Inhibition of recombinant N-type Ca(V) channels by the gamma 2 subunit involves unfolded protein response (UPR)-dependent and UPR-independent mechanisms

Abstract

Auxiliary gamma subunits are an important component of high-voltage-activated calcium (Ca(V)) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the gamma subunits on neuronal Ca(V) channel activity and expression. We found that coexpression of gamma2 or gamma3 subunits drastically inhibited macroscopic currents through recombinant N-type channels (Ca(V)2.2/beta3/alpha2delta) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by gamma. Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, gamma subunit expression altered the voltage dependence of channel activity. Second, gamma subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal gamma subunits significantly downregulate Ca(V)2.2 channel activity, (2) uncover a role for the gamma2 subunit in Ca(V)2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the gamma subunits, supporting the notion that Ca(V) channels constitute a class of difficult-to-fold proteins.

Figure 1.

The Ca_Vγ₂ and Ca_Vγ₃ auxiliary subunits specifically inhibit current through Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels heterologously expressed in HEK-293 cells. A, Representative current traces from control cells (−γ) or cells coexpressing either the Ca_Vγ₂ or Ca_Vγ₃ subunits or the 4TM protein sarcospan (SS). Currents were elicited by voltage steps to +10 mV from a holding potential of −80 mV. B, Comparison of I_Ba density in individual HEK-293 cells determined as the current amplitude at V_m of +10 mV normalized by cell capacitance (picoamperes per picofarad). The number of tested cells is listed in parentheses. C, Left, Representative traces of endogenous macroscopic K⁺ currents elicited by step changes in membrane potential from −80 to +60 mV in untransfected HEK-293 cells (−γ) or cells expressing the Ca_Vγ subunits. Right, Relationship between I_Ba [measured at its maximum amplitude after the step change in membrane potential (V_m) from a holding potential of −80 mV] and membrane voltage in control (n = 10) and Ca_Vγ-expressing cells (n = 10). D, Left, Representative traces of macroscopic currents through low-threshold channels of the Ca_V3.2 class. Currents were recorded from HEK-293 cells stably expressing Ca_V3.2 channels in the absence (−γ) or presence of the Ca_Vγ subunits in response to depolarizing steps to −30 mV. Right, Averaged peak currents plotted against the test potential under the indicated experimental conditions. Currents were activated from a holding potential of −80 mV by voltage steps applied at 0.05 Hz to various test potentials between −70 and +70 mV (n = 10–18). The asterisks denote significant differences (p < 0.05) compared with control. Error bars indicate SEM.

Figure 2.

The Ca_Vγ₂ subunit inhibits recombinant N-type Ca²⁺ channels in a concentration-dependent manner. A, Representative Ba²⁺ currents recorded (top) and comparison of averaged peak current density (bottom) in HEK-293 cells expressing Ca_V2.2(α_1B)/Ca_Vα₂δ/Ca_Vβ₃ channels. Currents were elicited by depolarizing pulses to +10 mV from a holding potential of −80 mV, in the absence (1:0) and the presence of different concentrations of Ca_Vγ₂ cDNA in the transfection mixture. B, I–V relationships for peak I_Ba density through Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels in control cells (−γ₂) and cells coexpressing the Ca_Vγ₂ cDNA in molar ratios ranging from 1:0.1 to 1:1.4 in relation to the Ca_V2.2 subunit (n = 12–14). Error bars indicate SEM, and the asterisks denote significant differences (p < 0.05) compared with control.

Figure 3.

The Ca_Vγ₂ subunit regulates whole-cell conductance. A, Voltage dependence of activation of recombinant N-type [Ca_V2.2(α_1B)/Ca_Vα₂δ/Ca_Vβ₃] channels in control cells (−γ₂) or cells cotransfected with different concentrations of the cDNA encoding the Ca_Vγ₂ subunit. Conductance data were derived from Figure 2B using the expression: G = I/(V_m − V_rev), where I is current, G is conductance, V_m is the test potential, and V_rev is the extrapolated reversal potential. The mean data were fitted with Boltzmann functions of the form G = G_max/(1 + exp[(V_m − V_1/2)/k]⁻¹), where G_max is maximum conductance, V_m is the test potential, V_1/2 is the potential for half-maximal activation of G_max, and k is a slope factor. B, Comparison of the mean half-activation voltage (V_1/2) for Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels alone (−γ₂) or coexpressed with the Ca_Vγ₂ subunit (n = 12–14). Error bars indicate SEM, and the asterisks denote significant differences (p < 0.05) compared with control.

