Zinc is both an intracellular and extracellular regulator of K_ATP channel function

Abstract

Extracellular Zn²⁺ has been identified as an activator of pancreatic K_ATP channels. We further examined the action of Zn²⁺ on recombinant K_ATP channels formed with the inward rectifier K⁺ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn²⁺, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of K_ATP channels. External Zn²⁺, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn²⁺ dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 μm, respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn²⁺ on the SUR subunit. Effects of internal Zn²⁺ were not due to Zn²⁺ leaking out, since they were unaffected by the presence of a Zn²⁺ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn²⁺. Therefore, Zn²⁺ is an endogenous K_ATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting K_ATP channels, and suggest a new role for Zn²⁺ as an intracellular signalling molecule.

Zinc is an essential component of many proteins. As a cofactor or as a key structural element, it is required for enzyme catalysis and is involved in gene transcription or metalloenzyme function (Vallee & Falchuk, 1993). Ionic zinc is also an important factor involved in cell–cell communication, notably in the nervous and endocrine systems. Indeed, Zn²⁺ has been shown to accmulate in synaptic vesicles or secretion granules and to be coreleased with neurotransmitters and hormones (Huang, 1997; Dodson & Steiner, 1998; Frederickson & Bush, 2001). In glutamatergic neurones, Zn²⁺ is secreted with glutamate during neuronal activity and is believed to act as a modulator of synaptic transmission (Huang, 1997; Lin et al. 2001).

In pancreatic beta-cells, Zn²⁺ is necessary to maintain the crystalline structure of insulin in secretory granules (Dodson & Steiner, 1998), and has been proposed to exert a negative feedback upon hormone release by reducing the electrical activity and insulin secretion of beta-cells (Ghafghazi et al. 1981; Ferrer et al. 1984; Aspinwall et al. 1997). These actions of Zn²⁺ are likely to result from direct interaction with membrane ion channels (Harrison & Gibbons, 1994; Smart et al. 1994; Frederickson & Bush, 2001).

Bloc et al. (2000) proposed that the inhibitory effect of Zn²⁺ on insulin secretion from pancreatic beta-cells could result at least partly from its potentiating action on ATP-sensitive potassium channels (K_ATP). These channels are widely distributed among most types of excitable cells, where they couple metabolic status to membrane excitability (Ashcroft & Ashcroft, 1990; Seino & Miki, 2003). In pancreatic beta-cells, K_ATP channels are involved in glucose-induced insulin secretion whereas in cardiac and neuronal cells, they could have a protective effect against ischaemic damage (Standen, 2002). The K_ATP channel is a hetero-octameric complex composed of two distinct proteins: the sulphonylurea receptor (SUR), an ATP-binding-cassette (ABC) protein, and an inward rectifier potassium channel, Kir6.x. In pancreatic and cardiac K_ATP channels, four Kir6.2 assemble to form a K⁺-selective pore, which is constitutively associated with four SUR subunits (Seino & Miki, 2003). SUR serves as a sensor of metabolic adenine nucleotide changes and is the target of pharmacological modulators of the channel: blockers, like the sulphonylureas, and potassium channel openers such as cromakalim and pinacidil. Pancreatic and neuronal K_ATP channels contain the SUR1 isoform, whereas the channel in cardiac and skeletal muscle cells is formed with the SUR2A isoform (Terzic & Vivaudou, 2001).

Apart from its extracellular action, Zn²⁺ can also enter cells via various routes and act as an intracellular messenger (Li et al. 2001; Frederickson & Bush, 2001). Zn²⁺ can permeate membranes either directly through ligand-gated channels such as glutamate or nicotinic receptors (Sensi et al. 1997; Ragozzino et al. 2000; Jia et al. 2002), voltage-activated Ca²⁺ channels (Kerchner et al. 2000; Sheline et al. 2002) and other channels (Monteilh-Zoller et al. 2003), or through transporters (Weiss et al. 2000; Gaither & Eide, 2001). Entry of Zn²⁺ may alter protein structure and function, as well as gene expression (Weiss et al. 2000; Frederickson & Bush, 2001), but may also modulate channels from the intracellular compartment (Tabata & Ishida, 1999; Wang et al. 2001).

We here report the potentiation of K_ATP channels by intracellular Zn²⁺, and compare it with that induced by extracellular application of Zn²⁺ (Bloc et al. 2000). We further investigate these effects of Zn²⁺ as a function of the subunit composition of the channel and show that, on either side of the membrane, SUR is the most probable target of Zn²⁺.

