In histograms, data are expressed as mean ± S.E. of zinc-induced changes in the holding current normalized to the control current, the control current being the level of holding current measured before zinc applications. Current values were determined by subtraction of tolbutamide- or glibenclamide-insensitive currents. n indicates the number of individual cells recorded for each conditions.

Chemicals—Diethylpyrocarbonate (DEPC) was obtained from Brunschwing, (Basel, Switzerland), glibenclamide was from RBI (Fluka Chemie AG, Buchs, Switzerland), and zinc chloride and other inorganic salts (with negligible listed zinc contamination) were from Sigma. All solutions were prepared in distilled-deionized water to minimize basal zinc levels.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Zinc Activates Native and Recombinant Kir6.2/SUR1 K_ATP Channels—Fig. 1 (A and B) shows that extracellular zinc at micromolar concentrations activates K_ATP current in two insulinoma cell lines (RINm5F and INS-1E), confirming our previous observation (19). The current enhanced by zinc reversed itself at approximately –80 mV and was fully blocked by tolbutamide. The rat Kir6.2/SUR1 K_ATP was reconstituted in HEK293T cells, a human embryonic kidney cell line, which does not express endogenous K_ATP channels (23). Like native insulinoma cell lines, Kir6.2/SUR1-transfected HEK293T cells displayed an outward current that reversed itself at about –80 mV. This current was activated by zinc (10 µM) and suppressed by tolbutamide (Fig. 1C). HEK293T cells transfected with the vector pEGFP alone or with only one K_ATP subunit, either Kir6.2 or SUR1, did not display K_ATP or any endogenous current sensitive to zinc (Fig. 1D).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Zinc activates KATP current of native insulinoma cells and cells co-expressing rat Kir6.2 and SUR1 channel subunits. Cells were held at –80 mV in voltage-clamp whole-cell configuration, and currents were recorded in response to 3-s voltage ramps from –120 to –40 mV, in the presence or absence of 10 µM zinc in the extracellular medium. (A and B) In native insulinoma RINm5F or INS-1E cells, zinc enhanced a basic membrane current which reversed itself at approximately –80 mV. Tolbutamide (200 µM) suppressed both the pre-existing KATP current and its enhancement by 10 µM zinc (bottom traces; the two traces in this recording cannot be distinguished). C, zinc exerted a clear activating effect on HEK293T cells co-expressing rat Kir6.2 and SUR1 channel subunits. D, no KATP current and no zinc effect were recorded under the same experimental conditions in HEK293T cells transfected with the SUR1 subunit alone (control and zinc traces cannot be distinguished).

View larger version (17K): [in this window] [in a new window]   FIG. 2. The action of zinc on KATP depends on the channel subunit composition. Cells were voltage-clamped at –40 mV in the whole-cell configuration. Drugs were perfused in the extracellular medium at the indicated concentrations during the periods of time marked by corresponding blocks. Zn, zinc 10 µM; D, diazoxide 100 µM; T, tolbutamide 200 µM; G, glibenclamide 5 µM; P, pinacidil 5 µM. Calibration: 10 s and 100 pA (200 pA in C and E). A, in native RINm5F cells, zinc enhanced a basic outward current. However, the initial increase in current amplitude (on effect) was followed by a slower inhibiting effect, which was clearly visible on zinc washout (off effect). Also diazoxide activated the outward current, an effect which was slightly additive to that of zinc. Tolbutamide fully blocked the current, attesting that it arose from opening of KATP channels. B, the same pharmacological pattern was obtained by using HEK293T cells co-expressing the rat Kir6.2 and SUR1 subunits. C, with the Kir6.1/SUR1 composition, zinc and diazoxide exerted a stronger activating action. D, in contrast, zinc and diazoxide did not activate, but rather inhibited, the KATP current of HEK293T cells co-expressing rat Kir6.2 and SUR2A subunits. E, finally, when HEK293T cells expressed the rat Kir6.1/SUR2A combination, pinacidil markedly activated the KATP current, but zinc inhibited both the basic current and even more so the pinacidil-enhanced current. In mutants containing the SUR2A subunit, glibenclamide was used instead of tolbutamide to suppress KATP currents. F, the rapid action of zinc (IZn), either positive (activation) or negative (inhibition), is expressed as the ratio to the corresponding basic outward current (IC). Mean ± S.E. values of the number or determinations indicated above histograms. The activating effect of zinc depended on the presence of SUR1; it was larger when Kir6.1 was co-expressed instead of Kir6.2.

