Copper and protons directly activate the zinc-activated channel

Abstract

The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively. The responses elicited by Zn(2+), Cu(2+) and H(+) through ZAC are all characterized by low degrees of desensitization. In contrast, currents evoked by high concentrations of the three agonists comprise distinctly different activation and decay components, with transitions to and from an open state being significantly faster for H(+) than for the two metal ions. The permeabilities of ZAC for Na(+) and K(+) relative to Cs(+) are indistinguishable, whereas replacing all of extracellular Na(+) and K(+) with the divalent cations Ca(2+) or Mg(2+) results in complete elimination of Zn(2+)-activated currents at both negative and positive holding potentials. This indicates that ZAC is non-selectively permeable to monovalent cations, whereas Ca(2+) and Mg(2+) inhibit the channel. In conclusion, this is the first report of a Cys-loop receptor being gated by Zn(2+), Cu(2+) and H(+). ZAC could be an important mediator of some of the wide range of physiological functions regulated by or involving Zn(2+), Cu(2+) and H(+).

Keywords: Calcium block; Copper; Cys-loop ligand-gated ion channels; Protons; Zinc.

Fig. 1

Zn²⁺ as a ZAC agonist. (A) Inward currents evoked by rapid applications of Zn²⁺ to ZAC-expressing COS-7 cells. Zn²⁺ was applied (solid bar) for 6 s to cells voltage-clamped at −60 mV Zn²⁺-mediated currents recorded from COS-7 cells transiently transfected with ZAC. (B) Concentration–response relationship for Zn²⁺ at ZAC-expressing COS-7 cells. Data shown as mean ± S.E.M. generated from 3 to 5 cells.

Fig. 2

Cationic permeability of ZAC. (A). Voltage ramps between −60 mV and 30 mV applied before and during agonist application. Non-specific currents recorded during voltage ramps in the absence of Zn²⁺ were subtracted from currents recorded during voltage ramps applied during the steady state inward current evoked by 1 mMZn²⁺ (solid bar). Insert; overlapping currents evoked by voltage ramps in the absence (gray current) and presence of Zn²⁺ (black current). (B) Representative Zn²⁺-mediated currents recorded from a ZAC-expressing COS-7 cell in extracellular solutions of varying ionic compositions. Zn²⁺ (1 mM) was applied 1/60 s (Zn²⁺-mediated currents are concatenated in the illustration). The major cationic species is indicated above each trace (see Section 2 for details). When all extracellular monovalent cations were replaced with either Ca²⁺ or Mg²⁺ no detectable Zn²⁺-mediated inward current was observed. (C) Current–voltage relationships from a representative ZAC expressing COS-7 cell obtained from voltage ramps applied during the application of 1 mM Zn²⁺. The current obtained with a NaCl-based extracellular solution was completely absent when Zn²⁺ was applied with a CaCl₂-based solution. The lack of outward current at positive potentials indicates that Ca²⁺ is inhibiting the outward flux of intracellular Cs⁺.

Fig. 3

Inhibition of ZAC by Ca²⁺ and Mg²⁺. (A) Overlaid representative Zn²⁺-evoked current in the absence (black current) or in the presence of 30 mM Ca²⁺ or Mg²⁺ (gray current). (B) Concentration–inhibition relationship of the Ca²⁺ block (closed circles) of ZAC. Block of Zn²⁺-evoked current by 30 mM Mg²⁺ is also shown (open circle). Data points are mean ± S.E.M., n = 3–9 cells. (C) Voltage-independence of Ca²⁺ block of ZAC. Left: Zn²⁺-evoked currents at holding potentials of 60 mV and −60 mV in the absence (black currents) and presence of 30 mM Ca²⁺ (gray currents). Right: bar graph showing mean inhibition at 60 mV (n = 4) and −60 mV (n = 9). (D) At a holding potential of −60 mV, application of 30 mM Ca²⁺ evoked a reversible outward current representing the inhibition of spontaneous current. Bar graph showing the mean outward Ca²⁺ (30 mM)-evoked current as a percent of the preceding Zn²⁺ (1 mM)-evoked current (n = 3 cells).

Fig. 4

The inactivity of other metal ions at ZAC. A selection of other transition metals and aluminum are all inactive at ZAC expressed in COS-7 cells. At 1 mM concentrations, iron (Fe²⁺), cobalt (Co²⁺), nickel (Ni²⁺), cadmium (Cd²⁺), and aluminum (Al³⁺) were either inactive or elicited negligible responses at ZAC. Zn²⁺ was used as reference agonist in the recordings.

Fig. 5

Activation of ZAC by Cu²⁺. (A) Inward currents evoked by rapid applications of Cu²⁺ to ZAC-expressing COS-7 cells. Cu²⁺ was applied (solid bar) for 6 s to cells voltage-clamped at −60 mV and compared to currents evoked by 1 mM Zn²⁺. (B) Cu²⁺ concentration–response relationship of ZAC relative to current evoked by 1 mM Zn²⁺. Data shown as mean ± S.E.M. generated from 3 to 14 cells.

Fig. 6

Activation of ZAC by H⁺. (A) Inward currents evoked by rapid application of H⁺ to ZAC-expressing COS-7 cells. Acidic extracellular solutions were rapidly applied (solid bar) for 6 s to cells voltage-clamped at −60 mV and compared to currents evoked by 1 mM Zn²⁺ (at pH 7.4). (B) H⁺ concentration (pH)–response relationship of ZAC relative to current evoked by 1 mM Zn²⁺. Data shown as mean ± S.E.M. generated from 4 to 6 cells.

Fig. 7

H⁺-evoked currents through ZAC are characterized by faster activation and decay components than Zn²⁺-evoked currents. (A) Time constants for activation and decay of 1 mM Zn²⁺, 30 µM Cu²⁺, and 10 µM H⁺ (pH 5)-evoked currents. The activation phase of Zn²⁺-evoked currents was significantly slower than Cu²⁺- and H⁺-evoked currents. Decay of H⁺-evoked currents was significantly faster than Zn²⁺- and Cu²⁺-evoked currents. n = 3,* = p < 0.05, ** = p < 0.01. (B) Example current traces from independent experiments with comparable peak values for Zn²⁺- (black), Cu²⁺- (light gray) and H⁺-evoked currents (dark gray), normalization to peak amplitudes at activation or the start of current decay, overlaid so that begin of application and wash off are in parallel for all three traces. Note the significant decay of H⁺-evoked currents.

Fig. 8

Whole-cell currents recorded from HEK293 cells expressing recombinant ZAC and endogenous ASICs. (A) Overlaid currents recorded from a HEK293 cell transfected with ZAC. Rapid application of Zn²⁺ or H⁺ (solid bar) produced different current characteristics. Zn²⁺ application produced slowly activating, non-desensitizing currents, whereas application of H⁺ produced ASIC-like faster activating currents with pronounced desensitization. (B) In the presence of amiloride, an ASIC blocker, application of H⁺ produced a typical ZAC-mediated current.