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Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Correspondence to Frank T. Horrigan: Horrigan{at}mail.med.upenn.edu

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Copper is an essential trace element that may serve as a signaling molecule in the nervous system. Here we show that extracellular Cu²⁺ is a potent inhibitor of BK and Shaker K⁺ channels. At low micromolar concentrations, Cu²⁺ rapidly and reversibly reduces macrosocopic K⁺ conductance (G_K) evoked from mSlo1 BK channels by membrane depolarization. G_K is reduced in a dose-dependent manner with an IC₅₀ and Hill coefficient of 2 µM and 1.0, respectively. Saturating 100 µM Cu²⁺ shifts the G_K-V relation by +74 mV and reduces G_Kmax by 27% without affecting single channel conductance. However, 100 µM Cu²⁺ fails to inhibit G_K when applied during membrane depolarization, suggesting that Cu²⁺ interacts poorly with the activated channel. Of other transition metal ions tested, only Zn²⁺ and Cd²⁺ had significant effects at 100 µM with IC₅₀s > 0.5 mM, suggesting the binding site is Cu²⁺ selective. Mutation of external Cys or His residues did not alter Cu²⁺ sensitivity. However, four putative Cu²⁺-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, S2, and S4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter Cu²⁺ and/or Cd²⁺ sensitivity. Consistent with the presence of acidic residues in the binding site, Cu²⁺ sensitivity was reduced at low extracellular pH. The three charged positions in S1, S2, and S4 are highly conserved among voltage-gated channels and could play a general role in metal sensitivity. We demonstrate that Shaker, like mSlo1, is much more sensitive to Cu²⁺ than Zn²⁺ and that sensitivity to these metals is altered by mutating the conserved positions in S1 or S4 or reducing pH. Our results suggest that the voltage sensor forms a state- and pH-dependent, metal-selective binding pocket that may be occupied by Cu²⁺ at physiologically relevant concentrations to inhibit activation of BK and other channels.

© 2008 Ma et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Copper and zinc are essential components of many enzymes and other proteins and, aside from iron, are the most abundant trace elements in the human body. Both metals are present in plasma at 15 µM and accumulate in the brain at concentrations on the order of 100 µM (Osterberg, 1980; Linder and Hazegh-Azam, 1996; Takeda, 2000; Mathie et al., 2006). While most of this metal is protein bound, Cu²⁺ and Zn²⁺ may function as signaling molecules that are concentrated in synaptic vesicles and coreleased with neurotransmitters (Frederickson et al., 2005). Nerve terminals in certain areas of the brain contain copper or zinc that is histochemically stainable and therefore present in a free or loosely bound form (Slomianka et al., 1990; Sato et al., 1994; Ono and Cherian, 1999; Takeda, 2000). Brain slice and synaptosome preparations release Cu²⁺ and Zn²⁺ in a Ca²⁺-dependent manner upon membrane depolarization (Howell et al., 1984; Hartter and Barnea, 1988), suggesting that total concentrations in the synaptic cleft may approach 100–250 µM Cu²⁺ (Kardos et al., 1989) or 300 µM Zn²⁺ (Assaf and Chung, 1984). The fraction of this release that can interact with synaptic proteins has not been clearly established, but indicator dye measurements suggest free synaptic concentrations on the order of 3 µM Cu²⁺ (Hopt et al., 2003) or 10 µM Zn²⁺ (Li et al., 2001; Thompson et al., 2002; Frederickson et al., 2006) are possible. Consequently there is considerable interest in understanding how low µM concentrations of Cu²⁺ or Zn²⁺ may interact with synaptic proteins such as ion channels. Such interactions could potentially play a role in normal synaptic physiology as well as disease states with neurological symptoms including inherited disorders of Cu²⁺ transport (Wilsons's and Menke's disease) and neurodegenerative diseases such as Alzheimers, Parkinsons, Creutzfeldt-Jakob disease, and amyotrophic lateral sclerosis, which are associated with altered Cu²⁺ and/or Zn²⁺ homeostasis (Bush, 2000; Frederickson et al., 2005; Mathie et al., 2006). In a broader sense, the ability of ion channels to detect trace metal ions is also relevant to understanding transition metal toxicity (Kiss and Osipenko, 1994).

Extracellular metal ions are well known to influence ion channel function by altering gating or blocking the pore (Elinder and Arhem, 2003). A variety of channels are extremely sensitive to Cu²⁺ and/or Zn²⁺ at concentrations <10 µM including subtypes of glutamate, glycine, Gaba(A), P2X, and acetylcholine receptors (Mathie et al., 2006; Huidobro-Toro et al., 2008), two-pore-domain K⁺ (K2P) channels (Gruss et al., 2004), and voltage-gated K⁺ (Kv1.3) (Teisseyre and Mozrzymas, 2006), Na⁺ (Nav1.5) (Mathie et al., 2006), and Ca²⁺ channels including a number of high voltage–activated types (Castelli et al., 2003) and Cav3.2 (Jeong et al., 2003). While the number of voltage-dependent channels identified as extremely sensitive to Cu²⁺ or Zn²⁺ is small, many voltage-gated channels exhibit responses to higher concentrations of metals (e.g., 100 µM) that have been studied in detail and provide a mechanistic basis for understanding of metal action (Elinder and Arhem, 2003).

Many transition metals at millimolar concentrations can shift the voltage dependence of channel activation in a nonspecific manner by screening surface charge on the channel and lipid bilayer. Similarly, metal occupancy of specific sites on channels outside of the voltage sensor can influence gating through electrostatic interaction with the voltage sensor (Elinder and Arhem, 2003; Yang et al., 2007). Metals can also bind directly to the voltage sensor and have been proposed to inhibit voltage sensor activation in squid axon K⁺ and Na⁺ channels by stabilizing the resting conformation (Gilly and Armstrong, 1982a,b). Acidic residues in the S2 and S3 segments of the voltage sensor domain of dEAG and hERG channels have been identified as putative coordinating sites for a variety of divalent cations (Silverman et al., 2000; Silverman et al., 2004; Fernandez et al., 2005). A key histidine in the S3–S4 loop of the voltage-sensor is also critical for the extreme metal sensitivity of Cav3.2 (Kang et al., 2006; Nelson et al., 2007). Finally, extracellular metals can enhance inactivation (Yellen et al., 1994), and the ability of Zn²⁺ to reduce the maximal conductance (G_KMAX) of Kv1.5 channels has been attributed to such a mechanism, involving putative coordinating residues in the pore domain (Kehl et al., 2002).

Although various mechanisms and sites of metal action in voltage-gated channels have been described, many fundamental questions remain unanswered. Most of the above examples involve channels with relatively low (mM) affinity and selectivity for metals. Which of these mechanisms, if any, apply to channels that are sensitive to metals at concentrations on the order of 1 µM? To what extent can channels distinguish between closely related metal ions like Cu²⁺ and Zn²⁺, and what mechanisms underlie selectivity? How are metals coordinated by voltage sensors, and how do such sites change with voltage sensor activation to interact with metals in a state-dependent manner?

Large conductance BK-type K channels (Slo1) are activated by voltage and intracellular Ca²⁺, play an important role in synaptic physiology (Faber and Sah, 2003), and are present in parts of the brain (Misonou et al., 2006) that contain synaptic Cu²⁺ and Zn²⁺ (Kozma et al., 1981; Slomianka et al., 1990; Sato et al., 1994; Ono and Cherian, 1999; Takeda, 2000). A previous study reported that BK channels from rat skeletal muscle are insensitive to 1–10 µM Cu²⁺ or 100 µM Zn²⁺, but their activity is reduced slowly and irreversibly by prolonged exposure to 20 µM Cu²⁺(Morera et al., 2003). This inhibitory effect of Cu²⁺ occurred with a substantial 20-min delay at 20 µM, was partially reversed by the reducing agent dithiothreitol (DTT), was reduced by mutation of extracellular Cys residues, and was attributed to a pro-oxidant effect of copper on amino acid sulfhydryl groups. By contrast, we demonstrate here an acute inhibitory effect of extracellular Cu²⁺ on the activation of heterologously expressed mSlo1 BK channels, characterized by an IC₅₀ of only 2 µM. The inhibition of G_K by Cu²⁺ is rapid, readily reversible, unaffected by removal of extracellular Cys residues, and therefore unlikely to involve channel oxidation. Instead, our results suggest that Cu²⁺ binds between transmembrane segments S1, S2, and S4 of the resting voltage sensor to inhibit voltage sensor activation. The binding site appears to be remarkably selective for Cu²⁺ over other transition metals (Zn²⁺, Cd²⁺, Mn²⁺, Fe³⁺, Co²⁺, and Ni²⁺). The state dependence and selectivity of Cu²⁺ action may explain why the extreme metal sensitivity of the BK channel has not been described previously. We also demonstrate a response of Shaker K⁺ channels to micromolar Cu²⁺ involving some of the same voltage sensor positions that are important in mSlo1. The ability of K⁺ channel voltage sensors to coordinate Cu²⁺ and Zn²⁺ has implications for understanding not only mechanisms of metal action in BK and related voltage-gated channels but also the conformational changes associated with voltage sensor activation.

