Functional roles of gamma2, gamma3 and gamma4, three new Ca2+ channel subunits, in P/Q-type Ca2+ channel expressed in Xenopus oocytes

Abstract

Stargazin or [gamma]2, the product of the gene mutated in the stargazer mouse, is a homologue of the [gamma]1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. [gamma]2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to [gamma]1. Two new members of the [gamma] family expressed in the brain have recently been identified: [gamma]3 and [gamma]4. We have co-expressed, in Xenopus oocytes, the human [gamma]2, [gamma]3 and [gamma]4 subunits with the P/Q-type (Ca(V)2.1) Ca2+ channel and different regulatory subunits ([alpha]2-[delta]; [beta]1, [beta]2, [beta]3 or [beta]4). Subcellular distribution of the [gamma] subunits confirmed their membrane localization. Ba2+ currents, recorded using two-electrode voltage clamp, showed that the effects of the [gamma] subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing [beta] subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both [beta] and [gamma] subunits increased the appearance of the slow-inactivating current. Our data support a role for the [gamma] subunit as a brain Ca2+ channel modulatory subunit and suggest that [beta] and [gamma] subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation.

Figure 1. Schematic representation of all the combinations of subunits used in this study

Between 5 and 35 oocytes were recorded for each combination (except β₄/γ₄, where n = 2 oocytes). ND, not determined.

Figure 2. Membrane expression of the γ₂, γ₃ and γ₄ subunits

Human γ₂, γ₃ or γ₄ subunit cDNA was transiently transfected into tsA-201 cells. Forty-eight hours after transfections, γ subunits were detected by immunofluorescence using an anti-γ_com antibody and a secondary antibody labelled with Cy3.

Figure 3. Modulation of the α_1A Ca²⁺ channel activation and inactivation properties by γ subunits

The different combinations of subunits used are indicated above the graphs, which show a comparison of current-voltage curves and steady-state inactivation curves in the absence and presence of γ subunits. A, current-voltage curves were calculated by measuring the peak current amplitudes recorded during test pulses to between -60 and +40 mV (+10 mV increments; holding potential was -80 mV). B, steady-state inactivation curves were obtained during a test pulse to +10 mV after 2.5 s conditioning depolarizations to between -60 and +30 mV (+10 mV increments; see Methods for equations).

Figure 4. Modulation of inactivation kinetics by γ subunits

Left panel in A, time course of normalized Ba²⁺ currents recorded from cells expressing different subunit combinations evoked by step deplarizations of 2.5 s duration to +10 mV. Right panel in A, non-inactivating current at 2.5 s (I₂/I₁) calculated for oocytes expressing different subunits during a depolarization to +10 mv (holding potential -80 mV). B, the inactivating phase of the current was adjusted to a bi-exponential decay. τ₁, fast time constant of inactivation; τ₂, slow time constant of inactivation; %τ₂, percentage of the slow part of the inactivation. * Significantly different from control (P < 0.05).

Figure 5. Two types of inactivation favoured by expression of the γ subunit

A, steady-state inactivation curves recorded from 20 different oocytes expressing the β₃/no γ (left) or β₃/γ₃ (right) subunits. Note the presence of oocytes with decreased inactivation in the β₃/γ₃ population. B, bar graph showing the probability of appearance of the slow inactivating currents for different combinations of subunits.

Figure 6. Separate analysis of fast- and slow-inactivating currents for β₃-containing channels

Averaged τ₁, τ₂ and %τ₂ calculated from oocytes expressing β₃/no γ (control), β₃/γ₂, β₃/γ₃ and β₃/γ₄ combinations. Left panels (fast β₃), analysis of the fast-inactivating current (I₂/I₁ < 0.2). Right panels (slow β₃), slow-inactivating currents (I₂/I₁ > 0.2).

Figure 7. Lack of direct β/γ interaction in tsA-201 cells

Cells were transfected with the β₁ subunit alone or with γ₂, γ₃ or γ₄ subunit cDNA. Forty-eight hours after transfection, cells were fixed and permeabilized. Localization of the β₁ subunit was done using an anti-β₁ subunit antibody and a secondary antibody coupled to Cy3. Immunofluorescence images show the cytoplasmic localization of the β₁ subunit, even in the presence of membrane-localized γ subunits, suggesting no direct interaction.