G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins

Abstract

1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.

Figure 4

Effects of RGS2 on the G protein modulation of α_1A/QQIEE mutant in the presence of GTPγS A, sample time courses of reinhibition are shown from cells expressing the α_1A/QQIEE mutant in the presence (control; •) or absence (○) of RGS2 and in the presence of RGS2 plus Gα_i1 subunit (+) with GTPγS in the intracellular solution. Currents were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a 10 ms conditioning prepulse to +100 mV was applied to completely relieve G protein inhibition, the cell was repolarized to −70 mV for a period of 1–427 ms, and a second 4 ms test pulse to +10 mV was applied. Peak currents were normalized to the smallest peak current from the second test pulse, and reinhibition (%) was plotted versus the time interval at −70 mV between the prepulse and second test pulse. B, time constants for I_Ba reinhibition are shown from cells expressing the α_1A/QQIER wild-type or α_1A/QQIEE mutant with (□) or without (control; ▪) RGS2. Time courses of I_Ba reinhibition were fitted with a single exponential, and mean time constants (τ_reinhib) ± s.e.m. were plotted. In some cases the Gα_i1 subunit was coexpressed as indicated. Numbers of cells tested are indicated in parentheses. * and † P < 0·05, Student’s t test. C, sample time courses of release from inhibition are shown from cells expressing the α_1A/QQIEE mutant in the presence (control; •) or absence (○) of RGS2 and in the presence of RGS2 plus Gα_i1 subunit (+) with GTPγS in the intracellular solution. Currents were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a conditioning prepulse to +100 mV for a period of 1–23 ms was applied, the cell was repolarized to −70 mV for a period of 10 ms, and a second 4 ms test pulse to +10 mV was applied. Peak currents were normalized to the largest peak current from the second test pulse, and recovery from inhibition (%) was plotted versus the time interval at +100 mV. D, time constants for I_Ba release from inhibition are shown from cells expressing the α_1A/QQIER wild-type or α_1A/QQIEE mutant with (□) or without (control; ▪) RGS2. Time courses of I_Ba release from inhibition were fitted with a single exponential, and mean time constants (τ_{release from inhib}) ± s.e.m. were plotted. In some cases a Gα_i1 subunit was coexpressed as indicated. Numbers of cells tested are indicated in parentheses. * and † P < 0·05, Student’s t test. E, relative means (± s.e.m.) of facilitation were determined from cells expressing the α_1A/QQIER wild-type or α_1A/QQIEE mutant with (□) or without (control; ▪) RGS2 as indicated. In some cases a G protein α_i1 subunit was coexpressed as indicated. Currents were normalized to the largest peak current of the second test pulse versus the first test pulse from the traces in B and D. Numbers of cells tested are indicated in parentheses. * and † P < 0·05, Student’s t test. F, schematic diagram of the α_1A subunit of the P/Q-type Ca²⁺ channel depicting the G protein-binding QXXER motif between loops I and II. The QXXER motif in the wild-type α_1A subunit QQIER was mutated to QQIEE (Herlitze et al. 1997).

