Fig. 2

Inclusion of exon 24a enhances the functional impact of S218L mutation on Cav2.1 activation gating. A. Macroscopic Ca²⁺ currents evoked by 90 ms voltage steps between -60 and + 35 from a holding potential of -90 mV, recorded from HEK293F cells expressing wt hCa_v2.1 channel splice isoform ΔSSTR (top left), wt +SSTR (top right), or S218L mutant channels in the corresponding splice variant background (bottom panels). Current density vs voltage relationships (B) show that alternatively spliced wild type +SSTR channel displays a negative shift in activation threshold, relative to the wild type ΔSSTR. I-V curves show the distinctive effects of the S218L mutation, such as reduced current density and a hyperpolarizing shift of the activation voltage. Mutation-induced effects were similar between the two splice isoforms; however, S218L +SSTR mutant channel (Panel B, right) displayed a shallower I-V curve slope factor and a further shift towards more negative potentials (see blue arrows), compared to the S218L ΔSSTR (panel B, Left). Bar graph (C) shows a comparison of the averaged current density (pA/pF) values at the peak of the I-V curve. Both S218L mutant channels display reduced current density, regardless of channel splice isoform. The number of cells recorded is given in parentheses; the asterisk indicates statistical significance < 0.0001 of mutant channel values, relative to their respective wt. Voltage dependence of activation (D) and inactivation (E) plots revealed that activation half-point (V₅₀) and slope factor (k) are significantly different between the two wild type splice isoforms (blue dots D), whereas steady-state inactivation curves were nearly identical (black dots vs blue dots, E) (Table 7). The activation curve is shallower for the mutant channels with a prominent hyperpolarizing shift of the activation voltage to even more negative potentials (D). S218L mutant channels display a similar difference (~ 15 mV) in half-maximal inactivation (E), relative to their corresponding splice isoform