pH Dependence of Zinc-induced K_ATP Activation—It has been shown that zinc ions can interact with proteins by direct binding to either histidine, cysteine, aspartate, or glutamate residues (24–26). The binding of zinc to these amino acids is expected to be differently affected by changes in H⁺ ion concentration. In the case of K_ATP, this should modify the zinc-induced activation of the channel. To test this, the action of zinc on the K_ATP current was examined as a function of extracellular pH on RIN-5F cells. As shown in Fig. 3A, zinc provoked a more pronounced activation of the K_ATP current at pH 8.0 than at pH 6.8. At pH 5.6, zinc was clearly inhibitory. By titrating the effect of zinc as a function of pH over the range of 5.6 and 8.0, we found a reverse point, which was close to pH 6.4 (Fig. 3B). This profile of pH dependence suggested a histidine side chain (pK_a 6–7) rather than a cysteine (pK_a 8–9) as the relevant target at the zinc binding site (27).

View larger version (14K): [in this window] [in a new window]   FIG. 3. The pH dependence of zinc action on KATP currents in insulinoma cells. A, outward currents were recorded from RIN-5F cells held at –40 mV at the indicated pH values and tolbutamide was added at the end to identify signals as KATP currents. Zinc activated KATP current when the pH was higher than 6.4 but inhibited it at lower pH values. B, the effect of zinc on KATP current (IZn) was measured as the ratio to basic current (IC) and expressed (mean ± S.E.) as a function of pH. The number of determinations is indicated.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Effects of DEPC and hydroxylamine on the activating action of zinc on KATP currents. HEK293T cells co-transfected with rat Kir6.2 and SUR1 subunits were recorded in whole-cell configuration. A, currents elicited by 3-s voltage ramps from –120 to –40 mV (holding potential –80 mV). The application of 10 µM zinc enhanced the outward current reversing at about –80 mV (traces in the top). After pre-incubation with 1 mM DEPC during 1–2 min, the effect of zinc was practically annihilated. B, in a series of 6 determinations (± S.E.), the activating effect of zinc was expressed as a ratio to the basic outward current. Pre-incubation with DEPC reduced zinc effect to 12%. Application of hydroxylamine (0.1 M) to DEPC-blocked cells for 1–2 min partly re-activated the action of zinc on the KATP current.

Binding Site for Zinc Ions on the K_ATP Channel SUR1 Subunit, Site-directed Mutagenesis—Comparison of the extracellular segments of SUR subunits of the rat K_ATP channel revealed that five histidine residues are present in SUR1 but not in SUR2A. These are His-11 and His-160, located in the first transmembrane domain (TMD-0), His-326 and His-332 in TMD-1, and His-1273 in TMD-2.

SUR1-specific extracellular histidines were individually mutated and replaced by the neutral amino acid, alanine. Fig. 5 illustrates the site of the different mutations (Fig. 5A) and the corresponding K_ATP currents recorded in the response to voltage ramps before, during, and after zinc application (Fig. 5B). Averaged zinc-induced changes in wild type and mutated channels are expressed in Fig. 5C as a percentage of the control K_ATP current measured before zinc application. Mutations H11A, H160A, and H1273A had no effect, as zinc enhanced the K_ATP current in these clones to the same extent as in the wild type Kir6.2/SUR1 channels. In contrast, both mutations H326A and H332A, and even more so the double mutation H326A/H332A, converted the activation into inhibition. Thus, the site of zinc activation of the rat K_ATP channel requires the His-326 and His-332 residues of SUR1, which are situated on the first extracellular loop of the second transmembrane domain (TMD-1).

View larger version (44K): [in this window] [in a new window]   FIG. 5. Determination of histidine residues involved in the activating action of zinc on KATP channels. The action of zinc on KATP currents was determined in series of rat SUR1 mutants, where each extracellular SUR1-specific histidine was replaced by alanine. SUR1 mutants were co-transfected in HEK293T cells with the rat Kir6.2 subunit. A, site of the SUR1 histidine mutation. B, effect of 10 µM zinc in the corresponding clones. Currents were measured in response to 3-s voltage ramps from –120 to –40 mV (holding potential –80 mV). Zinc (10 µM) was applied during the time indicated by the black bar. Calibration: 1 nA and 20 s C, percent KATP current increase, or decrease, caused by zinc in the corresponding clone. Mean ± S.E. of the indicated number of determinations. Mutations H326A and H327A and the double mutation H326A/H327A suppressed zinc activation of KATP. Mutations H11A, H160A, and H1273A did not affect the zinc activation of KATP current, whose amplitude remained equal to that of the wild type rat SUR1/Kir6.2 clone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of Zinc Binding Sites on Ion Channels—Zinc can interact with various ligand-gated and voltage-gated ion channels generally causing inhibition. Interestingly, the action of zinc on glycine receptors has been reported to be biphasic (potentiation and inhibition) corresponding to separate binding sites (29). Our present study suggests the same for K_ATP channels where activation or inhibition by zinc may occur at different sites of the Kir and SUR subunits.

