Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain

Abstract

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.

FIGURE 1.

C-terminal splice variants Ca_V1.3₄₂ and Ca_V1.3_42A. a, alternative usage of exon 42 results in either full-length (exon 42, black) or C-terminally truncated (exon 42A, gray) Ca_V1.3 channels. Arrows indicate the approximate position of forward (fwd) and reverse (rev) PCR primers used in Fig. 8. b, immunoblot of both splice variants expressed together with β3 and α2δ1 subunits in HEK-293 cells, as described under “Experimental Procedures.” Mock-transfected cells (mock) were used to show specificity of immunoreactivity. One representative experiment out of four is shown.

FIGURE 2.

Activation and steady-state inactivation properties of Ca_V1.3₄₂ compared with Ca_V1.3_42A channels. a, normalized mean I-V curves of I_Ca for Ca_V1.3₄₂ (black) and Ca_V1.3_42A (gray) channels. Activation parameters and statistics are given in Table 1. The dotted line describes the fit to a normalized I-V curve of I_Ca for Ca_V1.2 in comparison: V_0.5,act, 8.8 mV; k_act, –11.0 mV. Cell, 111d_16. b, representative current traces for I_Ca (300-ms depolarization to indicated test potentials). Cells: Ca_V1.3₄₂, 196l_23; Ca_V1.3_42A, 196l_32. c, V_0.5,act for Ca_V1.3_42A was independent from current density. d, exemplar ON-gating currents for Ca_V1.3₄₂ and Ca_V1.3_42A with similar I_Ba amplitudes measured by depolarizing cells to V_rev. One representative out of six experiments is shown. Cells: Ca_V1.3₄₂, 207l_34; Ca_V1.3_42A, 237k_26. e, steady-state inactivation curves for Ca_V1.3₄₂ and Ca_V1.3_42A. Activation curves were obtained from parameters in a. Solid lines are fits to the Boltzmann relationship (see “Experimental Procedures”). Statistics are given under “Results.” Error bars reflect S.E.

FIGURE 3.

Inactivation properties of Ca_V1.3₄₂ and Ca_V1.3_42A channel-mediated currents. a, representative current traces of Ca_V1.3₄₂ (black) and Ca_V1.3_42A (gray) I_Ca evoked by a 10-s depolarization to V_max. The inset illustrates inactivation during initial 30 ms. Cells: Ca_V1.3₄₂, 316h_51; Ca_V1.3_42A, 187g_46. b, percent I_Ca inactivation during 0.03-, 0.25-, 1-, and 5-s test pulses to V_max. ***, p ≤ 0.001, Student's t test. c, normalized representative I_Ca traces at physiologically relevant potentials (–20 mV; corresponding to ∼–35 mV at physiological Ca²⁺ concentrations; see “Results”). Cells: Ca_V1.3₄₂, 196l_23; Ca_V1.3_42A, 187g_17. Integrated current corresponding to the area under normalized I_Ca during a 300-ms pulse is shown. Statistically significant difference: ***, p ≤ 0.001, Unpaired t test. d, percent I_Ba inactivation during 0.03-, 0.25-, 1-, and 5-s test pulses to V_max. *, p < 0.05; **, p < 0.01, Student t test. e, voltage dependence of CDI for Ca_V1.3₄₂ (left) and Ca_V1.3_42A (right): r₂₅₀ corresponds to the fraction of I_Ca or I_Ba remaining after 250 ms; f is the difference between r₂₅₀ values at +10 mV (for statistics see “Results” and Table 3). Number of experiments is given in parentheses. Error bars reflect S.E.

FIGURE 4.

Gating properties of Ca_V1.3_ΔC116 channels and effect of the distal CTM peptide C₁₁₆. Data for Ca_V1.3₄₂ were taken from Fig. 2. a, representative normalized I-V curves for I_Ca. Activation parameters were as follows (in mV): Ca_V1.3₄₂: V_0.5,act, –4.2, and k_act, –8.6; Ca_V1.3_ΔC116: V_0.5,act: –12.8, and k_act, –7.7; Ca_V1.3_ΔC116+GFP-C₁₁₆: V_0.5,act, –4.7, and k_act, –8.8; Cells: 196l_23, 27g_57, and 27g_91, respectively. b, representative current traces of I_Ca evoked by a depolarization to V_max. Cells: Ca_V1.3_ΔC116, 27g_27; Ca_V1.3_ΔC116+GFP-C₁₁₆, 197f_26. c, percent I_Ca inactivation during test pulses to V_max: **, p < 0.01; ***, p < 0.001, compared with Ca_V1.3₄₂ (black); +, p < 0.05; +++, p < 0.001, comparison of Ca_V1.3_ΔC116 + GFP-C₁₁₆ (white) with Ca_V1.3_ΔC116 (gray) alone (one-way ANOVA followed by Bonferroni post-test). Number of experiments is given in parentheses. Error bars reflect S.E. d and e, representative I_Ca traces for Ca_V1.3_ΔC116 (d) and Ca_V1.3_ΔC116+GFP-C₁₁₆ (e). Cells: 27g_67 and 27g_91, respectively. For comparison to Ca_V1.3₄₂ see Fig. 2.

