Absence of modulation of the expressed calcium channel alpha1G subunit by alpha2delta subunits

Abstract

1. The modulatory action of the alpha2delta subunit on various high-voltage-activated calcium channels has been demonstrated previously. However, very little is known about auxiliary subunit modulation of low-voltage-activated (LVA) calcium channels. We have examined the modulation of the alpha1G subunit corresponding to the neuronal T-type calcium channel by the ubiquitously expressed alpha2delta-1 and brain-specific alpha2delta-3 subunits. 2. The alpha1G subunit was expressed alone or in combination with either the alpha2delta-1 or alpha2delta-3 subunit in human embryonic kidney (HEK 293) cells and whole-cell barium currents were measured. The current density-voltage relationships for peak and sustained current, kinetics of current activation and inactivation, voltage dependence of current inactivation and time course of the recovery from inactivation were analysed for each type of expressed channel. No significant difference was found for any of the examined parameters. 3. These results suggest that the LVA alpha1G channel is not regulated by known auxiliary alpha2delta subunits.

Figure 1. Effect of the α2δ-3 subunit on the voltage dependence of α1C channel activation

A, mean current density-voltage (I-V) relationships for 14 cells transfected with the α1C subunit only (○) and 14 cells cotransfected with α1C and α2δ-3 subunits (▪). The continuous lines are fits of mean data to the modified Boltzmann equation (Table 1). * P < 0.05, ** P < 0.01, *** P < 0.001, vs.α1C subunit only, Student's unpaired t test. B, examples of current records activated by voltage steps from the holding potential to voltages between -20 and +50 mV. ○, α1C channel, cell capacity 41 pF; ▪, α1C +α2δ-3 channel, cell capacity 79 pF. C, τ_act represents the time constant of a monoexponential fit to the ascending part of the inward current activated by voltage steps to the indicated membrane potentials (V_m). ○, α1C channel (n = 14 cells); ▪, α1C +α2δ-3 channel (n = 14 cells). * P < 0.05, ** P < 0.01, vs. α1C subunit only, Student's unpaired t test.

Figure 2. Voltage dependence of current activation

A, mean I-V relationships for 9 cells transfected with the α1G subunit only (□, ▪; a); 10 cells cotransfected with α1G and α2δ-1 subunits (○, •; b); and 11 cells cotransfected with α1G and α2δ-3 subunits (▵, ▴; c). Open symbols represent peak current amplitude, filled symbols represent sustained current amplitude measured at 39 ms. Continuous lines connecting open symbols are fits of mean data to the modified Boltzmann equation (Table 1). B, examples of current records activated by steps to voltages between -50 and +60 mV. □, α1G channel, cell capacity 25 pF; ○, α1G +α2δ-1 channel, cell capacity 58 pF; ▴, α1G +α2δ-3 channel, cell capacity 22 pF. C, I-V relationships for sustained current measured as described in A. Current amplitudes at 39 ms (I_end) were first normalized to the peak current amplitude (I_peak) of each trace and then averaged. Symbols as in B.

Figure 3. Time and voltage dependence of current activation, inactivation and deactivation

A, τ_act represents the time constant of a monoexponential fit to the ascending part of the current activated by voltage steps to the indicated membrane potentials. □, α1G channel (n = 9 cells); ○, α1G +α2δ-1 channel (n = 10 cells); ▴, α1G +α2δ-3 channel (n = 11 cells). B, τ_inact represents the time constant of a monoexponential fit to the descending part of the current activated by voltage steps to the indicated membrane potentials. □, α1G channel (n = 9 cells); ○, α1G +α2δ-1 channel (n = 10 cells); ▴, α1G +α2δ-3 channel (n = 11 cells). C, time constants of current decay evaluated by monoexponential fits of tail currents measured after repolarization to membrane voltages indicated. □, α1G channel (n = 7 cells); ○, α1G +α2δ-1 channel (n = 4 cells); ▴, α1G +α2δ-3 channel (n = 8 cells). D, examples of tail current records. □, α1G channel, cell capacity 33 pF; ○, α1G +α2δ-1 channel, cell capacity 36 pF; ▴, α1G +α2δ-3 channel, cell capacity 37 pF.

Figure 4. Voltage dependence of current availability and time course of recovery from voltage-dependent inactivation

A, steady-state inactivation curves for α1G (□, n = 8 cells), α1G +α2δ-1 (○, n = 9 cells) and α1G +α2δ-3 (▴, n = 10 cells) channels. Continuous lines represent the fits of experimental points to the Boltzmann equation. Resulting V_0.5 (potential of half-maximal current inactivation) and k values were -57.3 ± 0.6 mV and 5.6 ± 0.5 mV for the α1G channel, -55.9 ± 0.6 mV and 4.2 ± 0.4 mV for the α1G +α2δ-1 channel and -57.2 ± 0.5 mV and 4.6 ± 0.9 mV for the α1G +α2δ-3 channel. B, examples of currents measured during the steady-state inactivation protocol. □, α1G channel, cell capacity 25 pF; ○, α1G +α2δ-1 channel, cell capacity 92 pF; ▴, α1G +α2δ-3 channel, cell capacity 22 pF. C, proportion of current available during the second test pulse T2 plotted against recovery time (Δ). The voltage protocol is shown in the inset; V_h, holding potential. Continuous lines represent monoexponential fits to the experimental points. Individual time constants of recovery were 202 ± 17 ms for the α1G channel (□, n = 5 cells), 171 ± 15 ms for the α1G +α2δ-1 channel (○, n = 4 cells) and 206 ± 18 ms for the α1G +α2δ-3 channel (▴, n = 7 cells). D, examples of currents measured during both test pulses T1 and T2. The interval between the two test pulses was omitted. □, α1G channel, cell capacity 38 pF; ○, α1G +α2δ-1 channel, cell capacity 37 pF; ▴, α1G +α2δ-3 channel, cell capacity 23 pF.