Figure 5

Effect of different Ca²⁺ channel β subunits on the G protein modulation of P/Q-type Ca²⁺ channels in the presence of RGS2 and Gβ₁ subunit A, sample time courses of reinhibition are shown from cells expressing Gβ₁ subunit and Ca²⁺ channel β₃ subunit with (○) or without (control; •) RGS2. Currents were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a 10 ms conditioning prepulse to +100 mV was applied to completely relieve G protein inhibition, the cell was repolarized to −70 mV for a period of 1–427 ms, and a second 4 ms test pulse to +10 mV was applied. Peak currents were normalized to the smallest peak current from the second test pulse, and reinhibition (%) was plotted versus the time interval at −70 mV between the prepulse and second test pulse. B, sample time courses of release from inhibition are shown from cells expressing Gβ₁ subunit and Ca²⁺ channel β₃ subunits with (○) or without (control; •) RGS2. Currents were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a conditioning prepulse to +100 mV for a period of 1–23 ms was applied, the cell was repolarized to −70 mV for a period of 10 ms, and a second 4 ms test pulse to +10 mV was applied. Peak currents were normalized to the largest peak current from the second test pulse, and release from inhibition (%) was plotted versus the time interval at +100 mV. C, time constants for I_Ba reinhibition are shown from control (▪) and RGS2-expressing (□) cells with Gβ₁ subunit and Ca²⁺ channel β_1b, β_2a, β₃ or β₄ subunits as indicated. Time courses of reinhibition were fitted with a single exponential, and mean time constants (τ_reinhib) ± s.e.m. were plotted. Numbers of cells tested are indicated in parentheses. * and † P < 0·05, Student’s t test. D, time constants for I_Ba release from inhibition are shown from control (▪) and RGS2-expressing (□) cells with Gβ₁ subunit and Ca²⁺ channel β_1b, β_2a, β₃ or β₄ subunits as indicated. Time courses of I_Ba release from inhibition were fitted with a single exponential, and mean time constants (τ_{release from inhib}) ± s.e.m. were plotted. Numbers of cells tested are indicated in parentheses. * and † P < 0·05, Student’s t test. E, relative means (± s.e.m.) of facilitation were determined from control (▪) and RGS2-expressing (□) cells with Gβ₁ subunit and Ca²⁺ channel β_1b, β_2a, β₃ or β₄ subunits as indicated. Currents were normalized to the largest peak current of the second test pulse versus the first test pulse from the traces in C and D. Numbers of cells tested are indicated in parentheses. * and † P < 0·05, Student’s t test.

Figure 1

Effects of RGS2 on M2 mAChR-mediated inhibition and recovery from inhibition of P/Q-type Ca²⁺ channels A, sample time courses of M2 mAChR-mediated I_Ba inhibition in a control (top trace) and RGS2- expressing (middle trace) cell are shown following application and removal of 10 μM ACh. Sample time courses (bottom trace) from the control cell (○, grey line) and RGS2-expressing cell (•) were compared following application and removal of 10 μM ACh. ACh was applied for 15 s and then washed out with external Ca²⁺ solution as described in the Methods. Currents were recorded with 10 mM Ba²⁺ as the current carrier through P/Q-type Ca²⁺ channels and were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV every second. B, time constants for M2 mAChR-mediated I_Ba inhibition are shown after ACh application in control (▪) and RGS2-expressing (□) cells. Time courses for I_Ba inhibition were fitted with a single exponential, and mean time constants (τ_inhib) ± s.e.m. were plotted. C, time constants for recovery from M2 mAChR-mediated P/Q-type I_Ba inhibition are shown after ACh removal in control (▪) and RGS2-expressing (□) cells. Time courses for recovery from I_Ba inhibition were fitted with a single exponential, and mean time constants (τ_rec) ± s.e.m. were plotted. D, percentage means (± s.e.m.) of inhibition of I_Ba by 10 μM ACh in control (▪) and RGS2-expressing (□) cells were plotted. Currents were normalized to the largest current inhibition after ACh application compared to currents before ACh application. In B-D, numbers of cells tested are indicated in parentheses; * P < 0·05, Student’s t test (see ‘Data analysis’ for explanation of significance tests in figures).

