Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines

Abstract

L-type calcium channels regulate a diverse array of cellular functions within excitable cells. Of the four molecularly defined subclasses of L-type Ca channels, two are expressed ubiquitously in the mammalian nervous system (Ca(V)1.2alpha(1) and Ca(V)1.3alpha(1)). Despite diversity at the molecular level, neuronal L-type channels are generally assumed to be functionally and pharmacologically similar, i.e., high-voltage activated and highly sensitive to dihydropyridines. We now show that Ca(V)1.3alpha(1) L-type channels activate at membrane potentials approximately 25 mV more hyperpolarized, compared with Ca(V)1.2alpha(1). This unusually negative activation threshold for Ca(V)1.3alpha(1) channels is independent of the specific auxiliary subunits coexpressed, of alternative splicing in domains I-II, IVS3-IVS4, and the C terminus, and of the expression system. The use of high concentrations of extracellular divalent cations has possibly obscured the unique voltage-dependent properties of Ca(V)1.3alpha(1) in certain previous studies. We also demonstrate that Ca(V)1.3alpha(1) channels are pharmacologically distinct from Ca(V)1.2alpha(1). The IC(50) for nimodipine block of Ca(V)1.3alpha(1) L-type calcium channel currents is 2.7 +/- 0.3 microm, a value 20-fold higher than the concentration required to block Ca(V)1.2alpha(1). The relatively low sensitivity of the Ca(V)1.3alpha(1) subunit to inhibition by dihydropyridine is unaffected by alternative splicing in the IVS3-IVS4 linker. Our results suggest that functional and pharmacological criteria used commonly to distinguish among different Ca currents greatly underestimate the biological importance of L-type channels in cells expressing Ca(v)1.3alpha(1).

Fig. 1.

Two classes of L-type Ca channel activate at different voltages. A, Individual current traces measured from Xenopus oocytes transiently expressing Ca_v1.3α₁ (thick traces) and Ca_v1.2α₁ (thin traces), together with Ca_Vβ_1b, using 5 mm barium as the charge carrier. Currents were activated in response to voltage steps as indicated from a holding potential of −80 mV. B, Averaged, peak current–voltage plots for Ca_v1.3α₁ (●) and Ca_v1.2α₁ (○) channels using 5 mm barium as charge carrier. C, D, same as in A and B, except recordings were obtained using 5 mm calcium as the charge carrier. V_1/2 from peak current–voltage plot: with 5 mm barium as the charge carrier, Ca_v1.3α₁ and Ca_Vβ_1b, −36.8 ± 0.8 mV (n = 6); Ca_v1.2α₁ and Ca_Vβ_1b, −8.8 ± 0.8 mV (n = 6); with 5 mm calcium as the charge carrier, −20.0 ± 2.2 mV (n = 4) and 3.3 ± 2.0 mV (n = 4), respectively. Values are mean ± SE.

Fig. 2.

An increase in extracellular divalent cation concentration shifts the voltage dependence of Ca_V1.3α₁ activation to depolarized potentials. A, Individual current traces measured fromXenopus oocytes transiently expressing Ca_V1.3α₁ together with Ca_Vβ_1b, using 2 mm barium (thin trace) or 40 mm barium (thick trace) as the charge carrier. B, C, Ca_V1.3α₁ channel currents recorded with 2 mm barium (○), 5 mm barium (●), 10 mm barium (■), 20 mm barium (▪), and 40 mm barium (▴). Values plotted are averaged peak currents measured from each cell normalized to the maximum peak currents recorded with 5 mm barium (B) and averaged peak currents normalized to maximum peak current recorded at each barium concentration (C). V_1/2 from peak current–voltage plots: 2 mm barium, −42.7 ± 1.6 mV (n = 5); 5 mm barium, −33.8 ± 0.8 mV (n = 5); 10 mm barium, −29.6 ± 1.8 mV (n = 5); 20 mm barium, −24.7 ± 1.2 mV (n = 4); and 40 mm barium, −18.9 ± 1.6 mV (n = 4).

Fig. 3.

