Histidines in the IS1–IS2 and IS3–IS4 loops underlie the trace metal sensitivity of Ca_v2.3. (A) Alignment of the putative amino acid residues residing in the IS1–IS2 and IS3–IS4 regions of Ca_v2 channels. Triple histidines, responsible for the trace metal sensitivity in Ca_v2.3 (highlighted), were identified by their sequential substitution: H111A/H179E/H181A. (B) Representative current traces recorded from Ca_v2.3 channels bearing the H179E/H181A mutation in response to the I-V protocol depicted above (V_hold = −90 mV, depolarizing pulses from −30 to 10 mV). Highlighted current traces were recorded in response to a −10-mV voltage pulse. (C) Peak current I-V relationships for H179E/H181A mutant obtained in HEPES- or Tricine-based extracellular solutions and normalized to cell capacitance. Data were fitted with GHK Eq. 1 (Table 1). (D) Normalized and averaged Ca²⁺ currents ± SEM (shaded area) recorded in response to prolonged (3-s) depolarizing pulse to −10 mV in HEPES and Tricine solutions. Recordings were fitted with double-exponential function. Corresponding values and statistics are in Table 1. (E) Steady-state inactivation curves for H179E/H181A mutant measured in the HEPES- or Tricine-based extracellular solutions. Data were fitted with a Boltzmann equation (Table 1). (F) Currents through H111A/H179E/H181A mutant channels, measured in response to the voltage pulse depicted above in Tricine- and Cu²⁺-containing solutions. (G) Dose–response curves of the Cu²⁺ effect on currents through Ca_v2.3 channels bearing various point mutations. Smooth lines represent the fit to the average data with the Hill equation (H179E: IC₅₀ = 115 ± 11 nM and n_H = 1.4; n = 4; H183A: IC₅₀ = 142 ± 19 nM and n_H = 1.4; n = 4; H179E/H183A: IC₅₀ = 203 ± 27 nM and n_H = 1.4; n = 9; and H111A: IC₅₀ = 205 ± 75 nM and n_H = 0.6; n = 5). (H) Mean values of inhibition induced by the application of 123 nM Cu²⁺ (***, P < 0.0001; **, P < 0.001; *, P < 0.05 by one-way ANOVA with Bonferroni posthoc correction).