Current modulation and membrane targeting of the calcium channel alpha1C subunit are independent functions of the beta subunit

Abstract

1. The beta subunits of voltage-sensitive calcium channels facilitate the incorporation of channels into the plasma membrane and modulate calcium currents. In order to determine whether these two effects of the beta subunit are interdependent or independent of each other we studied plasma membrane incorporation of the channel subunits with green fluorescent protein and immunofluorescence labelling, and current modulation with whole-cell and single-channel patch-clamp recordings in transiently transfected human embryonic kidney tsA201 cells. 2. Coexpression of rabbit cardiac muscle alpha1C with rabbit skeletal muscle beta1a, rabbit heart/brain beta2a or rat brain beta3 subunits resulted in the colocalization of alpha1C with beta and in a marked translocation of the channel complexes into the plasma membrane. In parallel, the whole-cell current density and single-channel open probability were increased. Furthermore, the beta2a isoform specifically altered the voltage dependence of current activation and the inactivation kinetics. 3. A single amino acid substitution in the beta subunit interaction domain of alpha1C (alpha1CY467S) disrupted the colocalization and plasma membrane targeting of both subunits without affecting the beta subunit-induced modulation of whole-cell currents and single-channel properties. 4. These results show that the modulation of calcium currents by beta subunits can be explained by beta subunit-induced changes of single-channel properties, but the formation of stable alpha1C-beta complexes and their increased incorporation into the plasma membrane appear not to be necessary for functional modulation.

Figure 1. Changes in subcellular distribution patterns of calcium channel β_1a, β_2a and β₃ subunits coexpressed with α_1C or α_1CY467S

tsA201 cells were transfected with α_1C (wild-type or mutant) and β isoforms alone or in different subunit combinations and the subunits were localized with immunofluorescence or GFP labelling. Left column: when expressed alone α_1C (a) is localized in a tubular network - presumably the endoplasmic reticulum - that is very dense in the perinuclear region (saturated fluorescence) but can be resolved in the cell periphery (arrows); β_1a(d) and β₃ (o) are diffusely localized in the cytoplasm; and β_2a is localized in the plasma membrane (i; arrowhead). Centre column: when wild-type α_1C and β_1a are coexpressed, the two colocalize in clusters in the plasma membrane (b and e; examples indicated by arrows); α_1C and β_2a(g and j) are colocalized throughout the plasma membrane, diffusely (arrowheads) and in clusters (arrows); and α_1C and β₃ are either colocalized in clusters (l and p; arrows) or β₃ remains diffusely distributed in the cytoplasm (m and q). The α_1C+β pairs were double labelled by using GFP-α_1C (b, g, l and m) and anti-β_com (e, j, p and q). Right column: when coexpressed with the mutant α_1CY467S, translocation and colocalization fail and all subunits remain in the same compartments as when expressed individually; α_1CY467S in the endoplasmic reticulum (c, h and n; arrows); β_1a(f) and β₃ (r) in the cytoplasm; and β_2a in the plasma membrane (k; arrowhead). N, nucleus; scale bar, 20 μm.

Figure 2. Comparison of the time course of current traces for α_1C and α_1CY467S expressed without or with β_1a, β_2a or β₃ in tsA201 cells

The membrane potential was stepped for 400 ms from a holding potential of -80 mV to 0, +20 or +40 mV (upper, middle and lower traces, respectively). The magnitude of currents varied greatly between individual cells (see also error bars in Fig. 3A and B). Current inactivation was slowed down on coexpression of α_1C or α_1CY467S with β_2a. There was no apparent difference between currents recorded from α_1C- or α_1CY467S-transfected cells.

Figure 3. Voltage dependence of current activation for wild-type and mutant α_1C expressed with and without β subunits

A, I-V curves obtained by plotting the peak current density from voltage step measurements as shown in Fig. 2 against the test potential. Coexpression of α_1C (•) and α_1CY467S (○) with any one of the β subunits increased the current amplitude severalfold (means and s.e.m.). B, I-V curves for each individual recording were fitted by a modified Boltzmann function (see Methods) and the obtained parameters were subsequently averaged. □, α_1C; , α_1CY467S; error bars are s.e.m., and the number of cells recorded is shown. The specific conductance, g, was increased on β subunit coexpression whereas V₅₀ and k were decreased by β subunit coexpression. The decrease in k on coexpression with each of the β subunits compared with α_1C or α_1CY467S expression alone was significant with P < 0.05. The reversal potential, V_rev, was not significantly altered by the β subunits.

