Role of the C-terminal domain in modifying pH-dependent regulation of Ca v 1.4 Ca 2+ channels

Figures

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  Figure 1.. Schematic of CaV1.4 showing key modulatory regions in the C-terminal domain (CTD). Cav1.4 (UniProt: O60840) consists of four homologous domains (I-IV), each containing six transmembrane helices (S1-S6, with charged S4) and pore (P) loop containing glutamate (E) residue that contributes to selectivity filter. There is a large C-terminal domain containing an EF-hand domain (EF, L1459-K1478), pre-IQ domain (pre-IQ, N1524-E1568), IQ domain (IQ, V1569-R1597), and a distal C-terminal modulatory domain (CTM) containing exon 47 (e47). X depicts truncation after K1602 (equivalent to (K1591X) due to CSNB2-related mutation. Exon 47 comprises 65 residues, from G1836 to A1901.

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  Figure 2.. pH-dependent modulation of IBa in cells transfected with CaV1.4 FL. (a) Representative traces and I-V plot for IBa elicited by 50-ms pulses from −80 mV to various voltages. Current traces represent IBa in a single cell evoked by test pulses evoking the peak IBa at the indicated pH values (with increasing pH: +10, +5, +5, −5 and −10 mV). For each cell at the indicated pH (n = 7 cells), IBa was normalized to the peak amplitude of IBa at pH 7.3 and plotted against test voltage as the mean (line) ± SEM (shaded region). (b-e) Parameters from Boltzmann fits of I-V data in (a) plotted against extracellular pH (Vh, b; Gmax, c; k, d; and Vrev, e) as mean (symbols) ± SEM (bars) and fit by linear regression.

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  Figure 3.. pH-dependent modulation of IBa in cells transfected with CaV1.4Δe47. (a) Representative traces and I-V plot for IBa elicited by 50-ms pulses from −80 mV to various voltages. Current traces represent IBa in a single cell evoked by test pulses evoking the peak IBa at the indicated pH values (with increasing pH: −5, −5, −10, −15 and −15 mV). For each cell at the indicated pH (n = 5 cells), IBa was normalized to the peak amplitude of IBa at pH 7.3 and plotted against test voltage as the mean (line) ± SEM (shaded region). (b-e) Parameters from Boltzmann fits of I-V data in (a) plotted against extracellular pH (Vh, b; Gmax, c; k, d; and Vrev, e) as mean (symbols) ± SEM (bars) and fit by linear regression.

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  Figure 4.. pH-dependent modulation of IBa in cells transfected with K1591X. (a) Representative traces and I-V plot for IBa elicited by 50-ms pulses from −80 mV to various voltages. Current traces represent IBa in a single cell evoked by test pulses evoking the peak IBa at the indicated pH values (with increasing pH: +15, 0, −5, −5 and −15 mV). For each cell at the indicated pH (n = 4 cells), IBa was normalized to the peak amplitude of IBa at pH 7.3 and plotted against test voltage as the mean (line) ± SEM (shaded region). (b-e) Parameters from Boltzmann fits of I-V data in (a) plotted against extracellular pH (Vh, b; Gmax, c; k, d; and Vrev, e) as mean (symbols) ± SEM (bars). Line represents fit by linear regression.

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  Figure 5.. Weaker pH modulation of CaV1.4Δe47 and K1591X than CaV1.4 FL. (a) Representative traces for IBa elicited by 50-ms pulses from −80 mV to voltages evoking the peak IBa at the indicated pH values (with increasing pH: +10, +5 and −10 mV) in cells expressing CaV1.4 FL (n = 7 cells), CaV1.4Δe47 (n = 5 cells), or K1591X (n = 4 cells). (b) IBa evoked as in (a) was expressed as % difference from IBa at pH 7.3 (% ΔIBa) and plotted against extracellular [H+] in µM as mean (symbols) ± SEM (bars). Line represents fit by Hill equation. (c) IC50 and slope (α) determined by nonlinear fits of data in (b). Columns and bars represent mean ± SEM. Significant differences between groups (p-values shown above) were determined by one-way ANOVA (F(2,13) = 3.853, overall p = 0.0485 for IC50; F(2,13) = 18.53, overall p = 0.0002 for α) and post-hoc Dunnett’s test.

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  Figure 6.. Gmax for K1591X shows little pH-dependence compared to CaV1.4Δe47 and CaV1.4 FL. (a-d) Boltzmann parameters from I-V data in Figures 1–3 plotted against extracellular pH. Symbols and bars that represent mean ± SEM. Lines represent fit by linear regression. (e) Slopes (β) of the linear fits in (a-d). Bars represent mean ± SEM. By one-way ANOVA or Kruskal-Wallis test, there was a significant difference in β Gmax (F(2,13) = 5.532, p = 0.018) but no significant difference in β Vh (H(2) = 3.977, p = 0.1366) or β Vrev (F(2,13) = 0.6689, p = 0.5290). p-value for β Gmax shown for K1591X vs FL was determined by post-hoc Dunnett’s test. By one-way ANOVA, there was a significant overall difference in β k (F(2,13) = 5.041, p = 0.0240), but no difference by post-hoc Dunnett’s test.