Methods

Cell culture and heterologous expression

Clones

Rat Kir6.2 (GenBank accession X97041) and rat SUR1 (GenBank accession X97279) cDNAs were cloned from the rat insulinoma cell line RINm5F, using a RT-PCR-based strategy. Rat SUR2A cDNAs (GenBank accession D83598) and mouse Kir6.2 (GenBank accession D50581) were kindly provided by Dr S. Seino. Hamster SUR1 (GenBank accession L40623) was kindly provided by Dr J. Bryan.

Cell lines

For expression in COS-7 cells, rat Kir6.2, SUR1, and SUR2A cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen). Transfections were performed overnight by CaCl₂ precipitation with 4 μg per well of Kir6.2 + SURx combinations (ratio 1:3), together with 0.5 μg of vector pE-GFP (Invitrogen). Twenty-four hours after transfection, cells were plated and cultured in Dulbecco's modified Eagle's medium (DMEM) for 1–3 days before recordings. Cells expressing GFP were identified by fluorescence microscopy and used for electrophysiology.

Xenopus oocyte expression

Mouse Kir6.2, hamster SUR1 and rat SUR2A were subcloned in Xenopus oocyte expression vectors derived from the vector pGEMHE (Liman et al. 1992). Mutations were introduced in these plasmids by PCR (QuickChange Site-Directed Mutagenesis Kit, Stratagene). For the Kir6.2ΔC36 construct, a premature stop codon was introduced at the correct position. After amplification and linearization, plasmid DNAs were transcribed in vitro by using the T7 mMessage mMachine Kit (Ambion) to produce cRNA for subsequent oocyte microinjection.

Female Xenopus laevis were anaesthetized with 3-aminobenzoic acid ethyl ester (1 g l⁻¹ of water). Part of one ovary was removed with a minilaparotomy, the incision was sutured, and the animal was allowed to recover. Animal handling and experiments fully conformed with French regulations and were approved by local governmental veterinary services (authorization No. 28-03-15 delivered by the Ministère de l'Agriculture, Direction des Services Vétérinaires to M.V). Frogs were humanely killed by decapitation after final collection of oocytes from both ovaries.

Stage V or VI oocytes were defolliculated by a 60-min incubation at 19°C with 2 mg ml⁻¹ type A collagenase (Sigma). Selected oocytes were injected the next day with cRNAs encoding wild-type or truncated Kir6.2 (2 ng) and wild-type or modified SURs (6 ng). Injected oocytes were stored at 19°C in Barth's solution (in mm: 1 KCl, 0.82 MgSO₄, 88 NaCl, 2.4 NaHCO₃, 0.41 CaCl₂, 0.3 Ca(NO3)₂, 16 Hepes, pH 7.4) supplemented with 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 100 μg ml⁻¹ gentamycin. Three to five days after injection, oocytes were devitellinized and recombinant K_ATP channels were characterized by the patch-clamp technique in the excised inside-out or outside-out configuration.

Electrophysiology

All experiments were carried out at room temperature (20–22°C). ATP-containing solutions were prepared immediately before the experiments. Diazoxide, tolbutamide and glibenclamide (Sigma) were diluted to their final concentrations from stock solutions in DMSO at 100, 100, and 20 mm, respectively. At the concentrations used, DMSO had no effect on channel activity (D'hahan et al. 1999a). The indicated concentrations of Zn²⁺ (chloride salt; Fluka) represent free zinc as calculated by the program ALEX (Vivaudou et al. 1991). In our experimental conditions, ATP was the only Zn²⁺ complexant. Since Zn²⁺ binds to ATP with a dissociation constant ∼15 μm, we used Zn²⁺ concentrations low enough to limit effects due solely to interactions with ATP. Thus in our experimental conditions, the fraction of ATP complexed with Zn²⁺ was at most 11% (a value reached when 31 μm Zn was mixed with 100 μm ATP to yield 20 μm free Zn²⁺ and 11 μm ZnATP²⁻).

Cell lines

For whole-cell recording, the external bath solution contained (mm): 145 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 10 Hepes, 10 d-glucose, pH 7.2, and patch pipettes (2–3 MΩ) contained (in mm): 10 NaCl, 140 KCl, 1 MgCl₂, 10 Hepes, 1 EGTA, 1 Mg-ATP, pH 7.2. In the inside-out configuration, bath and pipette (5–10 MΩ) solutions were identical with (in mm): 10 NaCl, 140 KCl, 2 MgCl₂, 10 Hepes, pH 7.2. Applications of the various solutions were performed using a gravity solution-changer controlled by electrovalves. Capacitive transients and series resistance were compensated. Signals were analysed with pClamp8 software (Axon Instruments).