	MATERIALS AND METHODS

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Channel Expression and Molecular Biology
BK channels experiments were performed with the mbr5 clone of the mouse homologue of the Slo1 gene (mSlo1) (Butler et al., 1993). The clone was propagated and cRNA transcribed as described previously (Cox et al., 1997). The wild-type (WT) Shaker channel was Shaker H4 with residues 6–46 deleted to remove N-type inactivation and T449V to inhibit C-type inactivation. Xenopus oocytes were injected with 0.5–50 ng cRNA, incubated at 18°C, and studied 2–7 d after injection. Site-directed mutagenesis was performed with the QuikChange XL site-directed mutagenesis kit (Stratagene) and confirmed by sequencing.

Electrophysiology and Data Analysis
Currents were recorded using the patch clamp technique in the outside-out configuration (Hamill et al., 1981), at room temperature (20–22°C). For BK channels, the internal solution contained (in mM) 110 KMeSO₃, 20 HEPES, and 40 µM (+)-18-crown-6-tetracarboxylic acid (18C6TA) to chelate contaminant Ba²⁺ (Diaz et al., 1996; Neyton, 1996). In addition, "0 Ca²⁺" solution contained 5 mM EGTA, reducing free Ca²⁺ to an estimated 0.8 nM, and Ca²⁺ solutions contained 5 mM HEDTA. [Ca²⁺] reported as 1, 3, 5, and 10 µM correspond to concentrations of 0.87, 2.4, 3.98, and 8.7 µM measured with a Ca²⁺ electrode (Orion Research Inc.). Ca²⁺ was added as CaCl₂ and [Cl^–] was adjusted to 10 mM with HCl. The standard external solution contained 110 KMeSO₃, 2 MgCl₂, 6 HCl, 10 MES, 10 MOPS. MES and MOPS buffers were used rather than HEPES, which is known to chelate Cu²⁺ (Mash et al., 2003; Sokolowska and Bal, 2005). For Shaker channels, the internal solution contained (in mM) 160 KCl, 11 EGTA, 20 HEPES, and the standard external solution contained 155 NaCl, 5 KCl, 3CaCl₂, 1 MgCl₂, 10 MES, 10 MOPS. The pH of all solutions were adjusted to 7.2, except for pH experiments where the external pH was adjusted from 5.2 to 8.14. Solutions were exchanged by washing the chamber with 20 volumes of solution (5 ml), with the exception of experiments in Fig. 1 B and Fig. 3 (C and D), which used a rapid perfusion system (SF-77B Perfusion Fast-Step, Warner).

Metal stock solutions of 0.1 M or 1 M nitrate salt were prepared in distilled water, stored at 4°C, and diluted to their final concentration in standard external solution immediately before use. Metals were not buffered and are described by their nominal concentrations. All chemicals were Ultra-grade from Sigma-Aldrich including MES and MOPS (>99.5%) and transition metal nitrate salts (>99.99%) to avoid metal contamination. Metals were washed out using a 5 mM EGTA solution prepared from the standard external solution with a total of 2.43 mM Mg²⁺ to maintain free Mg²⁺ at 2 mM. Following application of EGTA solution, the chamber was washed with standard external solution before recording currents. We did not observe any effect on mSlo1 or Shaker channel function of switching between standard extracellular solutions and EGTA solution, suggesting any metal contamination is below the channel's detection limit.

Free metal concentrations can be influenced by the formation of chloride complexes and the limited solubility of metal hydroxides at neutral pH. Chloride is primarily a concern for Cd²⁺, estimated to reduce [Cd²⁺]_free to 49% of its nominal concentration in mSlo1 solution (10 Cl^–) based on equilibrium constants reported by Pivovarov (2005). By contrast, free Cu²⁺ and Zn²⁺ should only be reduced to 99% and 99.5% of their nominal concentration in mSlo1 solution or 86% and 90% in Shaker solution (160 Cl^–) (Pivovarov, 2005). The poor solubility of copper hydroxide (Cu(OH)₂) can limit [Cu²⁺]_free to equilibrium concentrations on the order of 100 µM, but hydroxide formation is slow and can require many hours to equilibrate (Hidmi and Edwards, 1999). Consequently, metal solutions were prepared and used within 30 min to minimize hydroxide formation, and discarded if any precipitate was observed. To evaluate the free copper concentration in our experimental solutions, [Cu²⁺] measured with a Cu²⁺ electrode (Orion Research Inc.) was compared in equivalent solutions at pH 7.2 and an acidic pH 5.8 where hydroxide formation is minimal. The results were indistinguishable after correcting for a constant shift in electrode potential due to pH, indicating that the concentrations used in experiments are very close to the nominal concentrations. In both cases, electrode potentials varied linearly with log[Cu²⁺] over a range of 0.1 to 1000 µM with slopes (30 mV/10-fold change) within the range specified by the manufacturer.

Data were acquired with an Axopatch 200B amplifier (Molecular Devices Corp.) in patch mode with Axopatch's filter set at 100 kHz. Currents were filtered by an 8-pole Bessel filter (Frequency Device, Inc.) at 20 kHz and sampled at 100 kHz with an 18-bit A/D converter (Instrutech ITC-18). A P/4 protocol was used for leak subtraction (Armstrong and Bezanilla, 1974) with a holding potential of –80 or –90 mV for mSlo1 or Shaker channels, respectively. Electrodes were made from thick-walled 1010 glass (World Precision Instruments, Inc.). The electrode's resistance in the bath solution (1.0–2.5 M) was used as an estimate of series resistance (R_S) for correcting the voltage at which macroscopic I_K was recorded. Series resistance error was <15 mV for all data presented. A Macintosh-based computer system was used in combination with Pulse Control acquisition software (Herrington and Bookman, 1995) and Igor Pro for graphing and data analysis (WaveMetrics, Inc.). A Levenberg-Marquardt algorithm was used to perform nonlinear least-squared fits. Data are presented as mean ± SEM.

For mSlo1 channels, open probability (P_O) was estimated over a wide voltage range by recording macroscopic I_K when NP_O was high (>5), and single channel currents in the same patch when NP_O was low (<10). Macroscopic conductance (G_K) was determined from tail currents at –80 mV following 30-ms voltage pulses (at 5-s intervals), and was normalized by G_Kmax in the absence of metals. At more negative voltages, NP_O was determined from steady-state recordings of 1–60-s duration that were digitally filtered at 5 kHz. NP_O was determined from all-points amplitude histograms by measuring the fraction of time spent (P_K) at each open level (k) using a half-amplitude criteria and summing their contributions NP_o = kP_k. P_o was then determined by estimating N from G_Kmax (N = G_Kmax/_K, where _K is the single channel conductance at –80 mV).

G_K-V and dose–response relations were characterized by Boltzmann functions and Hill equations, respectively, as data descriptor functions. Boltzmann functions were of the form G_K(V) = G_Kmax [1 + exp(–z_APP(V – V_0.5)/kT)]^–1, where z_APP is the apparent charge movement. Hill equations were of the form Y([L]) = Y₀ + (Y_Max – Y₀)*[L]^nH/([L]^nH + IC₅₀^nH), where [L] is the ligand concentration, Y₀ and Y_Max are values of the dependent variable (Y) at zero and saturating [L], and n_H is the Hill coefficient.

	RESULTS

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Extracellular Cu²⁺ Inhibits BK Channel Activation
Macroscopic BK channel potassium current (I_K) evoked by membrane depolarization is reduced markedly and rapidly by 100 µM extracellular Cu²⁺ (Fig. 1, A and B). I_K was recorded in the absence of intracellular Ca²⁺ (0 Ca²⁺) from outside-out patches expressing mSlo1 channels (Fig. 1 A). Steady-state inhibition of I_K was achieved within a fraction of a second of applying 100 µM Cu²⁺ to a patch (Fig. 1 B) and completely reversed by washing out Cu²⁺ with 5 mM EGTA (Fig. 1 A). Thus I_K inhibition does not reflect oxidation of BK channels by Cu²⁺, which reportedly proceeds with a 1-min delay in 100 µM Cu²⁺ and is not reversed by removal of Cu²⁺ (Morera et al., 2003). We did not observe any irreversible effects even after more than 20 min in 100 µM Cu²⁺.