Histidine residues have been identified as essential for the zinc inhibition of several subtypes of -aminobutyric acid receptors, depending on critical localization of histidines on definite subunits (28, 30–32). Also, in recombinant N-methyl-D-aspartate receptors critical histidines (His-42, His-44, and His-128) are required for high affinity, voltage-independent, pH-dependent zinc inhibition (33, 34).

Activation of acid-sensing ion channels by zinc implicates two critical histidines (His-162 and His-339) as well (35). In addition to histidine residues, cysteines have also been shown to participate in zinc binding to ion channels. As an example Cys-546 is critical for the zinc blockage of human skeletal muscle chloride channels (27).

The case of AMPAR is particularly interesting in connection with the present work, because it generates currents that are potentiated by micromolar concentrations of zinc (36). Histidine 412 seems to be critical for zinc binding on the AMPAR, because it is present on GluR2–4 (sensitive to zinc) but not on GluR1 (non-sensitive) subunits (37).

We also identified two histidines (His-326 and His-332) as critical amino acid residues for the activation action of zinc on the extracellular side of K_ATP SUR1 subunit. The site is localized on the short extracellular loop between the first and second segment of TMD1. The zinc binding site seems to be fully independent from the sulfonylurea binding site, which is localized in the intracellular side of SUR on TMD-2 (38, 39). Indeed, tolbutamide equally blocked K_ATP current in all mutated clones (Fig. 5), and the activation by zinc was additive to the activation induced by diazoxide (Fig. 2).

Physiological Significance of K_ATP Channel Activation by Zinc—By linking the cell metabolism with the membrane potential, and thereby electrical activity, K_ATP channels regulate important physiological functions in various tissues. In the pancreatic -cells, the K_ATP channels are critically involved in the control of glucose-induced insulin secretion. An increase in glucose metabolism in the vicinity of K_ATP channels depolarizes the membrane of the -cells, leading to the opening of voltage-dependent Ca²⁺ channels and thereby to an increase in insulin secretion (40). Zinc is co-stored with insulin in pancreatic granules and co-released by exocytosis (17). We found that micromolar concentrations of zinc hyperpolarized the -cells by activating K_ATP channels, reducing cell excitability and calcium spike firing (19). As a consequence, further release of insulin is reduced.² In this way, zinc could ensure a negative feedback on insulin and on its own secretion. However, a more relevant physiological role for the activation of pancreatic K_ATP channels by -cell-secreted zinc can be to regulate glucagon release from -cells. Indeed, -cells are also provided with SUR1-containing K_ATP channels (41), and because intra-islet circulation is expected to carry zinc from -cells toward -cells, the release of glucagon will be reduced when insulin and zinc co-release is intense. This hypothesis is supported by the work of Ishihara et al. (42) who demonstrated that zinc secreted by -cells is implicated in the suppression of glucagon secretion observed in response to -cell activation. In this view, zinc would amplify the overall effect of insulin by decreasing that of glucagon. Thereby, the same stimulus may control reversibly regulated secretion from the two main pancreatic cellular populations.

Similarly, in mossy fiber/CA3 neuron synapses of the hippocampus, zinc is concentrated in synaptic vesicles of nerve terminals. On stimulation, it is released (43–45), and recent experiments have shown that zinc at low concentrations activates presynaptic K_ATP channels, reducing further glutamate release (20). At this level, zinc could play a role of negative feedback on transmission.

Pathological Implications—Such data may be relevant to the pathological states of the pancreas, in view of the importance of -cell dysfunction in the pathogenesis of type-2 diabetes, or hyperinsulinism. Some studies (46–48), but not all (49–51), have reported disorganized oscillations of insulinemia in patients with type-2 diabetes and their near relatives with mild glucose intolerance. Insulin resistance of obese patients is also accompanied by a decrease in the regularity of pulsatile insulin secretion (52). Because oscillations of insulinemia favor optimal glucose homeostasis, one may speculate that a disturbance of this process in type-2 diabetes contributes to the disease. It would be most interesting to investigate whether the regulation of K_ATP channels and/or the feedback action of zinc might be perturbed in these affections.