Xenopus oocytes

For inside-out recordings, patch pipettes (2–10 MΩ) contained (mm): 136 KCl, 5 MgCl₂, 10 PIPES, 18 KOH, pH 7.1. DTPA (diethyle-netriaminepentaacetic acid; Sigma) and EGTA were added when specified. Patches were bathed in solutions that contained (in mm): 38 KCl, 92 KCH₃SO₃, 1 MgCl₂, 10 PIPES, 20 KOH, pH 7.1. Except where noted, EGTA was used at 1 mm. ATP (potassium salt; Sigma) was added as specified. For outside-out recordings, above pipette and bath solutions were switched to yield the same intra- and extracellular solutions. Unless otherwise specified, experiments were conducted at the Cl⁻ reversal potential (∼−30 mV) determined for each patch as the potential at which no current was induced by the removal of the Ca²⁺ chelator EGTA. Applications of the various solutions to the patch were performed using an RSC-100 rapid-solution-changer (Bio-Logic). Acquisition and analysis were performed with in-house software. Slow fluctuations of the baseline were removed by interactive fitting with a spline curve and subtraction of this fit from the signal. Single channel analysis (all-point histograms and fits) were performed with Origin software (OriginLab). Open probability was calculated as the overall time spent in open states over total recording time. Model fitting to the currents measured in 100 μm ATP normalized to the current measured in 0 ATP was done with Origin using a standard Hill equation:

where i₀ is the control normalized current in absence of Zn²⁺, K_½ the concentration for half-maximal inhibition and n the Hill coefficient.

Results are displayed as the mean ± s.e.m.

Results

Extracellular Zn²⁺ activates recombinant pancreatic K_ATP channels

Previous experiments showed that extracellular Zn²⁺ activates K_ATP channels in the pancreatic cell line RIN5mF (Bloc et al. 2000). In order to better understand the effects of Zn²⁺ on K_ATP channels, experiments were conducted on recombinant K_ATP channels formed with the pancreatic combination of subunits SUR1 and Kir6.2. Whole-cell voltage-clamp recordings were performed on COS-7 cells transfected with SUR1/Kir6.2 (Fig. 1). Large currents elicited by slow voltage ramps were suppressed in the presence of the SUR1-selective K_ATP blocker tolbutamide (300 μm) and reversed close to the K⁺ equilibrium potential. These currents were potentiated by bath application of Zn²⁺ (20 μm). Potentiation was absent in the presence of tolbutamide, confirming that Zn²⁺ activated K_ATP currents. On average, 20 μm Zn²⁺ potentiated the current measured at −40 mV by 42 ± 9% (Fig. 1C), a value similar to that reported for native channels of RIN5mF cells (Bloc et al. 2000). Similarly, outside-out patches excised from Xenopus oocytes expressing SUR1/Kir6.2 showed channel activity that was enhanced both by the SUR1 opener diazoxide (100 μm) and by bath application of Zn²⁺ (20 μm), and was inhibited by tolbutamide (200 μm; Fig. 2A). The increase in open probability (P_o) was variable from patch to patch, depending on the initial level of activity of the channels. On average, P_o increased from 0.22 ± 0.06 to 0.42 ± 0.07 (131 ± 40% increase; n = 6).

Figure 1. Differential effect of extracellular Zn²⁺ on SUR1/Kir6.2 and SUR2A/Kir6.2 whole-cell currents.

A, sulphonylurea-sensitive currents are activated by zinc in cells expressing SUR1/Kir6.2 but not in cells expressing SUR2A/Kir6.2. Whole-cell currents of COS-7 cells transfected with Kir6.2 and either SUR1 or SUR2A were elicited by voltage ramps from −120 to −40 mV (holding potential =−80 mV). Conductances were measured by fitting the ramp currents with a linear regression line between −100 mV and −60 mV and plotted as a function of time. The K_ATP channel blockers used here were tolbutamide for SUR1 and glibenclamide for SUR2A. B, relative effects of 20 μm extracellular Zn²⁺ on K_ATP currents as a function of subunit composition, measured at a holding potential of −40 mV. C and D, ramp protocol used for experiments of panel A and representative current recordings taken at the different times indicated by the circled numbers in A.

Figure 2. Activation by extracellular Zn²⁺ of single SUR1/Kir6.2 channels.