View larger version (26K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Extracellular Cu2+ inhibits mSlo1 activation. (A) Ik evoked in response to a pulse to +240 mV from a holding potential of –80 mV (0 Ca2+). Currents in 0 Cu2+ (control), 100 µM Cu2+, and washout with 5 mM EGTA were recorded from the same patch. Tail currents are shown on an expanded time scale, and have their own time scale bar. (B) The time course of inhibition by 100 µM Cu2+ was measured with a double pulse protocol. The ratio of steady-state IK during two 30-ms test pulses to +220 mV is plotted against the interval between pulses (t). Cu2+ was applied at –80 mV with rapid perfusion immediately following the first test pulse (t = 0). IK is inhibited rapidly ( = 0.2 s, dotted curve) following a delay of 0.1 s likely representing the time for Cu2+ to reach the patch. (C) Single channel IK-V relation is unaffected by 100 µM Cu2+ (top). Representative traces at +160 mV from a single channel patch show a marked decrease in PO (bottom). (D) Normalized GK-V relations determined in 0 Cu2+ (•), 100 µM Cu2+ (), and 1000 µM Cu2+ () for the same patch are fit by Boltzmann functions (0 Ca: GKmax = 1, V0.5 = 218 mV, Zapp = 0.82 e; 1000 Cu2+: GKmax = 0.81, V0.5 = 300 mV, Zapp = 0.57 e). (E) Normalized outward IK at +220 mV and (F) tail currents at –80 mV in 0 Cu2+ and 100 µM Cu2+ are fit by exponential functions. 100 µM Cu2+ increased the activation time constant 1.6-fold from 1.25 to 1.98 ms and decreased the deactivation time constant 1.4-fold from 153 to 108 µs.

BK Channels Are Sensitive to Low Micromolar Cu²⁺
The sensitivity of mSlo1 channels to Cu²⁺ is indicated by mean G_K-V relations measured in different [Cu²⁺], from 0 to 200 µM (Fig. 2 A). Currents were recorded in the presence of 3 µM intracellular Ca²⁺ to shift activation to more negative voltages and facilitate measurements in the presence of Cu²⁺. Similar to the results in 0 Ca²⁺, 100 µM Cu²⁺ shifted the G_K-V by +74 ± 1 mV and reduced G_Kmax by 27 ± 1% (Fig. 2 A). Normalized G_K-Vs in Fig. 2 B better illustrate the changes in V_0.5. I_K kinetics in 3 µM Ca²⁺ were altered by 100 µM Cu²⁺ more than in 0 Ca²⁺ (slowed 3.3 ± 0.1-fold at +220 mV, speeded 2.5 ± 0.2-fold at –80 mV). However, activation remained fast compared with the 30-ms voltage pulse duration (e.g., (I_K) = 3.1 ± 0.1 ms at +220 mV), indicating that G_K-V relations in Fig. 2 A should accurately represent the steady-state gating of Cu²⁺-bound channels.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 2. mSlo1 channels are extremely Cu2+ sensitive. (A) Mean GK-V relations with [Ca2+]i = 3 µM at different [Cu2+]: 0 (), 0.1 (•), 0.2 (), 0.5(), 1.0 (), 2.5 (), 5 (), 10 (), 20 (), 50 (), 100 () and 200 µM (), are normalized by GKmax in 0 Cu2+ and fit by Boltzmann functions. (B) Mean GK-V relations from panel A are normalized by GKmax at each [Cu2+] based on Boltzmann fits. (C) Cu2+ dose–response relations for V0.5 = (V0.5 [Cu2+] – V0.5[0]) (•) and GK[Cu2+] at 110 mV (), 150 mV () and 190 mV () were determined from fits in A. Solid lines are fits to a Hill equation. Thick dotted curve is a fit to Eq. 1 with most parameters set to previously determined values (zJ = 0.58 e, L0 = 10–6, zL = 0.3 e, KD(Ca2+) = 11 µM, C = 8, D = 25, E = 2.4) (Horrigan and Aldrich, 2002). VhC = 162 mV was adjusted to fit V0.5 of the 0 Cu2+ control, and Cu2+-dependent parameters (KDcu = 0.75 µM, Ecu = 0.124) were then varied to fit the V0.5-[Cu2+] relation. Thin dotted curve is a fit to Eq. 2 with KDcu = 2.1 µM, Ecu = 0.129, and all other parameters the same as for Eq. 1. (D) IC50 and (E) nH obtained by fitting GK-[Cu2+] relations in 3 µM Ca2+ (•) and 0 Ca2+ () are plotted at different voltages. Dashed lines are IC50 = 1.97 µM, nH = 1.01 from the V0.5-[Cu2+] relation fit in panel C. (F) Apparent charge (Zapp) from Boltzmann GK-V fits in A are plotted versus [Cu2+] and fit by a Hill equation (IC50 = 0.72 ± 0.12 µM, nH = 1.1 ± 0.2). Curves A and B are the predictions of Eqs. 1 and 2, respectively, using parameters from C.

The Mechanism of Cu²⁺ Action
Several different mechanisms could potentially account for a shift in the G_K-V relation by Cu²⁺, including inhibition of either the closed to open conformational change or voltage sensor activation. To help distinguish among these possibilities we examined the effects of saturating 100 µM Cu²⁺ on steady-state open probability (P_O) over a wide voltage range in 0 or 5 µM intracellular Ca²⁺ and plotted the results on a semi-log scale in Fig. 3 A. P_O was measured to values as low as 10^–7 by recording unitary current activity in macropatches (see Materials and methods). The mean log(P_o)-V relations in Fig. 3 A reveal several important features of Cu²⁺ action and suggest that Cu²⁺ acts in large part to inhibit voltage sensor activation.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 3. The mechanism and state dependence of Cu2+ action. (A) Mean log(Po)-V relations for mSlo1 for 0 and 5 µM [Ca2+]i in 0 Cu2+() and 100 µM Cu2+(), respectively. Dotted lines are exponential fits to the limiting slope of log(Po) with partial charge zL = 0.3 e. (B) Log(Po)-V relations from A are superimposed by shifting the 0 Cu2+ curves along the voltage axis by +45 mV. (C) IK evoked by 30-ms test pulses to +120 mV before (IP1) and after (IP2) application of 100 µM Cu2+ using the illustrated protocol (5 µM [Ca2+]i), without leak subtraction. IP1 was evoked from a holding potential of –80 mV in 0 Cu2+ following perfusion with standard external solution for 5 s. IP2 was recorded in 100 µM Cu2+ following perfusion with 100 µM Cu2+ for 500 ms at different prepulse voltages (VPRE). Following the second pulse the patch was washed at –80 mV for 5 s with 5 mM EGTA and then for 5 s with standard external solution before repeating the protocol. Maximal inhibition of IP2 was observed with VPRE = –80 to +40 mV (green traces) and minimal inhibition with VPRE = +180 to +220 mV (red traces). (D) The fraction of channels not inhibited fNI = (IP2[VPRE] – IP2[–80])/(IP1 – IP2[–80]) from C (, fNI[100 Cu2+]) is plotted against VPRE and compared with the mean steady-state Po-V relation () in 0 Cu2+ and 5 µM [Ca2+]i estimated as GK/GKmax. A control experiment (, fNI[0 Cu2+]) obtained from a different patch using the pulse protocol in C shows that fNI is voltage independent when the 100 µM Cu2+ solution is replaced with 0 Cu2+.

Cu²⁺ Action Is State Dependent
Cu²⁺ could potentially inhibit voltage sensor activation by binding in a state-dependent manner to the voltage sensor to stabilize the resting conformation, similar to the proposed mechanism of Zn²⁺ action on squid K⁺ channels (Gilly and Armstrong, 1982a). If so, then Cu²⁺ should interact better with the resting than the activated state. To test this hypothesis, we examined the effect on BK channel activation of applying Cu²⁺ at different membrane potentials (Fig. 3, C and D). I_K was recorded in response to brief test pulses to +120 mV before and after application of 100 µM Cu²⁺ (Fig. 3 C) with [Ca²⁺]_i = 5 µM. Cu²⁺ was applied during a 500-ms prepulse immediately preceding the second test pulse. When Cu²⁺ was applied at prepulse voltages (V_PRE) that do not activate the channel (–80 to +40 mV), the steady-state current recorded during the second test pulse (I_P2) in the presence of Cu²⁺ was reduced to 40% of the control (I_P1) (Fig. 3 C, green traces). However, at more depolarized V_PRE the inhibitory effect of 100 µM Cu²⁺ was reduced, and no difference between I_P1 and I_P2 was observed at V_PRE +180 mV (Fig. 3 C, red traces). The dependence of Cu²⁺ inhibition on prepulse voltage was analyzed by estimating the fraction of channels not inhibited as f_NI = (I_P2[V_PRE] – I_P2[–80])/(I_P1 – I_P2[–80]) and plotting this quantity versus V_PRE (f_NI[100 Cu²⁺], Fig. 3 D). These data superimpose on the mean P_o-V relation obtained in 0 Cu²⁺ (P_O, Fig. 3 D), suggesting that Cu²⁺ interacts poorly and/or is poorly accessible to its binding site when channels are activated. Indeed the failure of traces in Fig. 3 C to converge to the same current level at the end of the second test pulse suggests that Cu²⁺ binding does not equilibrate within 30 ms at the test pulse condition (+120 mV, 100 µM Cu²⁺), favoring the possibility that access of Cu²⁺ to its binding site is slow in the activated channel.

