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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 [Cu.sup.2+] is a potent inhibitor of BK and Shaker [K.sup.+] channels. At low micromolar concentrations, [Cu.sup.2+] rapidly and reversibly reduces macrosocopic [K.sup.+] conductance ([G.sub.K]) evoked from mSlo1 BK channels by membrane depolarization. [G.sub.K] is reduced in a dose-dependent manner with an [IC.sub.50] and Hill coefficient of ~2 [micro]M and 1.0, respectively. Saturating 100 [micro]M [Cu.sup.2+] shifts the [G.sub.K]-V relation by +74 mV and reduces [G.sub.Kmax] by 27% without affecting single channel conductance. However, 100 [micro]M [Cu.sup.2+] fails to inhibit [G.sub.K] when applied during membrane depolarization, suggesting that [Cu.sup.2+] interacts poorly with the activated channel. Of other transition metal ions tested, only [Zn.sup.2-] and [Cd.sup.2+] had significant effects at 100 DM with [IC.sub.50]s > 0.5 mM, suggesting the binding site is [Cu.sup.2+] selective. Mutation of external Cys or His residues did not alter [Cu.sup.2+] sensitivity. However, four putative [Cu.sup.2+]-coordinating residues were identified (D133, Q151, D153, and R207) in transmembrane segments S1, $2, and $4 of the mSlo1 voltage sensor, based on the ability of substitutions at these positions to alter [Cu.sup.2+] and/or [Cd.sup.2+] sensitivity. Consistent with the presence of acidic residues in the binding site, [Cu.sup.2+] sensitivity was reduced at low extracellular pH. The three charged positions in S1, $2, and $4 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 [Cu.sup.2+] than [Zn.sup.2+] and that sensitivity to these metals is altered by mutating the conserved positions in S1 or $4 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 [Cu.sup.2+] at physiologically relevant concentrations to inhibit activation of BK and other channels.

BK (Slol) potassium channels arc activated by millimolar intracellular [Mg.sup.2+] as well as micromolar [Ca.sup.2+] and membrane depolarization. [Mg.sup.2+] and [Ca.sup.2+] act ill all approximately additive manner m different binding sites to shift t conductance-voltage ([G.sub.K]-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we lind that the mechanism of [Mg.sup.2+] action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of [Ca.sup.2+]. Evidence that [Ca.sup.2+] acts independently of voltage sensor activation includes an ability to increase open probability ([P.sub.o]) at extreme negative voltages where voltage sensors are in the resting state; 2 [micro]M [Ca.sup.2+] increases [P.sub.o] more than 15-fold at -120 mV. However 10 mM [Mg.sup.2+] which has an effect on the [G.sub.K]-V relation similar to 2 [micro]M [Ca.sup.2+], has no detectable effect on [P.sub.o] when voltage sensors are in the resting state. Gating currents are only slightly altered by [Mg.sup.2+] when channels are closed, indicating that [Mg.sup.2+] does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by [Mg.sup.2+] in a mutam (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM [Mg.sup.2+] increases [P.sub.o] only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing [Mg.sup.2+] from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of [Ca.sup.2+]. The effects of [Mg.sup.2+] on steady-state activation and [I.sub.K] kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between [Mg.sup.2+] and voltage sensor that may underlie this mechanism.

The activation of large conductance [Ca.sup.2+]-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated [K.sup.+] ([K.sub.v]) channels. Yet BK and Kv channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mS1o1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation ([P.sub.o]) and [I.sub.K] kinetics ([tau] ([I.sub.K])) over an extended voltage range in 0-50 [micro]M [[Ca.sub.2+].sub.i]. mS1o1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of [P.sup.o]. The voltage dependence of [P.sub.o] and [tau]([I.sub.K]) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and [K.sub.v] channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to [K.sub.v] channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol- 1, indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.