A, recordings of K_ATP channels in outside-out patches excised from Xenopus oocytes expressing SUR1/Kir6.2 channels, at a holding potential of −43 mV with 300 μm ATP in the pipette. Single-channel activity was strongly enhanced by 20 μm extracellular Zn²⁺. Channels were also activated by diazoxide and inhibited by tolbutamide. B, amplitude histograms of the recordings shown in A, top traces, in the absence and in the presence of Zn²⁺. Zn²⁺ increased open probability with no change in single-channel current amplitude.

The effect of extracellular Zn²⁺ depends on the SUR subunit

In order to assess which subunit was responsible for the potentiating effect of Zn²⁺, we also tested Zn²⁺ on cells expressing the cardiac isoform of the channel composed of the SUR2A and Kir6.2 subunits. The cardiac SUR2A/Kir6.2 channel is characterized by a lower sensitivity to sulphonylureas than the pancreatic form of the channel (SUR1/Kir6.2), and is activated by openers such as cromakalim and pinacidil, but not by diazoxide (in absence of internal ADP; D'hahan et al. 1999b).

In contrast to its action on SUR1/Kir6.2 channel, application of 20 μm extracellular Zn²⁺ did not potentiate the SUR2A/Kir6.2 channel expressed either in COS-7 cells (n = 12; Fig. 1) or in Xenopus oocytes (n = 4 outside-out patches, data not shown). Rather, Zn²⁺ induced a small reversible inhibition of the K_ATP current (Fig. 1). This indicates that the SUR subunit is critical to extracellular Zn²⁺ action, with a specificity towards SUR1-containing channels.

Modulation of recombinant K_ATP channels by intracellular Zn²⁺

Unexpectedly, Zn²⁺ applied to the intracellular face of the channel also had profound effects on channel activity. Characterization of this effect was performed on Xenopus oocytes coinjected with SUR and Kir6.2, from which K_ATP currents of hundreds of pA can be recorded in a single patch (D'hahan et al. 1999a). In Xenopus oocytes, inside-out patch recordings are routinely performed in the presence of the Ca²⁺ chelator EGTA in the bath to prevent activation by residual Ca²⁺ of endogenous Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)). Because EGTA also chelates Zn²⁺, application of Zn²⁺ requires removal of EGTA from the bath solution, and experiments were therefore conducted at the Cl⁻ equilibrium potential (E_Cl) to prevent contamination by I_Cl(Ca). E_Cl was precisely measured in each patch as the membrane potential at which no current was induced by removal of EGTA. These precautions were necessary because Zn²⁺ was found to block the endogenous I_Cl(Ca) conductance, identified as such because it reversed at E_Cl and was activated by removal of EGTA. Intracellular Zn²⁺ (2 and 20 μm) had an inhibitory effect on Cl⁻ current, reducing both the outward current at 0 mV and the inward currents at −50 mV (Fig. 3). At E_Cl, the potential used to record K_ATP channels, Zn²⁺ had no effect in uninjected oocytes.

Figure 3. Effect of intracellular Zn²⁺ on endogenous Ca²⁺-activated Cl⁻ currents of Xenopus oocytes.

Currents recorded at a membrane potential of 0 mV (A) and −50 mV (B) from inside-out patches excised from non-injected oocytes. On the cytosolic side, in the continuous presence of 100 μm ATP, removal of EGTA triggered activation of large currents reversing at E_Cl (−30 mV) which were blocked by Zn²⁺.

In the presence of a concentration of ATP (100 μm) sufficient to produce ∼80% channel inhibition, bath application of 20 μm Zn²⁺ activated both SUR1/Kir6.2 and SUR2A/Kir6.2 K_ATP channels (Fig. 4A and B). Potentiation by Zn²⁺ was dose dependent (Fig. 4C), with a clearly greater affinity for SUR1/Kir6.2 than SUR2A/Kir6.2 channels (Fig. 4D). Since Zn²⁺ concentrations above 20 μm could not be used for fear of indirect effects due to ATP chelation (see Methods), the degree of maximal activation could not be ascertained. Assuming channel activity could be raised by Zn²⁺ to the level obtained in the absence of ATP, Hill equation fitting yielded K_½ of 1.8 and 60 μm for SUR1 and SUR2A, respectively (Fig. 4D). These values became 1.3 and 5.1 μm if the levels obtained in 20 μm Zn²⁺ were taken as maximal.

Figure 4. Effect of intracellular Zn²⁺ on recombinant pancreatic and cardiac K_ATP channels.