Ca_V1.3α₁ channels open at relatively hyperpolarized voltages independent of several factors that potentially affect the voltage dependence of activation. Shown are comparisons of normalized, averaged peak current–voltage plots of Ca_V1.3α₁ coexpressed with Ca_Vβ_1b (●), Ca_Vβ_2a (○), Ca_Vβ₃(▪), or Ca_Vβ₄ (■) subunits (A); with (●) or without (○) exon 32, which encodes a 15-amino acid sequence, PSDSENIPLPTATPG, in domain IVS3–IVS4 coexpressed with Ca_Vβ_1b(B); with (●) or without (○) exon 11, which encodes a 20-amino acid sequence, CWWKRRGAAKTGPSGCRRWG, in loop I–II coexpressed with Ca_Vβ_1b(C); and with exon 42 (●) or exon 42a (○) coexpressed with Ca_Vβ_1b(D), Ca channel currents were recorded with 5 mm barium as the charge carrier. Maximum, peak current amplitudes induced by coexpressing Ca_v1.3α₁with different Ca_Vβ subunits were −1.3 ± 0.1 μA (n = 7; Ca_Vβ_1b), −2.8 ± 0.2 μA (n = 8; Ca_Vβ_2a), −1.0 ± 0.1 μA (n = 7; Ca_Vβ₃), and −1.0 ± 0.5 μA (n = 4; Ca_Vβ₄). Ca_v1.3α₁ alone induced currents with amplitudes of −0.43 ± 0.05 μA (n = 6). Average V_1/2 values were: A, −33.0 ± 1.0 mV (n = 7; Ca_Vβ_1b), −32.1 ± 0.8 mV (n = 8; Ca_Vβ_2a), −28.4 ± 0.5 mV (n = 7; Ca_Vβ₃), and −30.0 ± 1.0 mV (n = 4; Ca_Vβ₄);B, −35.7 ± 0.5 mV (n = 8; Δexon 32) and −31.7 ± 0.7 mV (n = 5; +exon 32); C, −36.1 ± 1.6 mV (n = 8; Δexon 11) and −35.3 ± 0.6 mV (n = 5; +exon 11); and D, −33.9 ± 1.5 mV (n = 3; +exon42) and −32.9 ± 0.4 mV (n = 5; +exon 42a). Values are mean ± SE.

Fig. 4.

Ca_V1.3α₁ channels open at relatively hyperpolarized voltages independent of expression system. Shown are normalized, averaged peak-Ca_V1.3α₁channel current–voltage plots recorded from Xenopusoocytes expressing Ca_V1.3α₁ and Ca_Vβ₃ (5 mm Ba, ▪) and tsA-201 cells expressing Ca_V1.3α₁, Ca_Vβ₃, and Ca_Vα₂δ (5 mm Ba, ●; and 2 mm Ca, ○). Maximum peak currents were −1.0 ± 0.1 μA (n = 7, ▪), −1.2 ± 0.1 nA (n = 3, ●), and −3.3 ± 1.0 nA (n = 4, ○). Average V_1/2 values were −28.4 ± 0.5 mV (n = 7, ▪), −26.8 ± 0.7 mV (n = 3, ●), and −31.0 ± 0.8 mV (n = 4; ○), respectively. Values are mean ± SE.

Fig. 5.

Ca_V1.3α₁ currents were enhanced by Bay K 8644. A, Ca_V1.3α₁ currents in the absence (left) and presence (right) of 1 μm Bay K 8644. Currents were activated from a holding potential of −80 mV. B, Averaged, peak current–voltage plots of Ca_V1.3α₁ channels recorded with 5 mm barium in control (●) and in the presence of 1 μm Bay K 8644 (○). Maximum peak currents were −0.4 ± 0.1 μA (n = 3, ●) and −0.7 ± 0.1 μA (n = 3, ○). Average V_1/2 values were −32.2 ± 1.1 mV in control (n = 3, ●) and −39.0 ± 2.5 mV in the presence of Bay K 8644 (n = 3, ○).

Fig. 6.

Ca_V1.3α₁ currents are partially inhibited by dihydropyridines. A, Ca_V1.3α₁ (top) and Ca_V1.2α₁ (bottom) in the absence (thick traces) and presence (thin traces) of 1 μm nimodipine. Currents were activated by voltage steps as indicated, from a holding potential of −80 mV. B, Comparison of normalized, averaged peak current–voltage plots of Ca_V1.3α₁ and Ca_V1.2α₁ channels in the absence (●, ▪) and presence (○, ■) of 1 μm nimodipine, respectively.C, Peak Ca_V1.3α₁ (light gray) and peak Ca_V1.2α₁ (dark gray) currents remaining in the presence of nimodipine, plotted as percentage of control currents at various test potentials. Values are mean ± SE; n = 6 for Ca_V1.3α₁ and n = 3 for Ca_V1.2α₁.

Fig. 7.

Ca_V1.3α₁ and Ca_V1.2α₁ channels are pharmacologically distinct. A, Dose–response curve of nimodipine inhibition of Ca_V1.3α₁ (●) and Ca_V1.2α₁ (○) channel currents. Data were fit to the Hill equation: (I_contol −I_nimodipine)/I_control= 1/(1 + (IC₅₀/C_nimodipine)^h), where IC₅₀ is the concentration of nimodipine required to inhibit 50% of peak current, and h is the Hill coefficient. Ca_V1.3α₁, IC₅₀ = 2.7 ± 0.3 μm;h = 0.85 ± 0.04 (n = 5); Ca_V1.2α_1, IC₅₀ = 139 ± 12 nm;h = 0.63 ± 0.05 (n = 5). B, Inhibition of Ca_V1.3α₁ (Δexon 32, light gray, n = 8; +exon 32, dark gray, n = 6) and Ca_V1.2α₁ (white,n = 4) by nitrendipine are significantly different at concentrations of 1 and 10 μm. Values are mean ± SE.