FIGURE 5.

Ca_V1.3_ΔC473 channel properties in absence and presence of distal CTM peptide C₁₁₆. Data for Ca_V1.3₄₂ (black) and Ca_V1.3_42A (gray) were taken from Figs. 2 and 4. a, representative normalized I-V curves for I_Ca. Activation parameters (in mV): Ca_V1.3_42A: V_0.5,act, –14.3, and k_act, –6.8; Ca_V1.3_42A + GFP-C₁₅₈: V_0.5,act, –13.5 and k_act, –7.2; Cells: 196l_32 and 217c_3, respectively. b, representative current traces of I_Ca evoked a 10-s depolarization to V_max. Cells: Ca_V1.3_42A + GFP-C₁₅₈, 227k_19; Ca_V1.3₄₂, 316h_51; Ca_V1.3_42A, 187g_46. c, percent I_Ca inactivation during test pulses to V_max:*, p < 0.05; ***, p < 0.001 compared with Ca_V1.3₄₂; ++, p < 0.01; +++, p < 0.001, comparison of Ca_V1.3_42A + GFP-C₁₅₈ with Ca_V1.3_42A alone. d, representative normalized I-V curves for I_Ca. Activation parameters (in mV): Ca_V1.3_ΔC473: V_0.5,act, –14.4, and k_act, –7.1; Ca_V1.3_ΔC473 +GFP-C₁₁₆: V_0.5,act, –2.4, and k_act, –9.4; Cells: 177i_1 and 167j_24, respectively. e, same conditions as in b. Cells: Ca_V1.3_ΔC473, 247h_25; Ca_V1.3_ΔC473 +GFP-C₁₁₆, 167j_14. f, percent I_Ca inactivation during test pulses to V_max:*, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with Ca_V1.3₄₂; +, p < 0.05; ++, p < 0.01; +++, p < 0.001, comparison of Ca_V1.3_ΔC473 + GFP-C₁₁₆ with Ca_V1.3_ΔC473 alone. g–i, representative I_Ca traces for Ca_V1.3_42A + GFP-C₁₅₈ (g), Ca_V1.3_ΔC473 (h), and Ca_V1.3_ΔC473 + GFP-C₁₁₆ (i) correspond to I-V curves in a and d. For comparison to Ca_V1.3₄₂ and Ca_V1.3_42A see Fig. 2. For statistics: in a and d, Student's t test; c and f, one-way ANOVA followed by Bonferroni post-test. Number of experiments is given in parentheses. Error bars reflect S.E.

FIGURE 6.

Sequence alignment of LTCC C termini. A sequence alignment of neuronal human Ca_V1.3 (GenBank™ accession number NM_000720), Ca_V1.4 (GenBank™ accession number AJ224874), and Ca_V1.2 (uniprot accession number Q13936) α1 subunits is shown. Sequence identity (dark gray), similarity (light gray) and gaps (–) are indicated. PCRD, proximal C-terminal regulatory domain; DCRD, distal C-terminal regulatory domain; CTM, dotted line. Position of Ca_V1.3_42A and truncation mutants Ca_V1.3_ΔC473, Ca_V1.3_ΔC158, Ca_V1.3_ΔC116, and Ca_V1.3_ΔC76 are indicated by black arrows. Position of exon 44 is given.

FIGURE 7.

FRET analysis of the binding of peptide C₁₅₈ or C₁₁₆ to various fragments of the Ca_V1.3 C terminus. N_FRET values obtained from co-expression of CFP-C₁₅₈ or CFP-C₁₁₆ (probe) with the indicated YFP-tagged Ca_V1.3 C-terminal fragments in HEK-293 cells. All constructs showed a homogeneous intracellular distribution. As controls, N_FRET values are given for CFP co-expressed with YFP-EF-PreIQ-IQ-PCRD (CFP control) and YFP with probes CFP-C₁₅₈ (YFP control 1) and CFP-C₁₁₆ (YFP control 2). Controls are not significantly different from zero (one-sample t test against 0). Interaction between fragments was considered when significant difference to both the CFP- and the corresponding YFP control was observed (***, p < 0.001, one-way ANOVA followed by Bonferroni post-test).

FIGURE 8.

Tissue expression of Ca_V1.3 C-terminal splice variants. Qualitative RT-PCR experiments showing expression of both long (containing exon (Ex) 42) and short (containing exon 42A) Ca_V1.3 isoforms in different mouse (a) and human (b) tissues. GAPDH was used as a housekeeping gene. One representative out of at least three independent experiments is shown. Quantitative Taqman RT-PCR experiments in a (right), show the relative expression of exons 42 and 42A in percent of total signal in mouse whole-brain and several brain subregions (***, p < 0.001, unpaired Students t test, all significantly different from 0, one sample t test). Data are given as means ± S.E.