Channel activity was measured at −30 mV in inside-out patches excised from Xenopus oocytes coinjected with Kir6.2 and SUR1 or SUR2A. A, activation by 20 μm Zn²⁺ of SUR1/Kir6.2 channels. B, as for A but with SUR2A/Kir6.2. Zn²⁺-activated currents were blocked by 10 μm glibenclamide. C, dose-dependent effect of intracellular Zn²⁺ on SUR2A/Kir6.2 channels. D, average SUR1 and SUR2A/Kir6.2 currents in 100 μm ATP, measured in the presence of increasing concentrations of Zn²⁺. Currents are normalized to the current measured in absence of ATP. The data were collected from 17 (SUR1) and 9 (SUR2A) patches in which at least two concentrations of Zn²⁺ were tested sequentially. Best fits with a Hill equation (see Methods), represented by the dashed lines, yielded K_½ = 1.8 μm and n = 0.78 for SUR1 and K_½ = 60 μm and n = 0.74 for SUR2A. E, effect of intracellular Zn²⁺ on Kir6.2 modified to be expressed alone (Kir6.2ΔC36). F, average currents in 100 μm ATP (normalized to current obtained in 0 ATP) measured before or during application of 20 μm Zn²⁺. Numbers above bars indicate the number of patches included in the average.

To identify the subunit, SUR or Kir6.2, which is the target of intracellular Zn²⁺, experiments were conducted on a truncated form of Kir6.2, Kir6.2ΔC36, from which the last 36 amino acids were deleted (Tucker et al. 1997), removing the endoplasmic reticulum retention signal present on the C-terminal extremity (Zerangue et al. 1999). This allows the expression at the plasma membrane of functional channels formed solely by the Kir6.2 subunit, albeit at a lower density than wild-type K_ATP channels. Application of internal Zn²⁺ (20 μm) on inside-out patches from oocytes expressing Kir6.2ΔC36 channels, either in the absence (data not shown) or in the presence of ATP (100 μm), caused a slight reduction in current which was not statistically significant (P > 0.2; Student's paired t test). These results point to the SUR protein as the target of both extra- and intracellular Zn²⁺.

The bilateral effects of Zn²⁺ do not entail crossmembrane transit

Although it would be unlikely in our recording configurations, the possibility exists that Zn²⁺ could somehow cross the membrane to reach its site of action, thus explaining how internal and external Zn²⁺ can have similar effects. In order to test for such a mechanism, experiments were reproduced in the presence of the Zn²⁺ chelators EGTA (100 μm; n = 3) or DTPA (100 μm; n = 5) in the patch pipette. Neither chelator on the intracellular face prevented the extracellular effect of Zn²⁺, which still increased SUR1/Kir6.2 currents to a similar extent (Fig. 5A). Similarly, the presence of the chelators DTPA (100 μm; n = 3) or EGTA (100 μm; n = 9) on the extracellular side of the membrane did not prevent the increase in K_ATP currents induced by application of Zn²⁺ on the intracellular side of the membrane (Fig. 5B).

Figure 5. Activation by Zn²⁺ on either side of the membrane is unaffected by the presence of chelators on the opposite side.

A, whole-cell recording from a COS-7 cell expressing SUR1/Kir6.2 channels, in the presence of 100 μm DTPA added to the pipette solution. The Zn²⁺ chelator inside the cell did not prevent the potentiation by extracellular Zn²⁺. Holding potential is −40 mV. B, current recorded at −30 mV from an inside-out patch excised from a Xenopus oocyte coinjected with SUR1 and Kir6.2, with 100 μm EGTA added to the pipette solution. The presence of the chelator on the extracellular side of the membrane did not alter channel activation by intracellular Zn²⁺.

These observations make it unlikely that Zn²⁺ could cross the membrane and reach its target by diffusion. They do not dispel an alternative mechanism where a single target site would be accessible from either side of the membrane through a protein-delimited pathway. Indeed, SUR presents strong homologies with other ATP-binding-cassette (ABC) proteins like Ycf1p, which are known to transport heavy metals, including divalent cations such as Cd²⁺ (Li et al. 1997). Although SUR has no known transport activity, it might, like Ycf1p, possess metal-binding sites accessible from both sides of the membrane in a nucleotide-dependent manner. We therefore investigated the responses to Zn²⁺ of channels incorporating debilitating mutations in both nucleotide-binding domains of either SUR1 or SUR2A. The key residues targeted were either the Walker A lysines of SUR1 (K719 and 1385) or the Walker B aspartates of SUR2A (D832 and 1469). As is obvious in Fig. 6, both mutants retained the capacity to be activated by internal Zn²⁺ although the extent of activation was diminished for the SUR1 mutant.

Figure 6. K_ATP channels with impaired nucleotide binding domains are still activated by intracellular Zn²⁺.

