An extracellular Cu2+ binding site in the voltage sensor of BK and Shaker potassium channels

Abstract

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. GK is reduced in a dose-dependent manner with an IC50 and Hill coefficient of 2 microM and 1.0, respectively. Saturating 100 microM Cu2+ shifts the GK-V relation by +74 mV and reduces G(Kmax) by 27% without affecting single channel conductance. However, 100 microM Cu2+ fails to inhibit GK 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 microM with IC(50)s > 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.

Figure 1.

Extracellular Cu²⁺ inhibits mSlo1 activation. (A) I_k 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 I_K 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). I_K is inhibited rapidly (τ = 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 I_K-V relation is unaffected by 100 μM Cu²⁺ (top). Representative traces at +160 mV from a single channel patch show a marked decrease in P_O (bottom). (D) Normalized G_K-V relations determined in 0 Cu²⁺ (•), 100 μM Cu²⁺ (▵), and 1000 μM Cu²⁺ (▪) for the same patch are fit by Boltzmann functions (0 Ca: G_Kmax = 1, V_0.5 = 218 mV, Z_app = 0.82 e; 1000 Cu²⁺: G_Kmax = 0.81, V_0.5 = 300 mV, Z_app = 0.57 e). (E) Normalized outward I_K 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.

Figure 2.

mSlo1 channels are extremely Cu²⁺ sensitive. (A) Mean G_K-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 G_Kmax in 0 Cu²⁺ and fit by Boltzmann functions. (B) Mean G_K-V relations from panel A are normalized by G_Kmax at each [Cu²⁺] based on Boltzmann fits. (C) Cu²⁺ dose–response relations for ΔV_0.5 = (V_0.5 [Cu²⁺] − V_0.5[0]) (•) and G_K[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 (z_J = 0.58 e, L₀ = 10⁻⁶, z_L = 0.3 e, K_D(Ca²⁺) = 11 μM, C = 8, D = 25, E = 2.4) (Horrigan and Aldrich, 2002). V_hC = 162 mV was adjusted to fit V_0.5 of the 0 Cu²⁺ control, and Cu²⁺-dependent parameters (K_Dcu = 0.75 μM, E_cu = 0.124) were then varied to fit the V_0.5-[Cu²⁺] relation. Thin dotted curve is a fit to Eq. 2 with K_Dcu = 2.1 μM, E_cu = 0.129, and all other parameters the same as for Eq. 1. (D) IC₅₀ and (E) n_H obtained by fitting G_K-[Cu²⁺] relations in 3 μM Ca²⁺ (•) and 0 Ca²⁺ (□) are plotted at different voltages. Dashed lines are IC₅₀ = 1.97 μM, n_H = 1.01 from the ΔV_0.5-[Cu²⁺] relation fit in panel C. (F) Apparent charge (Z_app) from Boltzmann G_K-V fits in A are plotted versus [Cu²⁺] and fit by a Hill equation (IC₅₀ = 0.72 ± 0.12 μM, n_H = 1.1 ± 0.2). Curves A and B are the predictions of Eqs. 1 and 2, respectively, using parameters from C.

Figure 3.

The mechanism and state dependence of Cu²⁺ action. (A) Mean log(P_o)-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(P_o) with partial charge z_L = 0.3 e. (B) Log(P_o)-V relations from A are superimposed by shifting the 0 Cu²⁺ curves along the voltage axis by +45 mV. (C) I_K evoked by 30-ms test pulses to +120 mV before (I_P1) and after (I_P2) application of 100 μM Cu²⁺ using the illustrated protocol (5 μM [Ca²⁺]_i), without leak subtraction. I_P1 was evoked from a holding potential of −80 mV in 0 Cu²⁺ following perfusion with standard external solution for 5 s. I_P2 was recorded in 100 μM Cu²⁺ following perfusion with 100 μM Cu²⁺ for 500 ms at different prepulse voltages (V_PRE). 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 I_P2 was observed with V_PRE = −80 to +40 mV (green traces) and minimal inhibition with V_PRE = +180 to +220 mV (red traces). (D) The fraction of channels not inhibited f_NI = (I_P2[V_PRE] − I_P2[−80])/(I_P1 − I_P2[−80]) from C (▪, f_NI[100 Cu²⁺]) is plotted against V_PRE and compared with the mean steady-state P_o-V relation (○) in 0 Cu²⁺ and 5 μM [Ca²⁺]_i estimated as G_K/G_Kmax. A control experiment (□, f_NI[0 Cu²⁺]) obtained from a different patch using the pulse protocol in C shows that f_NI is voltage independent when the 100 μM Cu²⁺ solution is replaced with 0 Cu²⁺.

Figure 4.