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  Figure 7.. α2δ-4 enhances pH-dependent modulation of CaV1.4 FL. (a,b) Representative traces and I-V plot for IBa elicited by 50-ms pulses from −80 mV to various voltages for CaV1.4 FL expressed with α2δ-1 (a) or without any α2δ (b). Current traces correspond to voltages evoking the peak IBa at the indicated pH (with increasing pH: −5, −10, −15, −15 and −20 mV in (a) and 0, −10, −10, −15 and −15 mV in (b)). For each cell at the indicated pH (+α2δ-1, n = 6 cells; -α2δ, n = 6 cells), IBa was normalized to the peak amplitude of IBa at pH 7.3 and plotted against test voltage as the mean (line) ± SEM (shaded region). (c-g) % difference from IBa at pH 7.3 (% ΔIBa) (c) Boltzmann parameters were plotted against extracellular pH as mean (symbols) ± SEM (bars). Lines represent fit by Hill equation (c) or linear regression (d-g). (h) Slopes (α, β) of the fits in (c-g). Bars represent mean ± SEM. By one-way ANOVA, there was a significant difference in α for % ΔIBa (F(2,16) = 17.38, p < 0.0001), Vh (F(2,16) = 14.14, p = 0.0003), and Gmax (F(2,16) = 12.81, p = 0.0005) but not Vrev (F(2,16) = 3.674, p = 0.0486). By Kruskal-Wallis test, there was a significant difference in β for k (H(2) = 6.031, p =…

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  Figure 8.. α2δ subunits do not enhance pH-dependent modulation of CaV1.4 Δe47.Representative traces and I-V plot for IBa elicited by 50-ms pulses from −80 mV to various voltages for CaV1.4 Δe47 expressed with α2δ-1 (a) or without any α2δ (b). Current traces correspond to voltages evoking the peak IBa at the indicated pH (with increasing pH: −5, −10, −15, −15 and −20 mV in (a) and 0, −10, −10, −15 and −15 mV in (b)). For each cell at the indicated pH (+α2δ-1, n = 4 cells; -α2δ, n = 5 cells), IBa was normalized to the peak amplitude of IBa at pH 7.3 and plotted against test voltage as the mean (line) ± SEM (shaded region). (c-g) % difference from IBa at pH 7.3 (% ΔIBa) (c) and Boltzmann parameters were plotted against extracellular pH as mean (symbols) ± SEM (bars). Lines represent fit by Hill equation (c) or linear regression (d-g). (h) Slopes (α, β) of the fits in (c-g). Bars represent mean ± SEM. By one-way ANOVA, there was not a significant difference in α for % ΔIBa (F(2,11) = 1.240, p = 0.3269), Gmax (F(2,11) = 1.7, p = 0.2274), k (F(2,11) = 0.3577, p = 0.7071), and Vrev (F(2,11) = 3.415, p = 0.0702). By Kruskal-Wallis test, there was not a significant difference in β for Vh (H(2) =…

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  Figure 9.. α2δ-4 enhances pH-dependent modulation of K1591X. Representative traces and I-V plot for IBa elicited by 50-ms pulses from −80 mV to various voltages for CaV1.4 K1591X expressed with α2δ-1 (a) or without any α2δ (b). Current traces correspond to voltages evoking the peak IBa at the indicated pH (with increasing pH: −5, −10, −15, −15 and −20 mV in (a) and 0, −10, −10, −15 and −15 mV in (b)). For each cell at the indicated pH (+α2δ-1, n = 7 cells; -α2δ, n = 3 cells), IBa was normalized to the peak amplitude of IBa at pH 7.3 and plotted against test voltage as the mean (line) ± SEM (shaded region). (c-g) % difference from IBa at pH 7.3 (% ΔIBa) (c) and Boltzmann parameters were plotted against extracellular pH as mean (symbols) ± SEM (bars). Lines represent fit by Hill equation (c) or linear regression (d-g). (h) Slopes (α, β) of the fits in (c-g). Bars represent mean ± SEM. By one-way ANOVA, there was a significant difference in α for % ΔIBa (F(2,11) = 3.725, p = 0.0582), Vh (F(2,11) = 5.156, p = 0.0263), and Δk (F(2,11) = 6.740, p = 0.0123) but not Gmax (F(2,11) = 1.903, p = 0.1951) nor Vrev (F(2,11) = 2.575, p = 0.1210). p-values for significantly different groups are shown and…