Channel activity was measured at −30 mV in inside-out patches excised from Xenopus oocytes coinjected with Kir6.2 and SUR1(K719M,K1385M) or SUR2A(D832N,D1469N). A, activation by 20 μm Zn²⁺ of SUR1(K719M,K1385M)/Kir6.2 channels. B, average currents in 100 μm ATP measured before or during application of 20 μm Zn²⁺. Currents were normalized to the current measured in the absence of ATP. Numbers above bars indicate the number of patches included in the average.

Discussion

The results presented here identify Zn²⁺ as a unique endogenous regulator of K_ATP channels, able to activate them reversibly at low concentrations from either side of the membrane in an isoform-dependent manner. They suggest a new role for Zn²⁺ as an intracellular signalling molecule (Maret, 2001).

Inhibition of Xenopus oocyte Ca²⁺-activated Cl⁻ currents by Zn²⁺

During the course of this work, we discovered that intracellular Zn²⁺ inhibited the most prominent endogenous currents of Xenopus oocytes, Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)). Previous studies have reported that micromolar concentrations of extracellular divalent cations, including Zn²⁺, induced I_Cl(Ca) in Xenopus oocytes, but that internal injections of Zn²⁺ had no effect (Miledi et al. 1989). However, when applied onto excised patches, intracellular Zn²⁺ (2–20 μm) inhibited a current identified as I_Cl(Ca) by its activation upon EGTA removal and its reversal at the predicted Cl⁻ equilibrium potential. Although we did not further characterize this effect, which represented the only effect of Zn²⁺ on endogenous conductances under our experimental conditions, we did consider it carefully when recording effects of Zn²⁺ on K_ATP channels heterologously expressed in Xenopus oocytes. Ionic concentrations were adjusted to have E_Cl (∼−30 mV) and E_K (∼0 mV) sufficiently distant so that sizable K⁺ currents could be measured at E_Cl. Furthermore, the actual E_Cl was determined in each patch to within 1 mV, as the reversal potential of I_Cl(Ca). In this manner, effects of intracellular Zn²⁺ on endogenous Cl⁻ and exogenous K⁺ conductances could be characterized separately with minimal interference.

Pancreatic K_ATP channels are activated by extra- and intracellular Zn²⁺

Bloc et al. (2000) reported that micromolar concentrations of Zn²⁺ increased the open-state probability of endogenous K_ATP channels in the pancreatic RIN5mF cell line. We substantiated this observation by showing that extracellular Zn²⁺ also activated recombinant SUR1/Kir6.2 channels heterologously expressed in either COS-7 cells or Xenopus oocytes. Potentiation was rapid, reaching its maximum within a few seconds, and reversed with similarly fast kinetics.

Unexpectedly, application of Zn²⁺ to the intracellular side of the membrane also led to activation of K_ATP channels. Intracellular Zn²⁺ at submicromolar concentrations has been shown to inhibit Cl⁻ channels in neurones (Tabata & Ishida, 1999), but to our knowledge, this is the first observation of the agonist effect of an internal transition metal on identified potassium channels, (Miledi et al. (1989) did report activation by high external Zn²⁺ of a K⁺ conductance in Xenopus oocyte follicular cells), and the first reported case of a channel activated by a non-permeant ligand from both sides of the membrane. For SUR1/Kir6.2 channels expressed in oocytes, measurements over a number of experiments gave a half-maximal concentration of 1.8 μm but there were significant differences in the responses of individual patches. This value is remarkably close to the value of 1.7 μm measured under different experimental conditions as being the concentration for half-maximal activation by extracellular Zn²⁺ (Bloc et al. 2000). The amplitude of the effect was comparable to that of pharmaceutical potassium channel openers since, in most patches, 20 μm Zn²⁺ was sufficient to fully reverse the inhibition caused by the 100 μm ATP present during our tests.

Isoform-dependent modulation of K_ATP channels by extracellular and intracellular Zn²⁺

The potentiation of K_ATP channels by extracellular Zn²⁺ appeared specific to the pancreatic channel, since Zn²⁺ failed to activate the cardiac form of K_ATP channels (SUR2A/Kir6.2), and even elicited a weak inhibition of these channels at the highest concentration tested of 20 μm. Kwok & Kass (1993) showed that such a concentration also inhibited native channels but to a much greater extent. Since they recorded Zn²⁺ effects on pinacidil-activated currents, the discrepancy could arise from a possible antagonism between K⁺ channel openers and Zn²⁺, not unlike that between openers and protons (Forestier et al. 1996).