Figure 4.

Ca_Vγ₂ regulates Ca_V2.2(α_1B)/Ca_Vα₂δ/Ca_Vβ₃ channel steady-state inactivation. A, Voltage dependence of steady-state inactivation of N-type recombinant channels in the absence and presence of Ca_Vγ₂. Currents were recorded after conditioning pulses of 2 s duration, applied from a holding potential of −80 mV in 10 mV steps between −100 and +40 mV, followed by a 140 ms test pulse to +10 mV. Data are plotted against the conditioning potentials (n = 6–12). The mean data were fitted with a Boltzmann function of the following form: I_Ba = I_max/(1 + exp[(V_m − V_1/2)/k]), where the current amplitude I_Ba has decreased to a half-amplitude at V_1/2 with an e-fold change over k mV. B, Comparison of the mean half-inactivation voltage (V_1/2) for Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels alone (−γ₂) or coexpressed with the Ca_Vγ₂ subunit (n = 6–12). The asterisks denote significant differences (p < 0.05) compared with control. Error bars indicate SEM.

Figure 5.

Regulation of Ca_V2.2 channel currents by Ca_Vγ₂ is not dependent on the presence of the Ca_Vβ auxiliary subunits. A, Representative current traces from control cells [HEK-293 cells expressing Ca_V2.2(α_1B)/α₂δ channels in absence of the Ca_Vβ subunit] and cells coexpressing either the Ca_Vγ₂ or the Ca_Vγ₃ subunits. Currents were elicited by voltage steps to +10 mV from a holding potential of −80 mV. Bottom, Comparison of I_Ba density in HEK-293 cells determined as the current amplitude at V_m of +10 mV normalized by cell capacitance (picoamperes per picofarad). B, I_Ba density determined in cells cotransfected with Ca_V2.2/β₃ and varying molar ratios of the cDNA for the Ca_Vα₂δ subunit in absence (black bars) and presence of Ca_Vγ₂ (gray bars). The number of recorded cells is listed in parentheses. The asterisks denote significant differences (p < 0.05) compared with control.

Figure 6.

Regulation of Ca_V2.2 channel currents by Ca_Vγ₂ is not dependent on the presence of Ca_Vα₂δ. A, Superimposed whole-cell current traces from control cells [HEK-293 cells expressing Ca_V2.2(α_1B)/β₃ channels in the absence of the Ca_Vα₂δ subunit] and cells coexpressing either the Ca_Vγ₂ or the Ca_Vγ₃ subunits. Bottom, Comparison of I_Ba density in HEK-293 cells determined at V_m of +10 mV. B, I_Ba density determined in cells cotransfected with Ca_V2.2/α₂δ and varying molar ratios of the cDNA for Ca_Vβ₃ in absence (black bars) and presence of the Ca_Vγ₂ subunit (gray bars). The number of recorded cells is listed in parentheses. The asterisks denote significant differences (p < 0.05) compared with control.

Figure 7.

The actions of the Ca_Vγ₂ and Ca_Vγ₃ subunits are not prevented by inhibition of internalization. A, B, Mean I_Ba density in HEK-293 cells expressing Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃/Ca_Vγ_x subunits before (control) and after application of chloroquine (100 μm) and calpeptin (40 μm) at 37°C for 6 h, respectively. C, Mean I_Ba density in transfected HEK-293 cells before and after application of MG-132 (25 μm at 37°C for 6 h). Control cells (expressing Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels in absence of the Ca_Vγ subunits) were incubated for 6 h in the presence of the drug (MG-132) or the vehicle alone (dmso). D, Mean I_Ba density in nonexpressing Ca_Vγ HEK-293 cells (control), and cells cotransfected with Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃/Ca_Vγ₂ before (+γ₂) and after (+γ₂ E-64) acute exposure to the protease inhibitor E-64 (50 μm at 37°C for 6 h). In all cases, currents were elicited by depolarizing test pulses to +10 mV from a holding potential of −80 mV. Bars denote mean ± SE; the number of recorded cells is indicated in parentheses. The asterisks denote significant differences (p < 0.05) compared with control.

