The familial hemiplegic migraine mutation R192q reduces G-protein-mediated inhibition of p/q-type (Ca_v2.1) calcium channels expressed in human embryonic kidney cells

Abstract

Familial hemiplegic migraine is associated with at least 13 different missense mutations in the α1A Ca²⁺ channel subunit. Some of these mutations have been shown to affect the biophysical properties of α1A currents. To date, no study has examined the influence of such mutations on the G-protein regulation of channel function. Because G-proteins inhibit movement of the voltage sensor, we examined the effects of the R192Q mutation, which neutralizes a positive charge in the first S4 segment. Human wild-type (WT) or R192Q mutant channels were expressed in human embryonic kidney tsA-201 cells along with dopamine D2 receptors. Application of quinpirole induced fast (≈1 s), pertussis toxin-sensitive inhibition of α1A_WT and α1A_R192Q Ca²⁺ currents, consistent with the activation of a membrane-delimited pathway. α1A_WT Ca²⁺ currents were inhibited by 62.9 ± 0.9 % (n = 27), whereas α1A_R192Q Ca²⁺ currents were inhibited by only 47.9 ± 1.8 % (n = 35; P < 0.001). Concentration-response analysis showed that only the extent of inhibition was affected, with no change in agonist potency (EC₅₀ = 1 nm). Prepulse facilitation, which is a characteristic of voltage-dependent inhibition, was also reduced by the R192Q mutation. However, the kinetics of facilitation and slow activation were not affected, suggesting that G-protein-Ca²⁺ channel affinity was unchanged. These results show that the R192Q mutation reduces the G-protein inhibition of P/Q-type Ca²⁺ channels, probably by altering mechanisms by which Gβγ subunit binding induces a change in channel gating. Altered G-protein modulation and the consequent reduced presynaptic inhibition may contribute to migraine attacks by favouring a persistent state of hyperexcitability.

α1A (Ca_V2.1) is the pore-forming subunit of neuronal voltage-dependent P/Q-type Ca²⁺ channels. Together with N-type (Ca_V2.2) channels, P/Q-type channels are located in presynaptic nerve terminals (Westenbroek et al. 1995, 1998) and are responsible for the Ca²⁺ entry that triggers neurotransmitter release at most synapses (Dunlap et al. 1995). Voltage-dependent G-protein inhibition of N- and P/Q-type Ca²⁺ channels is a ubiquitous and powerful mechanism that underlies the receptor-mediated inhibition of neurotransmitter release (Lipscombe et al. 1989; Koh & Hille, 1997; Wu & Saggau, 1997). This form of inhibition is membrane delimited and develops rapidly, within 1 s of agonist binding to receptors (Jones, 1991; Zhou et al. 1997). Voltage-dependent inhibition is mediated by Gβγ subunits (Herlitze et al. 1996; Ikeda, 1996) through direct binding to multiple regions on α1A, α1B and α1E Ca²⁺ channel subunits (Zhang et al. 1996; DeWaard et al. 1997; Page et al. 1997; Qin et al. 1997; Zamponi et al. 1997; Simen & Miller, 1998, 2000). This shifts gating from a willing to a reluctant mode of opening (Bean, 1989). Inhibited channels exhibit slowed activation kinetics and can be transiently reconverted into willing channels by strong depolarization, leading to the prepulse facilitation of Ca²⁺ currents (Elmslie et al. 1990; Ikeda, 1991). Facilitation might reflect a state-dependent change in the affinity and dissociation of the Gβγ subunit from the channel at depolarized potentials. Facilitation can also be induced by trains of action potentials (Brody et al. 1997; Park & Dunlap, 1998; Currie & Fox, 2002), and relief of G-protein-mediated presynaptic inhibition by electrical firing can contribute to synaptic plasticity (Brody & Yue, 2000).

Mutations in α1A Ca²⁺ channel subunits are responsible for several human disorders including episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6) and familial hemiplegic migraine (FHM). FHM, which presents as ictal hemiparesis, and in some families as ataxia and cerebellar atrophy, is associated with at least 13 different missense mutations in conserved functional domains (Ophoff et al. 1996; Battistini et al. 1999; Carrera et al. 1999; Ducros et al. 1999). The effects of certain mutations on the biophysical properties of α1A have been characterized (see Pietrobon, 2002, for review). In terms of their biophysical effects and their predicted influence on Ca²⁺ entry, FHM mutations can paradoxically lead to both gain- and loss-of-function of recombinant P/Q-type Ca²⁺ channels.