Kwok & Kass (1993) also tested internal Zn²⁺ on ventricular myocyte channels and, even though they concluded a lack of inhibition, an activation, evident in their Fig. 4, could have been overlooked. In our hands, intracellular Zn²⁺ activated both the pancreatic and cardiac forms of K_ATP channels with a strong preference for the former. This dose-dependent effect, when modelled with sigmoidal functions, yielded K_½ in the range of 1–2 μm for SUR1/Kir6.2 and 5–60 μm for SUR2A/Kir6.2 channels. In both cases, the Hill coefficient was close to 1, indicative of a simple bimolecular reaction. This SUR-dependent difference, together with the lack of effects of Zn²⁺ on truncated Kir6.2 channels expressed alone, designates the sulphonylurea receptor as the primary target of intracellular Zn²⁺.

Possible mechanisms for Zn²⁺ potentiation of K_ATP currents

The first hypothesis that one could put forward about the effects of Zn²⁺ is that of an unspecific action related to the ionic nature of the effector. However, no such effects have been described for the other physiological divalent cations, Mg²⁺ and Ca²⁺. Furthermore, Zn²⁺ effects were recorded in the presence of Mg²⁺, present on both sides of the membrane throughout our experiments in large excess (>1 mm), and Ca²⁺, present outside in millimolar concentrations and inside in micromolar concentrations (as evidenced by the potent activation of I_Cl(Ca) upon removal of the chelator EGTA). A mechanism involving alterations of membrane surface charge (Vivaudou & Forestier, 1995) is therefore implausible. Likewise, interaction of intracellular Zn²⁺ with nucleotides could not lead to significant activation: first, Zn²⁺ will reduce free ATP by chelating it, possibly affecting the inhibitory potency of ATP on the Kir6.2 subunit, but our experimental conditions were designed to minimize this effect by keeping zinc-bound ATP at less than 11% of total ATP. Second, low concentrations of Mg–ATP have been shown to activate K_ATP channels through the SUR subunit (Gribble et al. 1998), but a similar effect of Zn–ATP would not be consistent with the available evidence demonstrating that Zn²⁺ cannot mimic Mg²⁺ in supporting nucleotide binding to ABC transporters (Cai et al. 2002).

This line of reasoning pleads for a more sophisticated mechanism involving Zn²⁺-specific receptor sites. The finding for SUR1 that the same molecule has the same, singular effects on an integral membrane protein when applied from either side of the membrane, intuitively suggests a single site of action. However, when we applied Zn²⁺ on one side, with a chelator on the other side, neither intra- nor extracellular effects were disturbed even though the concentration dependence of channel activation showed that Zn²⁺ has a 10⁴-higher affinity for the chelator, EGTA or DTPA, than for its receptor. This experiment suggests that the intracellular Zn²⁺ site is not seen from outside and vice versa. This evidence favours the existence of two distinct sites mediating the same effect. These sites would be located near or on the SUR protein itself since, first, the effects were dependent on the SUR isoform and were not seen with SUR-less channels, and second, the rapid onset and reversal of Zn²⁺ activation appears incompatible with long-range signalling mechanisms, such as a receptor-regulated metabolic process involving the zinc-sensing receptor ZnR (Hershfinkel et al. 2001). This G-protein-coupled receptor linked to phospholipase C is probably present in Xenopus oocytes as this would nicely explain the effects of external Zn²⁺ recorded by Miledi et al. (1989). Activation of phospholipase C by ZnR would result in a decrease in phosphatidylinositol 4,5-bisphosphate which would increase ATP sensitivity of the K_ATP channels and cause an inhibition of the channel (Xie et al. 1999; Kakei, 2003). Such a mechanism could sustain the delayed inhibition subsequent to application of external Zn²⁺ observed by Bloc et al. (2000), but cannot account for a potentiating effect of Zn²⁺. Cytosolic Zn²⁺ interacts nonetheless with a number of enzymes (Beyersmann & Haase, 2001) one of which is protein kinase C (PKC), which has been linked to K_ATP channel activation (Light, 1996). With a Zn²⁺-dependent PKC having been reported to downregulate a Cl⁻ conductance (Tabata & Ishida, 1999), a PKC role in intracellular Zn²⁺ activation of K_ATP channels cannot be ruled out but remains highly speculative because PKC activation of K_ATP channels depends on the Kir6.2 subunit (Light et al. 2000), but Zn²⁺ activation does not.