Figure 2

Effects of RGS2 on facilitation properties of P/Q-type Ca²⁺ channels during transmitter application A, time courses of I_Ba reinhibition during application of 10 μM ACh were determined by eliciting currents with a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a 10 ms conditioning prepulse to +100 mV was applied to relieve G protein inhibition, the cell was repolarized to −70 mV for a period of 1–427 ms, and a second 4 ms test pulse to +10 mV was applied. Sample I_Ba traces illustrating the reinhibition P/Q-type Ca²⁺ channels during 10 μM ACh application in a control and RGS-expressing cell are shown in the upper panel from every other time point. Peak currents were normalized to the smallest peak current from the second test pulse, and reinhibition (%) was plotted versus the time interval at −70 mV between the prepulse and second test pulse. The lower panel shows the time courses of current decrease from a control (▪) and RGS-expressing (□) cell during 10 μM ACh application. B, time courses of I_Ba release from inhibition during application of 10 μM ACh were determined by eliciting currents by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a conditioning prepulse to +100 mV for a period of 1–23 ms was applied, the cell was repolarized to −70 mV for a period of 10 ms, and a second 4 ms test pulse to +10 mV was applied. Sample I_Ba traces illustrating the release from G protein inhibition during application of ACh in a control and RGS-expressing cell are shown in the upper panel from every other time point. Peak currents were normalized to the largest peak current from the second test pulse, and release from inhibition (%) was plotted versus the time interval at +100 mV. The lower panel shows the time courses of current increase from a control (▪) and RGS-expressing (□) cell during application of ACh. C, time constants for M2 mAChR-mediated I_Ba reinhibition are shown during ACh application in control (▪) and RGS2-expressing (□) cells. Time courses of I_Ba reinhibition during 10 μM ACh application in control (▪) and RGS-expressing (□) cells were fitted with a single exponential, and mean time constants (τ_reinhib) ± s.e.m. were plotted. Numbers of cells tested are indicated in parentheses. D, time constants for I_Ba release from M2 mAChR-mediated inhibition are shown during ACh application in control (▪) and RGS2-expressing (□) cells. Time courses of I_Ba release from M2 mAChR-mediated inhibition in control (▪) and RGS-expressing (□) cells were fitted with a single exponential, and mean time constants (τ_{release from inhib}) ± s.e.m. were plotted. Numbers of cells tested are indicated in parentheses. * P < 0·05, Student’s t test. E, relative means (± s.e.m.) of facilitation were plotted from control (▪) and RGS2-expressing (□) cells. Currents were normalized to the largest peak current of the second test pulse versus the first test pulse from the traces in C and D. Numbers of cells tested are indicated in parentheses. * P < 0·05, Student’s t test.

Figure 3

Effects of RGS2 and RGS4 on prepulse facilitation properties of P/Q-type Ca²⁺ channels during coexpression of Gβ₁ subunit or activation of endogenous G proteins by GTPγS A, sample time courses of reinhibition are shown with Gβ₁ subunit (•) and GTPγS in the intracellular solution with (+) or without (○) RGS2. Currents were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a 10 ms conditioning prepulse to +100 mV was applied to completely relieve G protein inhibition, the cell was repolarized to −70 mV for a period of 1–427 ms, and a second 4 ms test pulse to +10 mV was applied. Peak currents were normalized to the smallest peak current from the second test pulse, and reinhibition (%) was plotted versus the time interval at −70 mV between the prepulse and second test pulse. B, sample time courses of release from inhibition are shown with Gβ₁ subunit (•) and GTPγS in the intracellular solution with (+) or without (○) RGS2. Currents were elicited by a 4 ms test pulse to +10 mV from a holding potential of −70 mV. After 1 s a conditioning prepulse to +100 mV for a period of 1–23 ms was applied, the cell was repolarized to −70 mV for a period of 10 ms, and a second 4 ms test pulse to +10 mV was applied. Peak currents were normalized to the largest peak current from the second test pulse, and release from inhibition (%) was plotted versus the time interval at +100 mV. C, time constants for I_Ba reinhibition from control (▪), RGS2- (□) and RGS4-expressing () cells are shown with Gβ₁ subunit or GTPγS in the intracellular solution as indicated. Time courses of I_Ba reinhibition were fitted with a single exponential, and mean time constants (τ_reinhib) ± s.e.m. were plotted. Numbers of cells tested are indicated in parentheses. * P < 0·05, Student’s t test. D, time constants for I_Ba release from inhibition from control cells (▪), and RGS2- (□) and RGS4-expressing () cells, are shown with Gβ₁ subunit or GTPγS in the intracellular solution as indicated. Time courses of I_Ba release from inhibition were fitted with a single exponential, and mean time constants (τ_{release from inhib}) ± s.e.m. were plotted. Numbers of cells tested are indicated in parentheses. * P < 0·05, Student’s t test. E, relative means (± s.e.m.) of facilitation were determined from control cells (▪), and RGS2- (□) and RGS4-expressing () cells, with Gβ₁ subunit or GTPγS in the intracellular solution as indicated. Currents were normalized to the largest peak current of the second test pulse versus the first test pulse from the traces in C and D. Numbers of cells tested are indicated in parentheses. * P < 0·05, Student’s t test.