It should be noted that the results in Fig. 3 (C and D) do not distinguish whether the state dependence of Cu²⁺ action reflects a failure to interact with the activated voltage sensor or the open conformation. In BK channels the voltage dependence of voltage sensor activation (Q-V relation) and P_o-V relation differ in 0 Ca²⁺ but are superimposable in saturating 70 µM Ca²⁺ (Horrigan and Aldrich, 2002). We did not measure the Q-V relation under the experimental conditions in Fig. 3 C (5 µM Ca²⁺). However, an allosteric gating scheme that reproduces the 0 and 70 µM Ca²⁺ data (Horrigan and Aldrich, 2002) predicts the half-activation voltages of Q-V and P_O-V in 5 µM [Ca²⁺] should differ by only 3 mV. Thus the similar voltage dependence of f_NI[100 Cu²⁺] and P_O in Fig. 3 D is compatible with a dependence of Cu²⁺ inhibition on either channel opening or voltage sensor activation.

BK Channel Inhibition Is Cu²⁺ Selective
To further characterize the metal sensitivity of mSlo1, we compared the effect on V_0.5 of different transition metals (Cu²⁺, Zn²⁺, Fe³⁺, Mn²⁺, Ni²⁺, Co²⁺, and Cd²⁺) at 100 µM (Fig. 4 A). Cu²⁺ produced by far the greatest shift in V_0.5 (+74.3 ± 2.7 mV), followed by Cd²⁺ (+19.0 ± 1.9 mV) and Zn²⁺ (+8.5 ± 1.5 mV). None of the other metals tested significantly altered V_0.5. These results indicate that, among the metals tested, the BK channel is primarily sensitive to Cu²⁺ at physiologically relevant concentrations.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Cu2+ selectively inhibits mSlo1 activation. (A) GK-V shift (V0.5) produced by 100 µM of different transition metals (Cd2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+) (0 [Ca2+]i). Stars indicate a significant change in V0.5 relative to the control (P < 0.05, paired t test). (B) GK-V relations in the absence of metal ions (), 100 µM Cu2+ (•), 100 µM Cd2+ (), or 4,000 µM Cd2+ () were normalized by the control and fit with Boltzmann functions (10 µM [Ca2+]i). (C) Dose–response relations for inhibition of GK[+220 mV] by different metal ions (0 [Ca2+]i) fit with Hill equations (Cu2+ () IC50 = 2.0 µM, nH = 0.98; Cd2+ (•) IC50 = 0.70 mM, nH = 1.0; Zn2+ () IC50 = 1.2 mM, nH = 0.87; Mn2+ () IC50 = 6.7 mM, nH = 1.4; Ni 2+ () IC50 = 7.8 mM, nH = 1.5).

The dose–response curves for Mn²⁺ and Ni²⁺ in Fig. 4 C are steeper than those of Cu²⁺, Cd²⁺, or Zn²⁺, possibly reflecting a different site or mechanism of action or an additional contribution to G_K inhibition from surface charge screening at millimolar concentrations. Charge screening has little effect on BK channel activation in neutral bilayers (MacKinnon et al., 1989), and is estimated in cell membranes to shift V_0.5 by only 3 mV in response to an increase in divalent cation concentration from 1 to 10 mM, and 9 mV from 10 to 100 mM (Hu et al., 2006). Thus, surface charge effects should be small compared with the 75-mV G-V shift caused by Cu²⁺ and can only account for a fraction of the G_K decrease by Mn²⁺ and Ni²⁺.

The Cu²⁺ Binding Site Does Not Include Cys or His
To locate the Cu²⁺ binding site we mutated individual amino acid residues to identify those that influence the IC₅₀ for Cu²⁺. Any side chain containing oxygen, nitrogen, or sulfur atoms can potentially interact with metal ions. However Cys and His are by far the most common coordinating residues in transition metal binding sites (Rulisek and Vondrasek, 1998). mSlo1 contains three Cys (C14, C141, and C277) and one His (H254) that are potentially accessible to the extracellular solution. A triple mutant lacking all three Cys (C14A/C141A/C277A) exhibited a G_K-V relation and inhibition by 100 µM Cu²⁺ similar to the WT (Fig. 5 A). Likewise, H254A shifted the G_K-V by approximately +60 mV but exhibited a similar V_0.5 shift as the WT in response to 100 µM Cu²⁺ (Fig. 5 B). Dose–response relationships for H254A and C14A/C141A/C277A were indistinguishable from the WT (Fig. 5 C). Therefore, we conclude that native Cys and His residues do not participate in Cu²⁺ binding.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Cu2+ action is independent of native Cys and His residues but is pH dependent. (A) GK-V relations for WT (: 0 Cu2+; •: 100 Cu2+) and C14A/C141A/C277A ("C3A", : 0 Cu2+; : 100 Cu2+) show similar inhibition by 100 µM Cu2+. (B) GK-V relations for WT (: 0 Cu2+; •: 100 Cu2+) and H254A (<: 0 Cu2+; : 100 Cu2+) also respond similarly to 100 µM Cu2+ as indicated by the shift in V0.5. (C) Mean dose–response relations (GK-[Cu]2+) measured at voltages near V0.5 for WT (), C14A/C141A/C277A (), and H254A () are indistinguishable. The solid line is a fit by the Hill equation (IC50 = 2.0 µM, nH = 0.98) (D) Dependence of Cu2+ action on extracellular pH (pHO) is fit by Hill equation (pH0.5 = 6.0, nH = 1.15). GK-pHO relation for WT channels at +220 mV in 0 Ca2+ represents GK in 100 µM Cu2+ normalized by GK in 0 Cu2+ at each pHO.

The pH dependence of 100 µM Cu²⁺ action was fit by a Hill equation (Fig. 5 D, solid line) with half-relief of inhibition at pH 6.0 (n_H = 1.1), suggesting Cu²⁺ is coordinated by one or more protonatable side chains with a pKa near 6. Since we have already ruled out the participation of histidine, this suggests that acidic residues may be involved. Although the mean pKa values for Asp and Glu in proteins are 3.4 and 4.1, respectively, values >6 have been reported, usually in enzyme active sites and ligand-binding sites (Forsyth et al., 2002).

An Acidic Residue in S2 Contributes to Cu²⁺ Sensitivity
To identify acidic residues that coordinate Cu²⁺ we focused initially on the S2 and S3 segments of the voltage sensor domain because acidic residues in these segments of dEAG and hERG channels have been implicated in metal binding. An amino acid sequence alignment of dEAG and hERG with mSlo1 and several other voltage-dependent channels (Fig. 6 A) illustrates that dEAG and hERG contain an unusually large number of acidic residues in S2 and S3, three of which (boxed) contribute to divalent cation sensitivity (Silverman et al., 2004; Fernandez et al., 2005). Only one of these putative coordinating residues is conserved in other Kv channels, corresponding to D153 in the S2 segment of mSlo1. Mutation of D153 in mSlo1 greatly alters the dose–response curve for Cu²⁺ (Fig. 6 B). Substitution of Lys at this position (D153K) reduced Cu²⁺ sensitivity (increasing IC₅₀ eightfold to 15.9 µM), whereas a His increased Cu²⁺ sensitivity (decreasing IC₅₀ eightfold to 0.26 µM). In both cases, mutant dose–response curves were shifted relative to the WT (dotted curve) with little change in shape, consistent with a change in the affinity of a single binding site. Thus D153 in S2 appears to contribute to Cu²⁺ binding in BK channels.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Acidic residues contribute to metal sensitivity of mSlo1 and other voltage-gated channels. (A) Multiple sequence alignment of voltage sensor domain from mSlo1, Kv channels (Shaker, Kv1.2, and Kv2.1), dEAG, and hERG, and other voltage-gated channels (Nav1.4, Nav1.5, Cav1.4, Cav3.2 Domain IV) (based on Liu et al., 2003; Long et al., 2007). Putative transmembrane segments are indicated by solid bars (Long et al., 2007). Charged residues that are highly conserved in mSlo1 and other voltage-gated channels are in red (acidic) or blue (basic). Sites that were mutated in mSlo1 are highlighted yellow with positions labeled. Putative metal coordinating residues in mSlo1, dERG, and hERG are indicated by boxes. (B) Dose–response relations (GK at +260 mV) fit by Hill equations for D153K (, IC50 = 16.7 µM, nH = 1.0) and D153H (, IC50 = 0.26 µM, nH = 0.73) (5 µM [Ca2+]i). Dashed curve represents fit to WT data from Fig. 5 C. (C) The dose–response relation (GK at +160 mV) for D186A (•, IC50 = 2.53 µM, nH = 0.97), D186H (, IC50 = 2.82 µM, nH = 0.95) (1 µM [Ca2+]i). (D) The dose–response relations (GK at +240 mV) for D133A (•, IC50 = 56.5 µM, nH = 0.97), D133C (<, IC50 = 1.5 µM, nH = 0.74) and D133H (, IC50 = 0.73 µM, nH = 0.63) (0 [Ca2+]i).

