The alpha 1E calcium channel exhibits permeation properties similar to low-voltage-activated calcium channels

Abstract

The physiological and pharmacological properties of the alpha 1E calcium (Ca) channel subtype do not exactly match any of the established categories described for native neuronal Ca currents. Many of the key diagnostic features used to assign cloned Ca channels to their native counterparts, however, are dependent on a number of factors, including cellular environment, beta subunit coexpression, and modulation by second messengers and G-proteins. Here, by examining the intrinsic pore characteristics of a family of transiently expressed neuronal Ca channels, we demonstrate that the permeation properties of alpha 1E closely resemble those described for a subset of low-threshold Ca channels. The alpha 1A (P-/Q-type), alpha 1B (N-type), and alpha 1C (L-type) high-threshold Ca channels all exhibit larger whole-cell currents with barium (Ba) as the charge carrier as compared with Ca or strontium (Sr). In contrast, macroscopic alpha 1E currents are largest in Sr, followed by Ca and then Ba. The unique permeation properties of alpha 1E are maintained at the single-channel level, are independent of the nature of the expression system, and are not affected by coexpression of alpha 2 and beta subunits. Overall, the permeation characteristics of alpha 1E are distinct from those described for R-type currents and share some similarities with native low-threshold Ca channels.

Fig. 1.

Comparison of macroscopic currents carried by 5 mm Ba, Ca, or Sr. Current traces obtained at the peak of the I–V relations for α_1A, α_1B, α_1C, and α_1E(coexpressed with α₂ and β_1b) are presented with their respectiveI–V relations. I–Vrelations were fitted as described in Materials and Methods. Note that Ba produces the largest currents through α_1A, α_1B, and α_1C, but the smallest currents through α_1E channels. Also note the pronounced Ca-dependent inactivation for α_1C.

Fig. 2.

Summary of the conductance properties of the different channels. Apparent maximum slope conductances obtained from fits to individual I–V relations are compared ina. Values are presented in the form of conductance ratios (G_ion/G_Ba).b, The peak current values from the same experiments, normalized to those seen in Ba (I_max(ion)/I_max(Ba)). Note the different permeation profile of the α_1E channel. Error bars are SE based on 5–14 determinations.

Fig. 3.

Comparison of macroscopic Ba, Ca, and Sr currents through α_1A and α_1E in the absence of ancillary subunits. a, Peak current traces with their respective current–voltage relationships as described in Figure 1 (○, Ba; △, Ca; ▿, Sr). b, Comparison of the maximum slope conductance ratios (G_ion/G_Ba) and peak current ratios (I_max(ion)/I_max(Ba)) in the presence and absence of α₂ and β_1b subunits. Note that the ancillary subunits do not significantly affect the permeation properties of the channels. Error bars are SE on 5–12 determinations.

Fig. 4.

Concentration–conductance relations obtained for the four α₁ subunits coexpressed with β_1b and α₂. The data were normalized to 1 at an ion concentration of 2 mm and superimposed. Note that the conductance depends similarly on both Ba and Ca concentration for each of the Ca channel subtypes. The α_1C channels seem to differ from the other subtypes in that the current saturates at lower concentrations. The data were obtained from fits to macroscopicI–V relations. Error bars indicate SE; thesolid lines are a smooth approximation of the Ba data based on the Hill equation.

Fig. 5.

Dependence of the single-channel conductance on the type of external permeant ion. The histogram in asummarizes the results obtained with the four α₁ subunits coexpressed α₂ and β_1b. The conductance of α_1E is not significantly different with 100 mm Ba, Ca, or Sr as permeant ion, whereas α_1C, α_1A, and α_1B exhibit larger conductances in Ba than in Ca. Error bars indicate SD. b, α_1E and α_1CI–V relations and current traces evoked by a step depolarization from −100 to 0 mV. Solid lines in the current–voltage relations are linear regressions through the data.

Fig. 6.

Comparison of Ba and Ca whole-cell currents of α_1E as expressed in HEK 293 cells.Left, Whole-cell Ba and Ca currents elicited by the indicated test depolarizations from a holding potential of −90 mV. The tail potential is −80 mV. Records have been leak-subtracted by a P/8 algorithm, filtered at 2 kHz (4-pole Bessel filter), and sampled at 10 kHz. Right, Peak current versus test voltage relation. Smooth curves are drawn by eye. Series resistance of 5 MΩ, compensated by 70%; cell capacitance of 34 pF. All recordings at room temperature.