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.