Alternative splicing of the beta 4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating

Abstract

Ca2+ channel beta subunits are important molecular determinants of the kinetics and voltage dependence of Ca2+ channel gating. Through direct interactions with channel-forming alpha1 subunits, beta subunits enhance expression levels, accelerate activation, and have variable effects on inactivation. Four distinct beta subunit genes each encode five homologous sequence domains (D1-5), three of which (D1, D3, and D5) undergo alternative splicing. We have isolated from human spinal cord a novel alternatively spliced beta4 subunit containing a short form of domain D1 (beta4a) that is highly homologous to N termini of Xenopus and rat beta3 subunits. The purpose of this study was to compare the gating properties of various alpha1 subunit complexes containing beta4a with those of complexes containing a beta4 subunit with a longer form of domain D1, beta4b. Expression in Xenopus oocytes revealed that, relative to alpha1A and alpha1B complexes containing beta4b, the voltage dependence of activation and inactivation of complexes containing beta4a were shifted to more depolarized potentials. Moreover, alpha1A and alpha1B complexes containing beta4a inactivated at a faster rate. Interestingly, beta4 subunit alternative splicing did not influence the gating properties of alpha1C and alpha1E subunits. Experiments with beta4 deletion mutants revealed that both the N and C termini of the beta4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are alpha1 subunit specific. Our data are best explained by a model in which distinct modes of activation and inactivation result from beta-subunit splice variant-specific interactions with an alpha1 subunit gating structure.

Fig. 1.

Sequence comparisons of human spinal cord Ca²⁺ channel β_4a and β_4bsubunits and other β subunit subtypes. Top, The amino acid sequence of domain 1 and a short segment of domain 2 of the human β_4a subunit (hβ_4a) is shown aligned with comparable domains of two Xenopusβ₃ subunits (xβ₃₂ andxβ₂₈) (Tareilus et al., 1997) and a human β₃ subunit (hβ₃). Amino acids identical to the hβ_4a sequence are boxed. Asterisks denote D to N amino acid conversions in the human β_4a sequence.Bottom, The amino acid sequence of domain 1 and a short segment of domain 2 of the human β_4b subunit (hβ_4b) is shown aligned with comparable domains of the human β_1b subunit. Identical amino acids are boxed. Dashed lines indicate gaps in the sequence. The bar denotes consensus sites for phosphorylation by protein kinase C.

Fig. 2.

Expression rates of α_1ACa²⁺ channel complexes with different β subunit compositions. Peak currents elicited by depolarization to +10 mV (α_1A/α₂δ), +5 mV (α_1A/α₂δ + β_4a), or 0 mV (α_1A/α₂δ + β_4b) from a holding potential of −80 mV are plotted against days after injection. Barium (5 mm) was the charge carrier. Oocytes were maintained in ND96 culture media at 18°C. Comparisons between experiments in which the β_4aor β_4b subunits were injected at 1:1 (1×) or 6:1 (6×) ratios relative to the α_1A are shown. Each data point represents a minimum of six recordings. The SEM for each point is shown unless the values were smaller than the symbol.

Fig. 3.

β_4a and β_4b subunits have α₁ subunit subtype-specific effects on the voltage dependence of activation. A, B,E, F, Normalized, averaged peak current–voltage (I–V) plots for α_1A (A), α_1B(B) α_1C (E), and α_1E (F) coexpressed with α₂δ and either β_4a or β_4b.A, B, The α_1A (BI-2) and α_1B (Δ21) subunits used in these and subsequent experiments are those described by Mori et al. (1991) and Pan and Lipscombe (2000), respectively. Currents were activated by 300 msec depolarizations to various test potentials (−40 to +40 mV in 5 mV increments) from a holding potential of −80 mV. Barium (5 mm) was the charge carrier for both α_1A and α_1B. C, D, Voltage dependence of activation up to +10 mV for α_1A(C) and +20 mV for α_1B(D) as determined from averagedI–V data in A andB. Data points represent the means of the normalized data at a given membrane potential. The SEM for each point is shown unless the values were smaller than the symbol. Smooth curves represent a single Boltzmann fit to the averaged data. Values for V_1/2 and k for α_1A and α_1B plus α₂/δ and either β_4a or β_4b are listed in Table 1. E,F, The α_1C (cardiac) and α_1E(doe-1) subunits used in these and subsequent experiments are those described by Mikami et al. (1989) and Horne et al. (1993), respectively. Currents were activated by 300 msec depolarizations to various test potentials (−40 to +40 mV in 5 mV increments) from a holding potential of −80 mV (α_1C + β_4a, n = 12; α_1C + β_4b,n = 13; α_1E + β_4a, n = 9; α_1E + β_4b,n = 9). Barium (40 mm) was the charge carrier.

Fig. 4.

β_4a and β_4b subunits have α₁ subunit subtype-specific effects on the voltage dependence of inactivation. A–D, Normalized, averaged isochronal inactivation curves for α_1A (A), α_1B(B), α_1C (C), and α_1E (D) coexpressed with α₂δ and either β_4a or β_4b. Curves were generated from peak currents elicited by a 300 msec test depolarization to +5 mV (α_1A + β_4a), 0 mV (α_1A + β_4b), +10 mV (α_1B + β_4a), +5 mV (α_1B + β_4b), or +20 mV (α_1C and α_1E with β_4a and β_4b) after a 20 sec conditioning prepulse to voltages ranging from −80 to +30 mV (A,C, D) or −100 to +10 mV (B). Barium (5 mm for α_1A and α_1B; 40 mm for α_1C and α_1E) was the charge carrier. Data points represent the means of the normalized data at a given membrane potential. The SEM for each point is shown unless the values were smaller than the symbol. Smooth curves represent a single Boltzmann fit to the averaged data. Values forV_1/2 and k for inactivation of α_1A and α_1B plus α₂δ and either β_4a or β_4b are listed in Table1.