The only other acidic residue in the S1–S4 segments of mSlo1 that is potentially accessible to Cu²⁺ is D133, at the extracellular end of S1. This position is highly conserved in most voltage-gated channels (Fig. 6 A), and the residues equivalent to D133 and D153 contact each other in the crystal structure of Kv1.2 (Long et al., 2005b), suggesting a likely role for D133 in Cu²⁺ binding. Consistent with this prediction, D133A increased IC₅₀ almost 30-fold relative to the WT, whereas D133C and D133H decreased IC₅₀ 1.4- and 3-fold (Fig. 6 D). Mutations at this site have similar small effects on gating (Table I) (Ma et al., 2006), supporting a direct role for D133 in Cu²⁺ binding.

Contribution of Nonacidic Residues to Cu²⁺ Sensitivity
To further characterize the Cu²⁺-binding site, we mutated 10 additional positions in S1–S4 segments plus E139 in the S1–S2 linker (Fig. 6 A). We tested all potential metal-coordinating residues in transmembrane segments that are close to D133 or D153 based on primary sequence and the structure of Kv1.2, as well as loci in S2 or S3 that interact with metals in dEAG and hERG. The effects of different mutations on the IC₅₀ for Cu²⁺ are summarized in Fig. 7 A.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Nonacidic residues also contribute to Cu2+ sensitivity. (A) IC50s of mutant channels are plotted at each position tested for Cu2+ (open symbols) or Cd2+ (filled symbols). Error bars were within the symbol size and therefore excluded. Symbols indicate different substitutions (WT: , ; Ala: , •; Lys: ; Gln: ; Asn: ; Cys: ,; His: , ). Dashed line indicates the IC50 of the WT for Cu2+. Vertical solid lines indicate the differences in IC50 between mutants at putative coordination sites. Dose–response relations (GK-[Cu2+]) for putative coordination sites 151 and 207 in 0 Ca2+ are plotted and fit by Hill equations in (B) Q151A (, IC50 = 23.8 µM, nH =1.0), Q151C (<, IC50 = 0.96 µM, nH = 0.97) at +240 mV and (C) R207Q (, IC50 = 85.8 µM, nH = 0.77) and R207C (<, IC50 = 0.52 µM, nH = 0.77) at +180 mV.

Altering the Selectivity of Metal Binding
Substitution of putative Cu²⁺-coordinating sites with Cys or His produced a substantial eightfold increase in Cu²⁺ sensitivity in the case of D153H, but a more modest 1.4–4-fold increase at positions 133, 151, and 207 (Fig. 7 A). Such a result is not unexpected because Cu²⁺ is not strongly selective for Cys or His over other side chains. However, to confirm that D133, Q151, and R207 participate directly in metal coordination we also looked at their Cd²⁺ sensitivity. Cd²⁺ interacts strongly with Cys or His, therefore Cys or His substitutions should have a much greater effect on the sensitivity of the channel for Cd²⁺ than Cu²⁺. Consistent with this prediction, D133C, Q151H, and R207C mutations produced more than 1,000-fold decreases in the IC₅₀ for Cd²⁺ relative to the WT (Fig. 7 A, filled symbols). Indeed, these mutants were all more sensitive to Cd²⁺ than Cu²⁺. The ability of Cys or His substitutions to enhance Cd²⁺ sensitivity and switch the selectivity of the channel for Cd²⁺ versus Cu²⁺ supports that D133, Q151, and R207 participate directly in metal binding.

The Role of R207 in Cu²⁺ Sensitivity
The identification of R207 in S4 as a potential Cu²⁺-coordinating residue is notable because this site plays an important role in voltage sensor activation (Ma et al., 2006) and because the presence of arginine in metal binding sites is rare. Therefore we investigated the contribution of R207 in more detail (Fig. 8).

The participation in metal binding of the Arg side chain, which is protonated and therefore positively charged at physiological pH, is unusual but not unprecedented (Bewley et al., 1999; Ferraroni et al., 2002; Berkovitch et al., 2004; Di Costanzo et al., 2006). Unlike Lys, the Arg side chain is bifurcated such that the proton is delocalized and two nitrogens are potentially available to interact with a metal ion. Nevertheless, two issues must be addressed to confirm the involvement of R207 in Cu²⁺ binding. First, the possibility that differences in IC₅₀ among mutant and WT channels reflect differences in gating rather than Cu²⁺ binding is of particular concern in the case of R207. Mutations of this site greatly facilitate voltage sensor activation (Ma et al., 2006), which could reduce Cu²⁺ sensitivity, given the state dependence of Cu²⁺ action (Fig. 3, C and D). Second, our analysis assumes that changes in IC₅₀ reflect modification of a native binding site; but the possibility must also be considered for any putative coordinating residue that high-affinity (Cys and His) substitutions modify Cu²⁺ sensitivity by introducing a new binding site.

The R207Q mutation shifts voltage sensor activation (i.e., the Q-V relation) to more negative voltages by >200 mV (Ma et al., 2006), such that the P_o-V relation for R207Q in 0 Cu²⁺ (Fig. 8 A) is left shifted and shallow compared with the WT (Table I). The possibility that this change in gating contributes to the increased IC₅₀ of R207Q relative to the WT is supported by the observation that mutations at position 210 (R210N and R210C), which also enhance voltage sensor activation (Ma et al., 2006), exhibit a P_o-V similar to R207Q (Table I) and increase IC₅₀ (Fig. 7 A). Thus the reduced Cu²⁺ sensitivity of R207Q cannot be taken as conclusive evidence that an Arg at this position normally interacts with Cu²⁺. That said, R207 is clearly in a position to interact with Cu²⁺ because the steady-state activation of R207Q and R207C are almost identical in the absence of Cu²⁺ (Fig. 8 A), implying that the 170-fold difference in IC₅₀ for these mutants (Fig. 7 A) must reflect a difference in Cu²⁺ binding rather than gating.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 8. The role of R207 in Cu2+ sensitivity. (A) GK-V relations fit by Boltzmann functions for R207C () and R207Q () in 0 Cu2+ (V0.5 = 149 mV, zAPP = 0.64 e) and in 100 µM Cu2+ (R207C, ) and 1000 µM Cu2+ (R207C: , V0.5 = 314.5 mV, zAPP = 0.64 e; R207Q: , V0.5 = 245.6 mV, zAPP = 0.46 e). (B) Log(Po)-V relation for R207C in 0 (<) and 100 µM Cu2+ (). (C) Cd2+ dose–response relations (GK-[Cd2+]) fit by Hill equations for WT (•, IC50 = 720 µM, nH = 0.98) at +220 mV and R207C (, IC50 = 0.038 µM, nH = 0.73) at +180 mV (0 [Ca2+]i). (D) GK-V relations for R207C in 0 Cd2+ (<, V0.5 = 149 mV, zAPP = 0.64 e), 100 µM Cd2+ (), and 1000 µM Cd2+ (, V0.5 = 316 mV, zAPP = 0.61 e) (0 [Ca2+]i).

Effects of Voltage Sensor Mutations on Cu²⁺ Efficacy
If Cu²⁺ inhibits BK channel activation by binding in a state-dependent manner to the voltage sensor, then mutations in the binding site may alter not only IC₅₀ but also efficacy. Efficacy can be defined as the extent to which a saturating concentration of Cu²⁺ shifts the G_K-V relation (V_0.5MAX), and is ultimately determined by the relative affinity (i.e., K_ds) of Cu²⁺ for different states (Colquhoun, 1998).

Why do we expect mutations to alter efficacy, and what do such effects reveal about the interaction of Cu²⁺ with coordinating residues? If Cu²⁺ binds in a state-dependent manner then the sum its energetic interactions with coordinating groups must be state dependent. Thus Cu²⁺ interaction with some individual coordinating groups must be state dependent while others may be state independent. Mutations of a state-dependent group that presumably abolish interaction with Cu²⁺, such as the substitution of a coordinating residue by Ala, must alter Cu²⁺ efficacy because a state-dependent component of the net binding energy will be eliminated. Mutations that enhance interaction with Cu²⁺ are also likely to alter efficacy, but are not certain to do so unless the site only interacts with Cu²⁺ in a single state such as the resting state. By contrast, Ala substitution at a state-independent site should reduce apparent affinity (increase IC₅₀) without altering efficacy, a so-called pure binding effect (Colquhoun, 1998). A high-affinity substitution at such a site is also likely but not certain to leave efficacy unchanged unless coordination geometry is identical in different states.

