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Figure S3. Mutations at the DCS locus do not affect channel gating. (A)Current-voltage curves obtained with the different Ca_V2.3 mutants coexpressed with α2-δ and β_2a subunits in the presence of 10 mM Ba²⁺ (left) or Ca²⁺ (right). Ca_V2.3(TEQE), square; Ca_V2.3(DEEE), circle; Ca_V2.3(DSED), triangle; Ca_V2.3(DEQN), inverted triangle; Ca_V2.3(TSQN), diamond; Ca_V2.3(AAAA), sideway triangle. See also Tables S2 and S3. (B) Isochronal inactivation curves (conditioning pulse of 2.5 s duration) obtained with the different Ca_V2.3 mutants coexpressed with α2-δ and β_2a subunits in the presence of 10 mM Ba²⁺ (left) or Ca²⁺ (right). See also Tables S2 and S3. The only noticeable difference is the larger inactivation recorded with the Ca_V2.3(AAAA) mutant. This modification cannot explain the monotonic decrease of the AMF because (a) this occurred without any modification of the activation parameters and the inactivation kinetics, and (b) the Ca_V2.3(GGGG), which has the same potentials for half activation and inactivation as the wild type, also displays monotonic AMF with smaller ICa/IBa ratio, suggesting that change in inactivation and cation selectivity are not related. (C) Time course of inactivation of these mutant channels recorded in Ba²⁺ or Ca²⁺ solutions and quantified as the fractional current at 2.5 s (current amplitude at the end of the depolarization divided by the current amplitude at the peak). None of the small differences shown in this graph, between the mutant Ca_V2.3 channels and between Ba²⁺ and Ca²⁺ currents, is statistically significant. The Ca_V2.1(TEQE) is also shown and has clearly no Ca²⁺-dependent inactivation.