To date, no study has examined the influence of such mutations on the G-protein regulation of channel function. Although no FHM mutation is located in a channel region described to be involved in G-protein inhibition, it was pertinent to examine the effects of the R192Q mutation, which neutralizes a positive charge in the amphipathic helix IS4 (one of the putative voltage sensors; Armstrong & Hille, 1998). Since G-proteins inhibit movement of the voltage sensor (Jones et al. 1997), the R192Q mutation may alter mechanisms by which binding of the Gβγ subunit induces a change in channel gating. Our results show that the extent of G-protein-mediated inhibition and prepulse facilitation of P/Q-type Ca²⁺ channels is reduced significantly by the R192Q mutation.

Methods

Cloning of the human α1A subunit

α1A cDNA was generated by RT-PCR from human cerebellum poly(A)⁺ RNA (no. 6543-1; Clontech, Palo Alto, USA) using proofreading Pfu DNA polymerase (no. 600154; Stratagene, Amsterdam, The Netherlands). First strand synthesis was performed by using the Ready-To-Go T-primed first-strand kit (27-9263-01; Amersham Pharmacia Biotech, Uppsala, Sweden). Six α1A fragments were amplified from first strands with PCR primers flanking suitable native restriction enzyme (RE) sites. PCR fragments were as follows (nucleotide numbers (nt) indicated in parentheses; asterisks indicate artificial RE sites of the out-of-frame 5′ and 3′ termini introduced by PCR): A, SalI*-NotI (nt −5-602); B1, NotI-EcoRI (nt 602-1568); B2, EcoRI-AatII (nt 1568-2875); C1, AatII-EcoRI (nt 2875-4319); C2, EcoRI- HindIII (nt 4319-5274); and D, HindIII-BamHI* (nt 5274-6794). Fragments A, B1 and D were cloned into the vector pBluescript SK⁺ (Stratagene) and fragments B2, C1 and C2 into pSport-1 (Invitrogen, Paisley, UK). The sequence integrity of all PCR fragments was checked by sequence analysis (MWG Biotech, Ebersberg, Germany). The completed α1A cDNA clone corresponds to the α1A-2 splice isoform, as described previously (Hans et al. 1999b; GenBank accession no. AF004883) with the exception of one splice deletion (Val⁷²⁶-Ala⁷²⁸) and was assembled as follows. Fragments B1 and B2 were coligated into the NotI/AatII RE sites of plasmid pSport-1. Subsequently, fragment A was added to the SalI/NotI cut subclone B1-B2 resulting in subclone A-B. Fragments C1 and C2 were coligated into the AatII/HindIII RE sites of plasmid pSport-1. Subsequently, fragment D was added to the HindIII/BamHI RE sites of subclone C1-C2 to gain subclone C-D. Finally, the SalI*-AatII fragment (nt −5-2875) of subclone A-B was coligated with the AatII-BamHI* fragment (nt 2875- 6794) of subclone C-D into the SalI/BamHI RE sites of the mammalian expression plasmid pGFP37 (Grabner et al. 1998) after removal of the cDNA coding for the green fluorescent protein tag via peripheral PstI RE sites.

Construction of mutant α1A_R192Q

Transmutation of the Arg-coding triplet CGG to CAG (nt 574- 576) was performed by using an antisense PCR primer extending from downstream of the NotI site (nt 602) to upstream of the mutational site (nt 612-562). This PCR-generated SalI*-NotI (nt −5-602) fragment replaced the corresponding cassette in the wild-type (WT) α1A cDNA clone. Finally the PCR-modified SalI*-NotI cassette was analysed for sequence integrity and for the existence of the introduced mutation by automated sequencing (MWG Biotech).