Zinc and K_ATP channels might also be involved in pathological conditions affecting the central nervous system. We recently found that micromolar amounts of zinc protect the nerve terminal from excessive depolarization, preventing massive transmitter release and neuronal death consecutive to anoxic insult. This neuroprotective effect was antagonized by tolbutamide, indicating K_ATP implication (20). Moreover, overexpression of SUR1 or Kir6.2 subunits in mice has been reported to render animals more resistant to ischemic insults (53, 54). Also, Kir6.2 knock-out mice are more sensitive to hypoxia-induced seizures (55).

In addition, zinc chelation or zinc deficiency is associated with an increased seizure susceptibility (56, 57). Mice knocked-out for the vesicular zinc transporter ZnT3 are more vulnerable to chemically induced seizures and consequent neuronal damage (58). These observations could be explained by the mechanism described herein, i.e. a failure of K_ATP activation when synaptic zinc is low or absent.

Conclusions—In pancreatic -cells and hippocampal mossy fibers, zinc is concentrated in large amounts and released on activity. These cells are also characterized by a high density of K_ATP channels composed of the SUR1/Kir6.2 subunits. Because the extracellular concentration of zinc following release probably reaches the range (1 µM) needed for activation of K_ATP of this type, zinc can act as a negative feedback regulator of secretion of both insulin and glutamate. This effect is mediated by two particular amino acids (His-326 and His-332) of the rat SUR1 subunit, located in the first extracellular loop of TMD-1.

	FOOTNOTES

 
^* This work was supported by Swiss Fonds National pour la Recherche Scientifique (FNRS) Grants 31-057135.99 (to Y. D. and A. B.) and the European Union project Lipidiet (QLK1-CT-2002-00172). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: DBCM, Université de Lausanne, Rue du Bugnon 9, CH-1005 Lausanne, Switzerland. Tel.: 41-21-692-5284; Fax: 41-21-692-5105; E-mail: Alain.Bloc{at}unil.ch.

¹ The abbreviations used are: K_ATP, ATP-sensitive K⁺; SUR, sulfonylurea receptor; HEK, human embryonic kidney; Kir, inward rectifier potassium channel; DEPC, diethyl pyrocarbonate; TMD, transmembrane domain; AMPAR, (aminoethyl)phosphonic acid-type glutamate receptor.