The effects of mutation on Cu²⁺ efficacy for mSlo1 (Fig. 9 A) suggest that some cooridinating residues interact with Cu²⁺ in a state-dependent manner while others do not. Fig. 9 A plots V_0.5MAX measured with saturating 1000 µM Cu²⁺ for various mutations of the four putative Cu²⁺-coordinating sites. Mutations in S2 (Q151, D153) that alter IC₅₀ (Fig. 7 A) have no effect on efficacy (Fig. 9 A), suggesting that S2 interacts with Cu²⁺ in a state-independent manner. By contrast, mutation of R207 (S4) or D133 (S1) had effects on efficacy that parallel their impact on IC₅₀ (Fig. 7 A). That is, mutations that increased apparent affinity (decreasing IC₅₀) also increased V_0.5MAX, suggesting that D133 and R207 interact with Cu²⁺ in a state-dependent manner. Although the magnitude of V_0.5MAX can potentially be influenced by the steepness of the G_K-V relation (Cui and Aldrich, 2000), mutants tested at each site had similar G_K-V shapes (Table I, Fig. 8 A), implying that variations in V_0.5MAX do not reflect effects of mutation on gating. In addition, R207C exhibits a much larger Cu²⁺-dependent shift in the log(P_o)-V relation (Fig. 8 B) than the WT (Fig. 3 A), providing more direct evidence for an enhanced shift in voltage sensor activation for this mutant.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 9. Effect of mutations on Cu2+-efficacy. (A) The maximal GK-V shift (V0.5MAX) in response to saturating 1000 µM Cu2+ are plotted for the WT () and mutants at the four putative Cu2+-coordinating sites (Ala: •; Lys: ; Gln: ; Cys: ; His: ). The dashed line indicates V0.5MAX for the WT. The vertical solid lines indicate the range of V0.5MAX for mutants at each position. (B) A speculative model of the Cu2+ binding site in the resting (R) and activated (A) state of the voltage sensor. In the resting state, Cu2+ is coordinated by D133(S1), Q151(S2), D153(S2), and R207(S4). The relative size and position of transmembrane segments correspond to those in the structure of a Kv1.2/Kv2.1 chimera (Long et al., 2007). During activation, the S2 residues interact with Cu2+ in a state-independent manner while interactions with D133 and R207 are weakened or disrupted, presumably by changes in the position or orientation of S1 and S4 relative to S2. In this way, mutation of any of these residues alter IC50, but only mutation of D133 or R207 alter efficacy.

The Cu²⁺ Binding Site Is Conserved in Shaker
Three of the four putative Cu²⁺-coordinating residues in mSlo1 are charged (D133, D153, and R207) and highly conserved among voltage-gated K⁺, Na⁺, and Ca²⁺ channels (Fig. 6 A). This raises the possibility that many voltage-gated channels contain a metal binding site homologous to that in mSlo1. To test this possibility we examined the Cu²⁺ and Zn²⁺ sensitivity of Shaker K⁺ channels (Fig. 10).

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 10. The Cu2+ coordination sites are conserved in Shaker channels. (A) IK evoked from WT Shaker channels in response to pulses to –10 mV from a holding potential of –90 mV at the indicated [Cu2+] (0–100 µM). (B) GK-V relations from the same patch in different [Cu2+] are normalized by GKmax in 0 Cu2+. (0 (), 1 (•), 5 (), 20 (), and 100 µM Cu2+ ()). (C) IK at 0 mV from WT channels at the indicated [Zn2+] (0–10,000 µM). (D) GK-V relations in different [Zn2+], normalized by GKmax in 0 Zn2+ (0 (), 100 (•), 1,000 (), 4,000 (), 10,000 µM Zn2+ ()). (E) IK at 0 mV from E247A channels in 0, 1, and 5 µM Cu2+. (F) IK at +30 mV from E247C in 0, 0.1, 1, and 5 µM Cu2+. (G and H) IK at +10 mV from E247A (G) or E247C (H) in 0, 100, and 1,000 µM Zn2+. (I and J) IK from R365A at 0 mV (I) or R365C at –40 mV(J) in 0, 1, and 5 µM Cu2+. All Shaker experiments were performed from a holding potential of –90 mV. Most data were obtained in standard Shaker internal and external solutions (see Materials and methods) with the exception of R365 mutant data in I and J, which were obtained in a low chloride external solution with all but 10 mM Cl– replaced by MeO3–, as in standard mSlo1 external solution.

Although Shaker can respond to low µM Cu²⁺ (e.g., 5 µM) it is evident from the small response to 1 µM Cu²⁺ (Fig. 10, A and B) that Shaker is less sensitive to Cu²⁺ than mSlo1. The G_K-V shift of Shaker by 100 µM Cu²⁺ (64 mV, Fig. 10 B) may be overestimated because the effect of Cu²⁺ on activation kinetics prevented achievement of steady-state activation during a 50-ms pulse at all but the most positive voltages tested. Given this caveat, the IC₅₀ of Shaker approximated by fitting V_0.5-[Cu²⁺] from Fig. 10 B with n_H = 1 was 18 µM. This difference compared with the BK channel is consistent with the fact that mSlo1 contains a putative coordinating residue Q151 that is not conserved in Shaker, and that the Q151A mutation increases mSlo1's IC₅₀ to 19.7 µM (Fig. 7 A).

To demonstrate that the metal binding site in Shaker is homologous to that in BK channels we examined the effects on Cu²⁺ and Zn²⁺ sensitivity of mutating S1 (E247) and S4 (R365) residues, corresponding to two of the three conserved coordinating sites in mSlo1 (Fig. 10, E–J). We tested for effects on activation kinetics since the kinetic changes complicated determination of steady-state activation. Mutation of E247 (D133 in mSlo1) to Ala (E247A) almost abolished the response to 5 µM Cu²⁺ and 1000 µM Zn²⁺ (Fig. 10, E and G), whereas E247C enhanced sensitivity to both Cu²⁺ (Fig. 10 F) and Zn²⁺ (Fig. 10 H). A similar result for Cu²⁺ was observed when R365 in S4 (R207 in mSlo1) was mutated to Ala (Fig. 10 I) or Cys (Fig. 10 J). In addition, similar to mSlo1, we observed that the response of WT Shaker to 5 µM Cu²⁺ was abolished at extracellular pH 5.2 (unpublished data). We did not investigate E283 in S2 (equivalent to D153 in mSlo1), because mutations at this position in Shaker expressed poorly in outside-out patches. Thus, we confirmed that two of the conserved Cu²⁺-coordinating residues in mSlo1 also contribute to the Cu²⁺/Zn²⁺ sensitivity of Shaker.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study describes a previously unreported, rapid, and reversible inhibitory effect of extracellular Cu²⁺ on BK channel (mSlo1) activation at low micromolar and submicromolar concentrations. Cu²⁺ acts primarily to shift the voltage dependence of steady-state activation, together with lesser effects on G_Kmax and I_K kinetics. The G_K-V relation is shifted by up to 75 mV (V_0.5) with an IC₅₀ of 2.0 µM Cu²⁺ and a Hill coefficient of 1.0 under our experimental conditions, and G_K can be reduced by >90% at voltages less than V_0.5. Thus BK channels are members of a small group of voltage-dependent channels that are extremely sensitive to physiologically relevant concentrations of Cu²⁺; and mSlo1 may be the most Cu²⁺-sensitive K⁺ channel identified so far.

Biophysical and mutagenesis results indicate that Cu²⁺ binds in a state-dependent manner to the voltage sensor domain where it interacts with transmembrane segments S1, S2, and S4, including acidic and basic residues that are highly conserved among most voltage-gated channels including Shaker. Native Cys and His residues do not contribute to the Cu²⁺ sensitivity of mSlo1. Likewise, native cysteines are not required for the Zn²⁺ sensitivity of Shaker (Boland et al., 1994). The binding site appears very selective for Cu²⁺ over closely related transition metals (Zn²⁺, Cd²⁺, Mn²⁺, Fe³⁺, Co²⁺, and Ni²⁺). Thus the voltage sensor can form a selective metal binding site with micromolar apparent affinity without requiring Cys or His residues that are often critical for the extreme metal sensitivity of other channels. We demonstrate here that Shaker K⁺ channels can also respond to low micromolar Cu²⁺ and that some of the putative coordinating sites in mSlo1 are important for the Cu²⁺ and Zn²⁺ sensitivity of Shaker. Thus it is possible that our results define a metal binding site of general importance in voltage-gated channels.

Despite extensive investigation of metal coordination in enzymes, relatively little is known about metal binding sites in ion channels outside of the permeation pathway. In many cases, individual residues critical for the extreme sensitivity of particular channel subtypes have been identified, such as Cav3.2 and Nav1.5 (Satin et al., 1992; Jeong et al., 2003). In some cases, metal bridges that coordinate Cd²⁺ or Ni²⁺ between a pair of Cys and/or His residues have been defined in native channels (Gordon and Zagotta, 1995) or introduced by mutation (Holmgren et al., 1998; Webster et al., 2004). However, in few cases have potential coordinating residues been screened in sufficient detail to characterize a multivalent binding site. Importantly, we screened many residues in mSlo1, making multiple substitutions at most sites to inhibit and enhance Cu²⁺ sensitivity and to control for effects of mutations on gating. To confirm the direct participation of some positions in metal binding we also demonstrated effects of Cys or His substitution on Cd²⁺/Cu²⁺ selectivity. In all, we identified four putative coordinating residues and 14 residues that do not contribute to Cu²⁺ sensitivity; both findings place constraints on the possible structure of the binding site. This detailed characterization together with the fact that putative coordinating sites are in transmembrane segments of the voltage sensor and in most cases highly conserved among Kv channels allows us to use Kv channel structures as a framework for understanding what the binding site may look like and how it may change with gating. These and other implications of our results are discussed below.