Figure 8.

Mutation at site N48 in the first extracellular loop does not disrupt the function of Ca_Vγ₂. A, Schematic representation of the Ca_Vγ₂ subunit. The position of the asparagine residue substituted is indicated. Bottom, Superimposed representative whole-cell Ba²⁺ current traces through Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ in HEK-293 cells in the absence of the Ca_Vγ subunit (control) and cells expressing the wild-type Ca_Vγ₂ (+γ₂) or the N → Q mutant (N48Q). Currents were generated by applying 140 ms activating pulses at +10 mV from a holding potential of −80 mV. B, Mean I_Ba density in HEK-293 cells as in A. Bars denote mean ± SE; the number of recorded cells is indicated in parentheses. *p < 0.05 compared with control.

Figure 9.

Reduction of macroscopic N-type current after Ca_Vγ₂ transfection may involve alterations in surface expression and interference with the synthesis of new channels. Flow cytometric analysis of the percentage of fluorescent cells and fluorescence intensity in untransfected HEK-293 cells, cells expressing Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels (control), and cells cotransfected with the recombinant N-type channels plus the Ca_Vγ₂ subunit (+γ₂). The histograms denote mean ± SEM values, and the data are representative of two independent experiments of channels probed with an anti-Ca_Vα₂δ affinity-purified antibody. Samples were run in triplicate and 1–2 × 10⁴ cells were measured per run. The asterisks denote significant differences (p < 0.05) compared with control. Ab, Antibody; Ctl, control untransfected cells.

Figure 10.

Extracellular application of genistein, an inhibitor of the UPR, antagonizes of the suppressive effect of Ca_Vγ₂ on the N-type current expressed in the HEK-293 cells. A, Representative Ba²⁺ currents through recombinant N-type channels obtained in HEK-293 cells expressing Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels either in the absence (control) or presence of Ca_Vγ₂ plus genistein (140 μm at 37°C for 6 or 24 h). Control conditions included exposure to ∼0.1% dimethyl sulfoxide (the vehicle for genistein). Currents were evoked by step depolarizations to +10 mV from a holding potential of −80 mV. B, Mean I_Ba density in transfected HEK-293 cells as in A. Statistical significance (p < 0.05) compared with respective control (*) or Ca_Vγ₂ plus vehicle alone (a) is indicated. Ctl, Control; GST, genistein.

Figure 11.

Coexpression of two dominant-negative constructs of the endoplasmic reticulum-resident RNA-dependent kinase (PERK) causes a reversal of current suppression observed after transfection of Ca_Vγ₂. A, Examples of Ba²⁺ currents elicited by 140 ms depolarizing pulses to +10 mV from a holding potential of −80 mV in HEK-293 cells expressing Ca_V2.2/Ca_Vα₂δ/Ca_Vβ₃ channels either in the absence (control) or presence of Ca_Vγ₂ plus wild-type PERK, or the mutant constructs PERK ΔC and PERK K618A. B, Mean I_Ba density in transfected HEK-293 cells as in A. Statistical significance (p < 0.05) compared with control (*) or wild-type PERK (a, b) is indicated.

Figure 12.

Ca_Vγ₂ transfection inhibits Ca²⁺ channel activity in neonatal DRG neurons. A, Representative traces of whole-cell I_Ba through Ca_V channels in cultured DRG neurons transfected with either GFP alone (control) and GFP plus Ca_Vγ₂. The currents were evoked by a single depolarizing step from a holding potential of −80 to +10 mV for 140 ms. B, Comparison of averaged peak current density in DRG cells expressing GFP alone (control) and the exogenous Ca_Vγ₂ subunit. Currents were elicited as in A.