² V. Bancila, P. Maeschler, Y. Dunant, and A. Bloc, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank Jonathan G. Gore and Robert Lyle for help with manuscript, Anne Baron and Laurianne van Bever for support and advice during this study, and Francoise Loctin for excellent technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Clement, J. P., Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L., and Bryan, J. (1997) Neuron 18, 827–838[CrossRef][Medline] [Order article via Infotrieve]
2. Inagaki, N., Gonoi, T., and Seino, S. (1997) FEBS Lett. 409, 232–236[CrossRef][Medline] [Order article via Infotrieve]
3. Shyng, S., and Nichols, C. G. (1997) J. Gen. Physiol. 110, 655–664[Abstract/Free Full Text]
4. Bryan, J., Vila-Carriles, W. H., Zhao, G., Babenko, A. P., and Aguilar-Bryan, L. (2004) Diabetes 53, Suppl. 3, 104–112
5. Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S., and Ashcroft, F. M. (1997) Nature 387, 179–183[CrossRef][Medline] [Order article via Infotrieve]
6. Inagaki, N., Gonoi, T., Clement, J. P., Wang, C. Z., Aguilar-Bryan, L., Bryan, J., and Seino, S. (1996) Neuron 16, 1011–1017[CrossRef][Medline] [Order article via Infotrieve]
7. Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar-Bryan, L., Seino, S., and Bryan, J. (1995) Science 270, 1166–1170[Abstract/Free Full Text]
8. Inagaki, N., Tsuura, Y., Namba, N., Masuda, K., Gonoi, T., Horie, M., Seino, Y., Mizuta, M., and Seino, S. (1995) J. Biol. Chem. 270, 5691–5694[Abstract/Free Full Text]
9. Sakura, H., Ammala, C., Smith, P. A., Gribble, F. M., and Ashcroft, F. M. (1995) FEBS Lett. 377, 338–344[CrossRef][Medline] [Order article via Infotrieve]
10. Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E., Gonzalez, G., Herrera-Sosa, H., Nguy, K., Bryan, J., and Nelson, D. A. (1995) Science 268, 423–426[Abstract/Free Full Text]
11. Isomoto, S., Kondo, C., Yamada, M., Matsumoto, S., Higashiguchi, O., Horio, Y., Matsuzawa, Y., and Kurachi, Y. (1996) J. Biol. Chem. 271, 24321–24324[Abstract/Free Full Text]
12. Sakura, H., Trapp, S., Liss, B., and Ashcroft, F. M. (1999) J. Physiol. (Lond.) 521, 337–350[Abstract/Free Full Text]
13. Aguilar-Bryan, L., and Bryan, J. (1999) Endocr. Rev. 20, 101–135[Abstract/Free Full Text]
14. Seino, S., and Miki, T. (2003) Prog. Biophys. Mol. Biol. 81, 133–176[CrossRef][Medline] [Order article via Infotrieve]
15. Karschin, C., Ecke, C., Ashcroft, F. M., and Karschin, A. (1997) FEBS Lett. 401, 59–64[CrossRef][Medline] [Order article via Infotrieve]
16. Frederickson, C. J. (1989) Int. Rev. Neurobiol. 31, 145–238[Medline] [Order article via Infotrieve]
17. Kristiansen, L. H., Rungby, J., Sondergaard, L. G., Stoltenberg, M., and Danscher, G. (2001) Histochem. Cell Biol. 115, 125–129[CrossRef][Medline] [Order article via Infotrieve]
18. Mourre, C., Ben Ari, Y., Bernardi, H., Fosset, M., and Lazdunski, M. (1989) Brain Res. 486, 159–164[CrossRef][Medline] [Order article via Infotrieve]
19. Bloc, A., Cens, T., Cruz, H., and Dunant, Y. (2000) J. Physiol. (Lond.) 529, 723–734[Abstract/Free Full Text]
20. Bancila, V., Nikonenko, I., Dunant, Y., and Bloc, A. (2004) J. Neurochem. 90, 1243–1250[CrossRef][Medline] [Order article via Infotrieve]
21. Kwok, W. M., and Kass, R. S. (1993) J. Gen. Physiol. 102, 693–712[Abstract/Free Full Text]
22. Prost, A. L., Bloc, A., Hussy, N., Derand, R., and Vivaudou, M. (2004) J. Physiol. (Lond.) 559, 157–167[Abstract/Free Full Text]
23. Yu, S. P., and Kerchner, G. A. (1998) J. Neurosci. Res. 52, 612–617[CrossRef][Medline] [Order article via Infotrieve]
24. Berg, J. M., and Shi, Y. (1996) Science 271, 1081–1085[Abstract]
25. Higaki, J. N., Fletterick, R. J., and Craik, C. S. (1992) Trends Biochem. Sci. 17, 100–104[CrossRef][Medline] [Order article via Infotrieve]
26. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257–287[Medline] [Order article via Infotrieve]
27. Kurz, L. L., Klink, H., Jakob, I., Kuchenbecker, M., Benz, S., Lehmann-Horn, F., and Rudel, R. (1999) J. Biol. Chem. 274, 11687–11692[Abstract/Free Full Text]
28. Horenstein, J., and Akabas, M. H. (1998) Mol. Pharmacol. 53, 870–877[Abstract/Free Full Text]
29. Harvey, R. J., Thomas, P., James, C. H., Wilderspin, A., and Smart, T. G. (1999) J. Physiol. (Lond.) 520, 53–64[Abstract/Free Full Text]
