The Ca2+ channel beta3 subunit differentially modulates G-protein sensitivity of alpha1A and alpha1B Ca2+ channels

Abstract

We have shown previously that the Ca2+ channel beta3 subunit is capable of modulating tonic G-protein inhibition of alpha1A and alpha1B Ca2+ channels expressed in oocytes. Here we determine the modulatory effect of the Ca2+ channel beta3 subunit on M2 muscarinic receptor-activated G-protein inhibition and whether the beta3 subunit modulates the G-protein sensitivity of alpha1A and alpha1B currents equivalently. To compare the relative inhibition by muscarinic activation, we have used successive ACh applications to remove the large tonic inhibition of these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the currents are temporarily relieved of tonic inhibition, they are still capable of undergoing inhibition through the muscarinic pathway. Using this rebound protocol, we demonstrate that the inhibition of peak current amplitude produced by M2 receptor activation is similar for alpha1A and alpha1B calcium currents. However, the contribution of the voltage-dependent component of inhibition, characterized by reduced inhibition at very depolarized voltage steps and the relief of inhibition by depolarizing prepulses, was slightly greater for the alpha1B current than for the alpha1A current. After co-expression of the beta3 subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both alpha1A and alpha1B currents; however, the reduction was significantly greater for alpha1A currents. Additionally, the difference in the voltage dependence of inhibition of alpha1A and alpha1B currents was heightened after co-expression of the Ca2+ channel beta3 subunit. Such differential modulation of sensitivity to G-protein modulation may be important for fine tuning release in neurons that contain both of these Ca2+ channels.

Fig. 1.

Co-expression of the M₂ muscarinic acetylcholine receptor does not modify the tonic inhibition of α_1A and α_1B Ca²⁺currents by a basally active PTX-sensitive G-protein population.A, Depolarizing prepulse protocol used to relieve voltage-dependent G-protein inhibition. B, Currents elicited using this protocol. The protocol consists of a voltage step to +10 mV both before (−PP) and after (+PP) a depolarizing prepulse to +100 mV for 75 msec.C, Mean facilitation (±SEM) of current amplitude using this prepulse protocol for α_1A (left) and α_1B (right) currents. Represented is the facilitation seen in control conditions (Con), after application of 200 μm NEM (NEM), and after incubation with 4 μg/ml PTX (PTX) for 72 hr. The number for each experiment is represented above the respective histogram. For these experiments RNA encoding either α_1A or α_1B was coinjected with RNA encoding the M₂ muscarinic ACh receptor.

Fig. 2.

Removal of ACh results in rebound potentiation of current amplitude for both α_1A and α_1BCa²⁺ currents. A, C, Time course of muscarinic-mediated G-protein inhibition for both α_1A(left) and α_1B (right) currents. The oocyte was held at a potential of −80 mV, and the oocyte was stepped to the test potential of +10 mV every 15 sec. Thecircles represent the peak current amplitude at the test potential of +10 mV. The black lines represent application of 50 μm ACh, whereas the gray lines represent application of 200 μm NEM. Spaces in which no circles are present indicate time periods in which other protocols were instituted. B, D, Current traces for various time points labeled on the graph in A andC. Note the similarity between the current kinetics and amplitude of the rebound current (3) and the current after treatment with NEM (4).

Fig. 3.

Rebound current facilitation is a result of temporary loss of tonic inhibition. A, α_1Bcurrents elicited using the prepulse protocol illustrated in Figure1A. Oocytes were stepped to +10 mV either with (+PP) or without (−PP) a depolarizing voltage step to +100 mV for 75 msec. The prepulse was given 20 msec before the test voltage step. Treatments: application of 50 μm ACh (+ ACH.); after the rebound of current amplitude on removal of ACh (Reb.); and after treatment of the oocyte with NEM (NEM). The representative currents were obtained from different oocytes.B, Summary of mean prepulse facilitation (±SEM) of current amplitude after the various treatments (* denotes significant difference from control; Student’s t test;p < 0.01). C, Summary of the mean potentiation (±SEM) of current by application of NEM (hatched) and by removal of acetylcholine (black). In all cases the control current was taken to be the initial stable current amplitude (trace 1, Fig.2A,C). The rebound potentiation (black) was the peak current amplitude attained after the removal of acetylcholine, both with (right) and without (left) previous incubation with PTX. The mean percent potentiation of the peak current amplitude after application of NEM (hatched) is also shown both with (right) and without (left) PTX pretreatment. D, Plot of current potentiation resulting from NEM application versus potentiation resulting from removal of acetylcholine. The white squares represent oocytes in which both rebound potentiation and NEM-induced potentiation were measured. The black squares represent oocytes that were pretreated with PTX and subsequently exposed to both treatments. The correlation coefficient is 0.87 (p < 0.001). The slope of the linear fit = 1.04.