Relation to Previous Studies
One reason that BK channels have not previously been recognized as extremely metal sensitive probably reflects their Cu²⁺ selectivity. Unlike Cav3.2, which is similarly sensitive to a number of transition metals, with IC₅₀s for Cu²⁺, Zn²⁺, and Ni²⁺ of 0.9, 2.1, and 4.9 µM (Kang et al., 2006), the BK channel responds poorly to metals other than Cu²⁺. Another factor that may have masked the effect of Cu²⁺ is the state dependence of its action. Contrary to our results, a previous study failed to observe an acute response of single BK channels from rat skeletal muscle to 1–100 µM Cu²⁺ (Morera et al., 2003). This difference is unlikely to reflect that Morera et al. used a native channel reconstituted in lipid bilayers because they reported observing similar preliminary results with cloned hSlo1 channels expressed in Xenopus oocytes; the same preparation used here with a Slo1 homologue that is virtually identical to mSlo1. However, Morera et al. tested for the effect of Cu²⁺ under steady-state recording conditions where channels were maximally activated with P_o 0.9 before Cu²⁺ was applied (V = +40 mV, [Ca]_i = 60 µM). It is possible that these recording conditions prevented the acute effect of Cu²⁺ since mSlo1 channels that are maximally activated for 0.5 s are also insensitive to 100 µM Cu²⁺ (Fig. 3, C and D).

The IC₅₀ for Cu²⁺ inhibition of mSlo1 G_K varied with voltage (0.53–2.24 µM, Fig. 2 D) but was in any case comparable to that reported previously for the most Cu²⁺-sensitive Kv channel Kv1.3 (IC₅₀ = 5.3 ± 0.5 µM) (Teisseyre and Mozrzymas, 2006) or the K2P "leak" channels Task-3 (IC₅₀ = 2.7 ± 0.4 µM) and Trek-1 (EC₅₀ = 3.0 ± 1.0 µM) (Gruss et al., 2004). However, the Cu²⁺ dose–response relation for mSlo1, characterized by a Hill coefficient 1.0, is much shallower than that of Kv1.3 (n_H = 3.8), Trek-1 (n_H = 1.7 ± 0.8), or Task-3 (n_H = 1.8 ± 0.4). Therefore, with the caveat that these channels have not been studied under identical conditions, the BK channel is potentially the most Cu²⁺-sensitive K⁺ channel in that it has a lower threshold for response to Cu²⁺ (submicromolar), owing to its shallow dose–response relation.

We identified D153 as a coordinating site in mSlo1 because the corresponding residue in hERG is important for Cd²⁺ sensitivity (Fernandez et al., 2005). However, additional acidic coordinating sites in the S2 and S3 segments of hERG and dEAG are not conserved in mSlo1 (Fig. 6 A), suggesting the binding site in hERG and dEAG may be distinct from that in Slo1. Our results provide further support for this conclusion. First, we tested sites in S2 and S3 close to the positions that are important in hERG/dEAG and none were important for Cu²⁺ sensitivity in mSlo1. This included substituting Cys and Ala residues at a position in S2 (N158) that corresponds to a coordinating residue in hERG and dEAG. Conversely, the coordinating sites we identified in mSlo1 are largely absent from hERG or dEAG. The equivalent of D133 and Q151 in mSlo1 are aliphatic (I, V) in dEAG and hERG, and R207 is replaced by a lysine. Thus the binding sites of mSlo1 and dEAG may be distinct while mSlo1 and hERG share only a single acidic residue in S2.

Physiological Implications
The physiological importance of BK channel inhibition by Cu²⁺ is unknown. However, it is certainly intriguing that Slo1 appears poised to respond specifically to Cu²⁺ at concentrations estimated to be achieved in the synaptic cleft (Hopt et al., 2003). BK channel activation in nerve terminals is important for limiting action potential duration, Ca²⁺ entry, and neurotransmitter release (Faber and Sah, 2003). BK channels and Cu²⁺ are both present in glutamatergic synapses in the hippocampus (Kozma et al., 1981; Mathie et al., 2006; Misonou et al., 2006; Schlief and Gitlin, 2006). Thus the possibility exists that Cu²⁺ release could exert a positive feedback effect to enhance neurotransmitter and Cu²⁺ release by inhibiting BK channels in the same nerve terminal. Whether or not this could occur may depend on several factors including the metal sensitivity of Ca²⁺ channels in the nerve terminal as well as the time course of Cu²⁺ elevation. Because BK channels are less sensitive to Cu²⁺ when fully activated than at rest, it is possible they would respond less to release during an action potential than residual Cu²⁺ from preceding synaptic events. Another factor that could conceivably be important to the physiological response of BK channels is the effect of accessory β subunits. However, preliminary experiments indicated no appreciable effects on Cu²⁺ sensitivity of coexpressing mSlo1 with an excess of bovine β₁ (IC₅₀ = 2.8 µM, n_H = 0.84, +220 mV, 0 Ca²⁺) or human β₄ (IC₅₀ = 2.7 µM, n_H = 0.95, +240 mV, 0 Ca²⁺).

Shaker K⁺ channels were found to be roughly 10-fold less sensitive to Cu²⁺ than mSlo1 in terms of IC₅₀. However the response of Shaker could also be physiologically relevant because, unlike mSlo1, Shaker activation kinetics were greatly slowed by Cu²⁺. For example, 5 µM Cu²⁺ slowed activation kinetics threefold at –10 mV (Fig. 10 A) and might therefore greatly reduce the response to a brief action potential even though the effect of this concentration on steady-state activation was modest.

Mechanisms of Cu²⁺ Action
Several lines of evidence suggest that Cu²⁺ binds to the voltage sensor in a state-dependent manner to inhibit voltage sensor activation. First, Cu²⁺ shifts steady-state activation (e.g., V_0.5) to more positive voltages, consistent with a shift in voltage sensor activation. Conversely, Cu²⁺ does not affect P_o at extreme negative voltages in the presence or absence of intracellular Ca²⁺ (Fig. 3 A), indicating that Cu²⁺ has little or no direct effect on channel opening or Ca²⁺-dependent activation. Second, mutagenesis results define a site of action in the voltage sensor domain (Fig. 7). Third, mutation of some putative coordinating sites (D133 and R207) alter Cu²⁺ efficacy (V_0.5MAX), indicating that Cu²⁺ action does not depend merely on occupancy of its binding site, and consistent with a state-dependent interaction that favors Cu²⁺ binding to the resting state (Fig. 9). Fourth, the insensitivity of fully activated channels to Cu²⁺ (Fig. 3 B) also implies a state-dependent change in the affinity and/or access of the binding site. Finally, the dose–response relation for Cu²⁺ can be reproduced by a model that assumes Cu²⁺ binds in a state-dependent manner to the voltage sensor, as discussed below.

To a first approximation, the effect of Cu²⁺ on steady-state activation can be reproduced by a gating scheme illustrated in Fig. 11 A (Scheme 1), which assumes that Cu²⁺ binds with higher affinity to the resting than activated state of the voltage sensor. The best fit of this model to the V_0.5-[Cu²⁺] relation (Fig. 2 C, thick dotted curve) is almost identical to the Hill equation fit (solid curve) and yields Cu²⁺ dissociation constants for resting and activated states of 0.75 µM and 6.03 µM, respectively (see Fig. 2 legend for parameters).

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 11. Mechanism and site of Cu2+ action. (A) Scheme 1 (see text), green indicates Cu2+-dependent mechanisms, black represents HA model (Horrigan and Aldrich, 2002). (B and C) Residues mutated in mSlo1 are mapped onto the structure of a Kv1.2/Kv2.1 chimera (Long et al., 2007). B and C show side and top views, respectively. Only the S1–S4 segments from one subunit are shown with S1–S2 linker removed for clarity. Putative Cu2+-coordinating residues were changed to the corresponding residue from mSlo1: D133(red), Q151(yellow), D153(red), R207(cyan). The backbone was colored dark blue at other positions that were tested. Dotted spheres indicate the position of water molecules in the structure. The green sphere illustrates a possible position for Cu2+.

(1)

Scheme 1 requires only an 8.0-fold difference in affinity between resting and activated states (E_cu = 0.124) to account for the G_K-V shift by Cu²⁺. Thus, the estimated K_ds of both the resting and activated states are much less than 100 µM, suggesting that the inability of 100 µM Cu²⁺ to inhibit fully activated channels in Fig. 3 C may reflect poor access to the binding site rather than a weak interaction with the activated state. This conclusion is supported by the observation that mutation of sites in S2 (Q151 and D153) altered IC₅₀ but not efficacy (V_0.5MAX, Fig. 9 A), suggesting these residues can interact with Cu²⁺ in the activated state.

Although Scheme 1 reproduces the V_0.5-[Cu²⁺] relation it does not account for all effects of Cu²⁺. Scheme 1 predicts that the G_K-V in 3 µM Ca²⁺ should shift without decrease in slope (Fig. 2 F, curve A) or G_Kmax. However we observed that 100 µM Cu²⁺ reduced G_Kmax by 27% and reduced the slopes of the G_K-V and log(P_o)-V relations by 39% and 12%, respectively. Several mechanisms that could account for these effects are discussed below, but cannot currently be distinguished based on our data.