Cu²⁺ selectively inhibits mSlo1 activation. (A) G_K-V shift (ΔV_0.5) produced by 100 μM of different transition metals (Cd²⁺, Mn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺) (0 [Ca²⁺]_i). Stars indicate a significant change in V_0.5 relative to the control (P < 0.05, paired t test). (B) G_K-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 G_K[+220 mV] by different metal ions (0 [Ca²⁺]_i) fit with Hill equations (Cu²⁺ (○) IC₅₀ = 2.0 μM, n_H = 0.98; Cd²⁺ (•) IC₅₀ = 0.70 mM, n_H = 1.0; Zn²⁺ (□) IC₅₀ = 1.2 mM, n_H = 0.87; Mn²⁺ (▪) IC₅₀ = 6.7 mM, n_H = 1.4; Ni ²⁺ (▵) IC₅₀ = 7.8 mM, n_H = 1.5).

Figure 5.

Cu²⁺ action is independent of native Cys and His residues but is pH dependent. (A) G_K-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) G_K-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 V_0.5. (C) Mean dose–response relations (G_K-[Cu]²⁺) measured at voltages near V_0.5 for WT (○), C14A/C141A/C277A (▵), and H254A (▾) are indistinguishable. The solid line is a fit by the Hill equation (IC₅₀ = 2.0 μM, n_H = 0.98) (D) Dependence of Cu²⁺ action on extracellular pH (pH_O) is fit by Hill equation (pH_0.5 = 6.0, n_H = 1.15). G_K-pH_O relation for WT channels at +220 mV in 0 Ca²⁺ represents G_K in 100 μM Cu²⁺ normalized by G_K in 0 Cu²⁺ at each pH_O.

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 (G_K at +260 mV) fit by Hill equations for D153K (▪, IC₅₀ = 16.7 μM, n_H = 1.0) and D153H (▴, IC₅₀ = 0.26 μM, n_H = 0.73) (5 μM [Ca²⁺]_i). Dashed curve represents fit to WT data from Fig. 5 C. (C) The dose–response relation (G_K at +160 mV) for D186A (•, IC₅₀ = 2.53 μM, n_H = 0.97), D186H (▴, IC₅₀ = 2.82 μM, n_H = 0.95) (1 μM [Ca²⁺]_i). (D) The dose–response relations (G_K at +240 mV) for D133A (•, IC₅₀ = 56.5 μM, n_H = 0.97), D133C (<, IC₅₀ = 1.5 μM, n_H = 0.74) and D133H (▴, IC₅₀ = 0.73 μM, n_H = 0.63) (0 [Ca²⁺]_i).

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 (G_K-[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, n_H =1.0), Q151C (<, IC₅₀ = 0.96 μM, n_H = 0.97) at +240 mV and (C) R207Q (⋄, IC₅₀ = 85.8 μM, n_H = 0.77) and R207C (<, IC₅₀ = 0.52 μM, n_H = 0.77) at +180 mV.

Figure 8.

The role of R207 in Cu²⁺ sensitivity. (A) G_K-V relations fit by Boltzmann functions for R207C (⋄) and R207Q (▿) in 0 Cu²⁺ (V_0.5 = 149 mV, z_APP = 0.64 e) and in 100 μM Cu²⁺ (R207C, ▾) and 1000 μM Cu²⁺ (R207C: ○, V_0.5 = 314.5 mV, z_APP = 0.64 e; R207Q: ♦, V_0.5 = 245.6 mV, z_APP = 0.46 e). (B) Log(P_o)-V relation for R207C in 0 (<) and 100 μM Cu²⁺ (▾). (C) Cd²⁺ dose–response relations (G_K-[Cd²⁺]) fit by Hill equations for WT (•, IC₅₀ = 720 μM, n_H = 0.98) at +220 mV and R207C (▴, IC₅₀ = 0.038 μM, n_H = 0.73) at +180 mV (0 [Ca²⁺]_i). (D) G_K-V relations for R207C in 0 Cd²⁺ (<, V_0.5 = 149 mV, z_APP = 0.64 e), 100 μM Cd²⁺ (▾), and 1000 μM Cd²⁺ (○, V_0.5 = 316 mV, z_APP = 0.61 e) (0 [Ca²⁺]_i).

Figure 9.

Effect of mutations on Cu²⁺-efficacy. (A) The maximal G_K-V shift (ΔV_0.5MAX) 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 ΔV_0.5MAX for the WT. The vertical solid lines indicate the range of ΔV_0.5MAX 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.

Figure 10.

The Cu²⁺ coordination sites are conserved in Shaker channels. (A) I_K 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) G_K-V relations from the same patch in different [Cu²⁺] are normalized by G_Kmax in 0 Cu²⁺. (0 (○), 1 (•), 5 (▾), 20 (▴), and 100 μM Cu²⁺ (▪)). (C) I_K at 0 mV from WT channels at the indicated [Zn²⁺] (0–10,000 μM). (D) G_K-V relations in different [Zn²⁺], normalized by G_Kmax in 0 Zn²⁺ (0 (○), 100 (•), 1,000 (▾), 4,000 (▴), 10,000 μM Zn²⁺ ()). (E) I_K at 0 mV from E247A channels in 0, 1, and 5 μM Cu²⁺. (F) I_K at +30 mV from E247C in 0, 0.1, 1, and 5 μM Cu²⁺. (G and H) I_K at +10 mV from E247A (G) or E247C (H) in 0, 100, and 1,000 μM Zn²⁺. (I and J) I_K 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.

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⁺.