Considering a more direct pathway, the hypothesis of metal binding sites on SUR seems plausible because other IIb transition metals, such as Cd²⁺, can be transported by Ycf1p and Mrp1, ABC proteins that present a high degree of homology with SUR (Li et al. 1997; Tommasini et al. 1996), and because some prok aryotic ABC proteins are known to transport Zn²⁺ (Hantke, 2001). Zn²⁺ could even act as a substrate of SUR, not unlike the hypothesis raised for K⁺ channel openers (Moreau et al. 2000). However, SUR having mutations known to disable nucleotide-dependent transport activity of ABC transporters, retained sensitivity to Zn²⁺. This experiment argues against the necessity of a transport of Zn²⁺ for activating the channel, but it leaves open for further studies the possibility that a unique site of action of Zn²⁺ could lie within a transport pathway where chelators would not have access.

Physiological significance

In neurones, Zn²⁺ is concentrated in synaptic vesicles and cosecreted with neurotransmitters upon stimulation, and it is believed to act as a neuromodulator at excitatory synapses (Huang, 1997). In pancreatic beta-cells, Zn²⁺ accmulates in secretory granules, where it forms hexamers with insulin. These microcrystals dissolve during exocytosis and large amounts of Zn²⁺ are coreleased with insulin. It has been proposed that by activating K_ATP channels and hyperpolarizing cells, Zn²⁺ could exert a negative feedback on insulin secretion (Bloc et al. 2000). Aspinwall et al. (1999) have shown that released insulin stimulates insulin secretion via a positive feedback mechanism, and they suggested that some mechanism should take over to counteract this feedback. Stimulating K_ATP channels by Zn²⁺ coreleased with insulin could constitute one such mechanism. Zn²⁺ was also postulated to be responsible for the cross-talk between beta-cells and alpha-cells in pancreatic islets, whereas insulin secretion by beta-cells induces suppression of glucagon release by alpha-cells (Ishihara et al. 2003).

If extracellular Zn²⁺ can reach the micromolar levels used in this study (Weiss et al. 2000), intracellular free Zn²⁺ concentration is tightly controlled and maintained in the low nanomolar range (Sensi et al. 1997; Beyersmann & Haase, 2001). Since we have measured half-maximal activation of pancreatic/neuronal and muscle K_ATP channels at 1.8 and 60 μm, respectively, one could confidently conclude that cytosolic Zn²⁺ activation is a laboratory artefact that will never be seen under physiological conditions. The same could be said of ATP which inhibits K_ATP channels with micromolar affinities, but never goes below millimolar concentrations in living cells. Cook et al. (1988) settled that controversy by demonstrating that K_ATP channels can efficiently modulate membrane potential by operating at the very bottom of the dose–response curve for ATP, where inhibition is 99% or more. This argument applies to cytosolic Zn²⁺ activation as well: using the model curves fit to the data, it is predicted that 1% activation above control of SUR1/Kir6.2 and SUR2A/Kir6.2 can be achieved with 0.5 nm and 4 nm Zn²⁺, respectively. These concentrations are well within the limits of the feasible, without even postulating the existence of Zn²⁺-enriched microdomains which might well play a role due to tight coupling between K_ATP channels and Zn²⁺-permeant Ca²⁺ channels (Shibasaki et al. 2003).

What could then be the physiological role of intracellular Zn²⁺ potentiation of K_ATP channels? One possible role would be to protect cells against the cytotoxic effects of elevated internal Zn²⁺ (Kim et al. 2000; Frederickson & Bush, 2001). Depolarizing stimuli can drive Zn²⁺ entry through voltage-dependent Ca²⁺ channels in the presence of physiological amounts of other cations, including Ca²⁺ in cardiac myocytes (Atar et al. 1995) or in neurones (Sensi et al. 1997; Kerchner et al. 2000). Maintaining cells hyperpolarized by opening K_ATP channels could thus prevent further entry of Zn²⁺ through Ca²⁺ channels, by keeping them from being activated. Such a scheme could reinforce the negative feedback effect of external Zn²⁺ on insulin secretion discussed above (Bloc et al. 2000). In cardiac myocytes, Zn²⁺ could act as an endogenous opener of K_ATP channels, reducing the force of contraction of the myocardium, and thus preventing ischaemia–reperfusion injury as do classical potassium channel openers (Terzic & Vivaudou, 2001).

In pancreatic beta-cells or neurones, an increase in cytosolic Zn²⁺ could potentially occur during perturbation of the Zn²⁺-accmulating machinery of secretory granules. This could happen during metabolic distress or after sustained secretion leading to penury of secretory granules and reduction in the cytosolic Zn²⁺-storage capacity of cells. In both cases, opening of K_ATP channels would provide a resting period for the cell by decreasing its excitability, thus reducing metabolic demand, and at the same time would decrease ambient Zn²⁺ by reducing secretion.