30. Fisher, J. L., and Macdonald, R. L. (1998) J. Neurosci. 18, 2944–2953[Abstract/Free Full Text]
31. Wang, T. L., Hackam, A., Guggino, W. B., and Cutting, G. R. (1995) J. Neurosci. 15, 7684–7691[Abstract]
32. Wooltorton, J. R., McDonald, B. J., Moss, S. J., and Smart, T. G. (1997) J. Physiol. (Lond.) 505, 633–640[Abstract/Free Full Text]
33. Choi, Y. B., and Lipton, S. A. (1999) Neuron 23, 171–180[CrossRef][Medline] [Order article via Infotrieve]
34. Low, C. M., Zheng, F., Lyuboslavsky, P., and Traynelis, S. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11062–11067[Abstract/Free Full Text]
35. Baron, A., Schaefer, L., Lingueglia, E., Champigny, G., and Lazdunski, M. (2001) J. Biol. Chem. 276, 35361–35367[Abstract/Free Full Text]
36. Dreixler, J. C., and Leonard, J. P. (1994) Brain Res. Mol. Brain Res. 22, 144–150[Medline] [Order article via Infotrieve]
37. Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165–181[CrossRef][Medline] [Order article via Infotrieve]
38. Ashfield, R., Gribble, F. M., Ashcroft, S. J., and Ashcroft, F. M. (1999) Diabetes 48, 1341–1347[Abstract]
39. Uhde, I., Toman, A., Gross, I., Schwanstecher, C., and Schwanstecher, M. (1999) J. Biol. Chem. 274, 28079–28082[Abstract/Free Full Text]
40. Seino, S., Iwanaga, T., Nagashima, K., and Miki, T. (2000) Diabetes 49, 311–318[Abstract]
41. Bokvist, K., Olsen, H. L., Hoy, M., Gotfredsen, C. F., Holmes, W. F., Buschard, K., Rorsman, P., and Gromada, J. (1999) Pfluegers Arch. Eur. J. Physiol. 438, 428–436[CrossRef][Medline] [Order article via Infotrieve]
42. Ishihara, H., Maechler, P., Gjinovci, A., Herrera, P. L., and Wollheim, C. B. (2003) Nat. Cell Biol. 5, 330–335[CrossRef][Medline] [Order article via Infotrieve]
43. Assaf, S. Y., and Chung, S. H. (1984) Nature 308, 734–736[CrossRef][Medline] [Order article via Infotrieve]
44. Howell, G. A., Welch, M. G., and Frederickson, C. J. (1984) Nature 308, 736–738[CrossRef][Medline] [Order article via Infotrieve]
45. Li, Y., Hough, C. J., Suh, S. W., Sarvey, J. M., and Frederickson, C. J. (2001) J. Neurophysiol. 86, 2597–2604[Abstract/Free Full Text]
46. Lang, D. A., Matthews, D. R., Burnett, M., and Turner, R. C. (1981) Diabetes 30, 435–439[Abstract]
47. Matthews, D. R., Lang, D. A., Burnett, M. A., and Turner, R. C. (1983) Diabetologia 24, 231–237[CrossRef][Medline] [Order article via Infotrieve]
48. O'Rahilly, S., Turner, R. C., and Matthews, D. R. (1988) N. Engl. J. Med. 318, 1225–1230[Abstract]
49. Laedtke, T., Kjems, L., Porksen, N., Schmitz, O., Veldhuis, J., Kao, P. C., and Butler, P. C. (2000) Am. J. Physiol. 279, E520–E528
50. Mao, C. S., Berman, N., Roberts, K., and Ipp, E. (1999) Diabetes 48, 714–721[Abstract]
51. Nyholm, B., Porksen, N., Juhl, C. B., Gravholt, C. H., Butler, P. C., Weeke, J., Veldhuis, J. D., Pincus, S., and Schmitz, O. (2000) Metabolism 49, 896–905[CrossRef][Medline] [Order article via Infotrieve]
52. Zarkovic, M., Ciric, J., Stojanovic, M., Penezic, Z., Trbojevic, B., Dresgic, M., and Nesovic, M. (1999) Eur. J. Endocrinol. 141, 494–501[Abstract]
53. Hernandez-Sanchez, C., Basile, A. S., Fedorova, I., Arima, H., Stannard, B., Fernandez, A. M., Ito, Y., and LeRoith, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3549–3554[Abstract/Free Full Text]
54. Heron-Milhavet, L., Xue-Jun, Y., Vannucci, S. J., Wood, T. L., Willing, L. B., Stannard, B., Hernandez-Sanchez, C., Mobbs, C., Virsolvy, A., and LeRoith, D. (2004) Mol. Cell. Neurosci. 25, 585–593[CrossRef][Medline] [Order article via Infotrieve]
55. Yamada, K., Ji, J. J., Yuan, H., Miki, T., Sato, S., Horimoto, N., Shimizu, T., Seino, S., and Inagaki, N. (2001) Science 292, 1543–1546[Abstract/Free Full Text]
56. Mitchell, C. L., and Barnes, M. I. (1993) Neurotoxicol. Teratol. 15, 165–171[CrossRef][Medline] [Order article via Infotrieve]
57. Noebels, J. L., and Sidman, R. L. (1989) J. Neurogenet. 6, 53–56[Medline] [Order article via Infotrieve]
58. Cole, T. B., Robbins, C. A., Wenzel, H. J., Schwartzkroin, P. A., and Palmiter, R. D. (2000) Epilepsy Res. 39, 153–169[CrossRef][Medline] [Order article via Infotrieve]

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