One mechanism that cannot account for effects of 100 µM Cu²⁺ on G_Kmax and G_K-V shape is the possibility raised by Fig. 3 C that Cu²⁺ cannot equilibrate with its binding site during a 30-ms depolarization. First, it is important to note that Cu²⁺ is equilibrated with the resting channel by the start of each voltage pulse, since the effect of 100 µM Cu²⁺ reaches a steady state within 1 s of application at –80 mV (Fig. 1 B) while the interval between pulses is 5 s. Second, 100 µM Cu²⁺ has a maximal effect on V_0.5 (Fig. 1 D and Fig. 2 C), implying that channels have a maximal number of Cu²⁺ bound when the G_K-V is measured, regardless of when or how fast Cu²⁺ reaches its binding site. Thus we conclude that decreases in G_Kmax and G-V slope in 100 µM Cu²⁺ reflect an equilibrium gating property of Cu²⁺-bound channels.

Although Cu²⁺ equilibration kinetics cannot account for changes in G_K-V shape observed in saturating Cu²⁺, they can affect the predictions of Scheme 1 and our estimate of K_dCu. To show this, we considered the extreme case that Cu²⁺ equilibrates at the holding potential but cannot bind or dissociate during a voltage pulse. Thus, the probability that a voltage sensor is Cu²⁺ bound (P_Cu[Cu²⁺]) is determined only by the K_d of the resting state (K_dCu), and the distribution of channel species with different numbers of Cu²⁺ bound (i = 0–4) is determined by a binomial distribution where each species has a different open probability (P_O[i]) specified by Scheme 1. Mean open probability in this case is given by Eq. 2 (see below).

(2)

Eq. 2 can fit the V_0.5-[Cu²⁺] relation (Fig. 2 C, thin dotted curve) with a value of E_Cu = 0.129 similar to that determined from Eq. 1 (0.124). However, the value of K_dCu from Eq. 2 (2.1 µM) is comparable to the IC₅₀ and therefore greater than predicted by Eq. 1 (0.75 µM). An interesting property of Eq. 2 is it predicts a decrease in G_K-V slope (z_APP) at intermediate [Cu²⁺] (Fig. 2 F, curve B). However, this slope decrease is a consequence of heterogeneity in the number of Cu²⁺ bound to each channel rather than a property of any one species (P_o[i]), and therefore does not persist in saturating Cu²⁺.

The decreased slope of the log(P_o)-V relation observed in saturating Cu²⁺, according to the HA model, can only be caused by a decrease in gating charge or a decrease in voltage sensor/gate coupling (D-factor) (Ma et al., 2006). Either of these mechanisms is possible. One of the putative coordinating sites for Cu²⁺, D153, is thought to be a voltage-sensing residue in Slo1 in part because a charge reversal at this position (D153K) reduces the slope of log(P_o)-V by almost 50% (Ma et al., 2006). If Cu²⁺ remains associated with D153 during voltage sensor activation it might in effect reverse the net charge of this residue and reduce gating charge. It would also be possible for Cu²⁺ to reduce voltage sensor/gate coupling if its binding depends on the open state of the channel as well as the activation state of the voltage sensor. Intracellular ligands in Slo1 are known to have such effects on voltage sensor/gate coupling (Horrigan et al., 2005; Horrigan and Ma, 2008).

The decrease in G_Kmax by Cu²⁺ was observed at all [Ca²⁺] tested (0–10 µM) and is not caused by a decrease in single channel conductance (Fig. 1 C) or a failure to measure steady-state activation since Cu²⁺ had a relatively minor effect on I_K kinetics in mSlo1 (Fig. 1 F). These results cannot be accounted for by Scheme 1 or the HA model because any mechanism that decreases the C-O equilibrium constant (L) to reduce G_Kmax in 0 Ca²⁺ should be overcome by the ability of Ca²⁺ to increase L more than 100-fold at 5–10 µM concentrations (Horrigan and Aldrich, 2002). That Cu²⁺ reduced G_Kmax by a similar amount in different [Ca²⁺]_i suggests that a fraction of channels enter a nonconducting state that is not included in our model. Metal binding to the pore domain of Kv1.5 has been proposed to reduce G_Kmax by stabilizing a slow inactivated state (Kehl et al., 2002). Such a mechanism seems unlikely since Slo1 is not known to inactivate in the absence of β subunits. However, BK channels have been reported to enter brief "flicker" closed states that are not included in the HA model and limit the maximum open probability of BK channels to 0.9 in all [Ca²⁺]_i(Rothberg and Magleby, 1998). Therefore it is conceivable such states could be stabilized by Cu²⁺ to reduce G_Kmax in a Ca²⁺-independent manner.

Structure and Function of the Cu²⁺ Binding Site
The detailed structure of the BK channel is unknown. However, the putative Cu²⁺-coordinating residues in mSlo1 are for the most part conserved in other voltage-gated channels, including Kv channels of known structure (Fig. 6 A). We have also confirmed that S1 and S4 residues, corresponding to D133 and R207 in mSlo1, are important for the Cu²⁺ and Zn²⁺ sensitivity of Shaker (Fig. 10). Thus it is likely we can gain insight into the properties of this site by examining the structure of Kv channels.

The crystal structures of several Kv channels have been solved in the activated conformation (Jiang et al., 2003; Long et al., 2005a; Long et al., 2007). In Fig. 11 (B and C) we mapped the putative Cu²⁺-coordinating residues from mSlo1 onto the highest resolution example, a Kv1.2/Kv2.2 chimera (Long et al., 2007), based on the sequence alignment in Fig. 6 A. Although our results suggest this structure in the activated conformation should not be ideal for Cu²⁺ binding, it does provide some important insight. First, a cavity is present between the S1–S4 transmembrane segments that contains water molecules (dotted spheres) and therefore can presumably accommodate metal ions. Second, the three most highly conserved putative Cu²⁺-coordinating residues in S1(D133), S2(D153), and S4(R207) face each other in the crystal structure to form what looks like a reasonable binding pocket (Fig. 11 C). Although the fourth putative Cu²⁺-coordinating residue (Q151) faces away from the others in the crystal structure (toward lipid), it is conceivable that S2 in mSlo1 might adopt a different orientation to allow both Q151 and D153 to participate in Cu²⁺ binding (as illustrated in Fig. 9 B). Third, all four putative coordinating residues are distributed around the cavity, near the top of the voltage sensor, and roughly define a plane when viewed from the side (Fig. 11 B). This distribution may help explain the Cu²⁺ selectivity of the BK channel since the most favored coordination geometry for Cu²⁺ is square planar (Rulisek and Vondrasek, 1998). That many sites above and below the putative coordinating residues did not contribute to Cu²⁺ sensitivity in mSlo1 (backbone colored dark blue) is also consistent with a planar coordination geometry. However it should be noted that other than Cys, His, and acidic residues, extracellular loops were not tested in detail and could potentially participate in metal binding through backbone interactions. Finally, the acidic coordinating residues D133 and D153 are present on antiparallel helices (S1 and S2) and contact each other (in Kv1.2), consistent with the possibility that their pKa's are perturbed to account for the pH sensitivity of Cu²⁺ action (Fig. 5 D). Such an interaction would tend to increase the pKa of one partner and decrease the other. Additional factors that could increase pKa include the presence of D133 at the C terminus of a helix and the location of these residues near an aqueous crevice (Forsyth et al., 2002).

Our results suggest that Cu²⁺ should bind better in the resting than the activated conformation. Therefore it is reasonable to expect that the relative position of the four putative coordinating residues may change during activation. Recent studies based on a variety of approaches suggest that S4 may undergo a large motion relative to S1 and S2 during activation in Kv channels (Campos et al., 2007; Long et al., 2007; Pathak et al., 2007). Such a motion is qualitatively consistent with the formation of a state-dependent binding site between S1, S2, and S4. However, it should be noted that this gating motion is likely larger in Kv channels than in BK channels, which are weakly voltage dependent. Indeed we have suggested previously that the resting conformation of mSlo1 may be similar to an intermediate activated state of Shaker (Ma et al., 2006). It is possible that Cu²⁺ slowed the activation of Shaker (Fig. 10 A) more than mSlo1 (Fig. 1 E) by stabilizing an intermediate state in Shaker. However another reason for this difference is that voltage sensor activation is rate limiting for I_K activation in Shaker, whereas the closed–open transition is rate limiting in Slo1 (Horrigan and Aldrich, 2002). Thus a slowing of voltage sensor activation by Cu²⁺ could increase the time constant of I_K activation in Shaker while altering primarily the delay in activation of Slo1 (Horrigan and Aldrich, 1999).

	ACKNOWLEDGMENTS

 
This work was supported by a grant from the National Institutes of Health (NS42901) to F.T. Horrigan. An abstract of this work was presented at the 50th Annual Meeting of the Biophysical society (2006).

Olaf S. Andersen served as editor.

Submitted: 4 February 2008
Accepted: 1 April 2008

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