Fig. 4.

Comparison of muscarinic M₂receptor-induced G-protein inhibition of α_1A and α_1B currents in the absence of tonic inhibition.A, B, Inhibition of current amplitude by reapplication of acetylcholine to oocytes during the rebound phase of current amplitude, which resulted from removal of previous application of acetylcholine. The current during the rebound period is taken as control (Control), whereas the current after application of acetylcholine is represented as + ACh.C, Slowing of the activation kinetics after application of acetylcholine. D, Inhibition of peak current amplitude by activation of the M₂ receptor in the absence of tonic inhibition at various test voltages for α_1A(gray) and α_1B(black) currents. (* represents significant difference in inhibition of α_1A and α_1B currents at a given test pulse voltage; Student’s independent t test;p ≤ 0.01).

Fig. 5.

Voltage-dependent component of M₂-mediated inhibition of α_1A and α_1B in the absence of the Ca²⁺ channel β₃ subunit. A, B, Facilitation of α_1A and α_1B Ca²⁺currents using the prepulse protocol illustrated in Figure1A. C, D, Normalized current versus voltage plots of rebound current (▪), subsequent inhibition by application of 50 μm ACh (•), and facilitation of the inhibited current by a depolarizing prepulse to +100 mV for 75 msec (▵). E, Facilitation of current amplitude by the prepulse voltage protocol illustrated previously, for α_1A(gray) and α_1B(black) currents. Facilitation is measured as the percentage of current inhibited by application of ACh, which is subsequently relieved by the prepulse voltage protocol.

Fig. 6.

Co-expression of the Ca²⁺channel β₃ subunit differentially modulates the inhibition induced by activation of the muscarinic M₂receptor. A, B, Currents elicited by a voltage step to +10 mV from a holding potential of −80 mV before (Control) and after (+ACh) application of 50 μm acetylcholine. C, Slowing of activation kinetics by application of acetylcholine after co-expression of the Ca²⁺ channel β₃subunit (voltage step to +10 mV). D, Inhibition of current amplitude at various test potentials for both α_1Aβ₃ (gray) and α_1Bβ₃ (black) currents. (* represents significant difference between inhibition of α_1Aβ₃ and α_1Bβ₃currents at a given test pulse voltage; Student’s independentt test; p ≤ 0.01)

Fig. 7.

Co-expression of the Ca²⁺channel β₃ subunit differentially modulates the voltage-dependent inhibitory characteristics associated with M₂-induced G-protein inhibition. A, B, Facilitation of α_1Aβ₃ and α_1Bβ₃ Ca²⁺ currents using the prepulse protocol illustrated in Figure1A. C, D, Normalized current versus voltage plots of rebound current (▪), subsequent inhibition by application of 50 μm ACh (•), and facilitation of the inhibited current by a depolarizing prepulse to +100 mV for 75 msec (▵). E, Facilitation of α_1Aβ₃ (gray) and α_1Bβ₃ (black) current amplitude. Facilitation was measured as the percentage of inhibited current, which was reversed by the depolarizing prepulse (* represents significant difference between facilitation of α_1Aβ₃ and α_1Bβ₃currents at a given test pulse voltage; Student’s ttest; p ≤ 0.01).

Fig. 8.

Prepulse facilitation of α_1Aβ₃ and α_1Bβ₃currents as a function of prepulse duration. A, B, Facilitation of current with a prepulse to +100 mV for varying durations, in a representative experiment. The data were fit with a single exponential, and the time constant of this fit is shown. In both cases, facilitation of current was normalized to the values of facilitation seen in the absence of G-protein inhibition, to eliminate any contribution of desensitization produced by these protocols.