Fig. 5.

α_1A and α_1B complexes containing β_4a inactivate faster than those containing β_4b. A, B, Representative current traces of α_1A (A) and α_1B (B) plus α₂δ and either β_4a (top) or β_4b(bottom). Currents were elicited by step depolarizations to a range of test potentials (−10 to +30 mV in 10 mV increments) from a holding potential of −80 mV. Barium (5 mm) was used as the charge carrier. Traces were fit with a single exponential from 25 msec beyond the peak inward current to the end of the depolarization. Averages of τ_inactivation at the peak current potential were α_1A + β_4a, 226.6 ± 12.5 msec (n = 12); α_1A + β_4b, 307.2 ± 19.2 msec (n = 10); α_1B + β_4a, 160.1 ± 20.0 msec (n = 10); α_1A + β_4b, 213.9 ± 15.6 msec (n = 10). C, D, Current remaining at the end of a 300 msec test pulse (R₃₀₀), elicited as in the protocol above, for α_1A (C) and α_1B (D) plus α₂δ and either β_4a or β_4b. The SEM for each bar is shown. Asterisks denote statistical significance (p < 0.05) as determined by a Student's two-sample equal variance t test.

Fig. 6.

Effects of β₄ subunit N- and C-terminal deletions on the voltage dependence of activation of α_1A and α_1B Ca²⁺channels. A, Schematic diagrams of the wild-type and artificial β₄ subunits used in this series of experiments. The 15 amino acid β_4a and 49 amino acid β_4b N termini (alternatively spliced forms of domain 1) are denoted by filled and open bars, respectively. Domains 2–4 are represented by a singlecross-hatched bar. The C terminus (domain 5) is denoted by a diagonally striped bar. B, C, Voltage dependence of activation up to +10 mV for α_1A(B) and +20 mV for α_1B(C) as determined from averagedI–V data. Data points represent the means of the normalized data at a given membrane potential. The SEM for each point is shown unless the values were smaller than the symbol.Smooth curves represent a single Boltzmann fit to the averaged data. Broken curves represent activation data shown in Figure 3, C and D, and are included in this figure for reference. Values forV_1/2 and k for α_1A and α_1B plus α₂/δ and each of the six β₄constructs are grouped according to curve similarities in Table1.

Fig. 7.

Effects of β₄ subunit N- and C-terminal deletions on the voltage dependence of inactivation of α_1A and α_1B Ca²⁺channels. A, B, Normalized, averaged steady-state inactivation curves for α_1A(A) and α_1B(B) coexpressed with α₂δ and one of the six β₄ constructs shown in Figure6A. Curves were generated from peak currents elicited by a 300 msec test depolarization to −5 mV (α_1A+ β₄ΔNΔC), 0 mV (α_1A + β_4b, β_4aΔC), +5 mV (α_1A + β_4a, β₄ΔN, and β_4bΔC; α_1B + β_4b and β₄ΔN), +10 mV (α_1B + β_4a, β₄ΔNΔC), or +15 mV (α_1B + β_4aΔC and β_4bΔC) after a 20 sec conditioning prepulse to voltages ranging from −80 to +10 mV (A) or −100 to +10 mV (B). Barium (5 mm) was the charge carrier for both α_1A and α_1B. Data points represent the means of the normalized data at a given membrane potential. The SEM for each point is shown unless the values were smaller than the symbol. Smooth curves represent a single Boltzmann fit to the averaged data. Values forV_1/2 and k for inactivation of α_1A and α_1B plus α₂δ and each of the six β₄ constructs are grouped according to curve similarities in Table 1.

Fig. 8.

Potential α_1A and β subunit domain interactions as viewed from inside the cell looking out through the pore. Top, α_1A alone. Transmembrane domains I–IV are represented as gray circlesand intracellular domains as white circles. Middle, α_1A + β_4b. The β_4b subunitA–E domains are shown as black circlessuperimposed on α_1A. Bottom, α_1A + β_4a. The radius of eachcircle was calculated from the spherical volume (V = 4/3 πr³) of each subunit domain, where V = [(0.73 cm³/gm × 10²⁴ų/cm³ × molecular weight)/6.02 × 10²³] and the average molecular weight of an amino acid is 120 Da. For α_1A(BI-2): N terminus, 98 aa; transmembrane domains I–IV, 229–268 aa; I–II linker, 127 aa; II–III linker, 537 aa; III–IV linker, 54 aa; C terminus, 604 aa. For β_4b [nomenclature as in Hanlon et al. (1999)]: A domain, 92 aa; B domain, 61 aa; C domain, 37 aa; D domain, 210 aa; E domain, 144 aa. For β_4a: A domain, 44 aa. Interactions of the β₄ D domain with the α_1A I–II linker (Pragnell et al., 1994) and β₄ E domain with α_1A N and C termini have been well documented (Walker et al., 1998, 1999). Dashed arrow in the bottomdiagramindicates the potential for a conformational change when the β_4a N terminus is substituted for the β_4b N terminus.