Figure 4. Calcium current kinetics in tsA201 cells transfected with α_1C or α_1CY467S with and without β subunits

□, α_1C; , α_1CY467S. A, activation kinetics of calcium currents are expressed as the time from the onset of a voltage step to +40 mV (holding potential, -80 mV) to 70% of the total rise in current amplitude, τ_70%. β_1a slowed down current activation, β_2a accelerated it and β₃ did not affect activation kinetics. In all cases α_1CY467S was slightly faster than the wild-type α_1C. B, inactivation kinetics were determined by fitting a single exponential function with a time constant τ_inact to the decay phase of the current during a voltage step to +40 mV from a holding potential of -80 mV. For α_1C and α_1CY467S coexpressed with β_2a, test pulse duration was 4 s; for all other conditions test pulse duration was 400 ms. Only β_2a severely slowed down current inactivation, by a factor of ≈10.

Figure 5. Steady-state inactivation in tsA201 cells transfected with α_1C or α_1CY467S with and without β subunits

A, normalized current amplitude during a voltage step from -100 to +40 mV after 10 s prepulses to various potentials with an interpulse interval of 2 ms (•, α_1C; ○, α_1CY467S; means and s.e.m. of individual recordings are shown). There were only minor differences in steady-state inactivation between the different subunit combinations. B, steady-state inactivation curves for each individual recording were fitted by a Boltzmann function: i = 1/(1 + exp((V - V₅₀)/k)), where i is the test pulse current amplitude, V is the prepulse potential, V₅₀ is the prepulse potential of half-maximal inactivation and k is the slope factor, and the obtained parameters were subsequently averaged (□, α_1C; , α_1CY467S; error bars are s.e.m.). The apparent difference in the slopes of the averaged curves shown in the upper left figure in A is not reflected in the slope factor, k, shown in the right panel of B, due to the large variability of V₅₀ for α_1C expression (see error bar in left panel of B). This analysis does not indicate statistically significant differences in steady-state inactivation parameters between cells transfected with different subunit combinations.

Figure 6. Single-channel recordings from tsA201 cells transfected with α_1C (A), α_1C+β_1a (B), α_1CY467S (C) and α_1CY467S +β_1a (D)

Voltage pulses of 200 ms duration from a holding potential of -70 mV to a test potential of 0 mV were applied every 3 s. Coexpression of α_1C or α_1CY467S with the β_1a subunit resulted in an increased frequency of channel openings. The right panel shows open-state dwell time histograms. The mean open times, τ, obtained by fitting single exponential functions to the histograms, were increased by a factor of about 2 by β_1a coexpression.

Figure 7. Determination of the number of channels in a patch, N, and the single-channel open probability, P_o

Open probabilities for individual patches, NP_o, were calculated from recordings from tsA201 cells transfected with α_1C (○), α_1C+β_1a (□), or α_1CY467S +β_1a (▿) and plotted against the number of channels in each patch, N. N values were determined by two different approaches. (i) Open symbols in A represent recordings in which at the end of an experiment 5 μm (±)-Bay K 8644 was added to the bath solution to visualize simultaneous openings of multiple channels in the patch. C shows an example of the short and long openings before and after addition of (±)-Bay K 8644, respectively, in a cell transfected with α_1CY467S and β_1a (upper trace, test pulse duration 200 ms at 0 mV; lower trace, test pulse duration 400 ms at +10 mV). The all-points amplitude histogram in D corresponding to the lower trace in C shows the number of conductance levels. (ii) Filled symbols in A represent recordings for which N values were estimated by finding the best fit of all NP_o values in one experimental group to a linear regression crossing the abscissa at zero. Multiple data points with the same or similar NP_o marked with 1 (▪, ▾, ▾) and 2 (•, •). The slope corresponds to the mean single-channel open probability. B, the P_o values determined experimentally with (±)-Bay K 8644 (□) closely match the values from the fitting procedure (▪; error bars give standard deviation). Coexpression of β_1a resulted in a 7- to 8-fold increase of P_o.