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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

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.

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