Figure 6

High potency of Zn²⁺ and Ni²⁺ block of Ca_v3.2 channels is conferred onto LCa_v3 channels by replacement of cysteines in D2L5 extracellular loops. (A,B) Representative current traces of LCa_v3b channels with and without D2L5 extracellular loop cysteine replacements with alanines, in response to Zn²⁺ doses. T-type currents were generated by step depolarizations from − 110 mV to peak voltage (− 40 mV) (left panel). Peak currents per sweep (middle panel) in Ca²⁺ containing external solution (Table 1). Tau mono-exponential fits of mean inactivation kinetics (right panel). Normalized, overlapping currents illustrating kinetic rate differences (right panel, inset). (C) Zn²⁺ and (D) Ni²⁺ blocking effects. Dose response curves (left panel) and box plot of 50% inhibitory concentrations (IC50) (right panel), with human Ca_v3.1, Ca_v3.2 and Ca_v3.3 shown for comparison. Human Ca_v3.x channel response to Zn²⁺ taken from^17,18 and human Ca_v3.x response to Ni²⁺ taken from^15,16. The graphs in (C,D) represent mean ± s.e.m. with replicates illustrated in grey diamonds. IC50 block with Zn²⁺ dose16s of wild type channels (LCa_V3–12b/LCa_V3–12a), as well as cysteine mutant pairs (LCa_V3–12b ΔCys/LCa_V3–12b ΔCys) are not statistically significant from each other. Snail LCa_v3 channels possess the weak Zn²⁺ and Ni²⁺ block of Ca_v3.1 and Ca_v3.3 channels, but are conferred the high potency of Zn²⁺ and Ni²⁺ block of Ca_v3.2 channels as well as the characteristic Ca_v3.2 behavior with a loss of the property where inactivation kinetics slows with increasing Zn²⁺ doses, after alanine replacement of cysteines in D2L5 extracellular loops. Color coding of differing Ca_v3 channels: Ca_v3.1 (light blue), Ca_v3.2 (dark blue), Ca_v3.3 (green), LCa_V3–12b (orange), LCa_V3–12a (red), LCa_v3 Δcys mutants (striped orange or red bars or dotted lines). Data contained in this figure were analyzed and illustrated using OriginPro 2018 (64-bit) SR1 b9.5.1.195.