Culture and transient transfection of tsA-201 cells

The human embryonic kidney (HEK) tsA-201 cell line was maintained under standard conditions at 37 °C in a humidified atmosphere containing 5 % CO₂. The culture medium contained 90 % Dulbecco's modified Eagle's medium (no. 41966-029; Invitrogen), 10 % fetal bovine serum (no. F9423; Sigma, Dorset, UK) and 50 μg ml⁻¹ gentamicin (no. 15750-037; Invitrogen). Once a week, cells were replated at low density on 60 mm culture dishes and transfected within 3-5 days using CaPO₄ precipitation. Cells were transfected with cDNAs encoding WT or the R192Q mutant human α1A-2 subunit along with human α2/δ (Williams et al. 1992; GenBank accession no. M76559), human β1b (referred to as β2 in Williams et al. 1992; M94173) or rabbit β2a (Hullin et al. 1992; X64297) at 2.5 μg each per dish, the short form of the human D2 dopamine receptor (McAllister et al. 1993; X51645) at 1.25 μg per dish and enhanced green fluorescent protein plasmid (pEGFP, Clontech, Cambridge, UK; U55763) at 0.25 μg per dish. The day following transfection, cells were briefly trypsinized (no. T4049, Sigma) and replated on glass coverslips. Patch-clamp recording was performed 16-36 h later from fluorescent cells.

Patch-clamp recordings

Glass coverslips were transferred to a recording chamber superfused with an external solution containing 145 mm NaCl, 40 mm CaCl₂, 2 mm KCl and 10 mm Hepes (pH 7.4 with NaOH). Patch pipettes were filled with a solution containing 155 mm CsCl, 4 mm Mg-ATP, 0.32 mm Tris-GTP and 10 mm Hepes (pH 7.4 with CsOH), and had a DC resistance of 2-2.5 MΩ. Whole-cell patch-clamp recordings (Hamill et al. 1981) were performed using an Axopatch 200B amplifier linked to a personal computer equipped with pCLAMP 8.0 software. Data were sampled using a Digidata 1322A analog-to-digital board. Residual pipette capacitance was compensated in the cell-attached configuration using the patch-clamp amplifier. Series resistance compensation was adjusted to just below the point of oscillation to minimize voltage errors (typically < 5 mV). All currents were corrected for linear cell capacitance and leakage currents using -P/4 subtraction. The steady holding potential was −90 mV. Quinpirole-induced inhibition was calculated by measuring the reduction of Ca²⁺ current at a time corresponding to the peak of the control current (before drug application). Agonist application was by local superfusion using a macropipette positioned close to the cell under study. Recordings were made at room temperature (20-22 °C).

Curve fits and figures were made with Origin 6.1. Statistical comparisons were made using Student's unpaired two-tailed t test with P < 0.05 considered significant. Error bars in figures represent ±s.e.m.

Results

Expression of WT and R192Q mutant α1A in tsA-201 cells

Figure 1A illustrates representative families of whole-cell Ca²⁺ currents recorded from tsA-201 cells expressing either WT or R192Q mutant α1A Ca²⁺ channels. Maximum current densities resulting from the expression of α1A_R192Q (51.7 ± 6.6 pA pF⁻¹, n = 42) were comparable (P = 0.2) to current densities of the WT α1A channels (40.5 ± 5.0 pA pF⁻¹, n = 32). Consistent with previous biophysical studies (Hans et al. 1999a), the R192Q mutation produced a slight but significant (P = 0.04) negative shift in the voltage dependence of activation without changing the steepness of the activation curve or the apparent reversal potential (Fig. 1B and C). Thus, the half-maximal voltage for activation (V_1/2) was 21.1 ± 1.1 mV (n = 10) for WT and 16.7 ± 1.8 mV (n = 6) for R192Q channels. The maximum current amplitude was elicited for both current types by depolarizing pulses to +30 mV (Fig. 1A and B).

Figure 1. Expression of α1A Ca²⁺ channels in human embryonic kidney (HEK) tsA-201 cells.

A, whole-cell Ca²⁺ currents recorded from HEK tsA-201 cells expressing wild-type (WT) α1A channels (α1A_WT, top) or R192Q mutant channels (α1A_R192Q, bottom) along with α2/δ, β1b Ca²⁺ channel subunits and the D2 receptor. The steady-state holding potential was −90 mV and test potentials ranged from −20 to +70 mV. Scale: 500 pA, 10 ms. B, average current-voltage (I-V) relationships for WT (filled circles) and R192Q (open circles) Ca²⁺ currents (I_Ca). Averaged voltage errors and current densities were 2.7 ± 0.7 mV and 35.5 ± 13.8 pA pF⁻¹, respectively, for α1A_WT (n = 10 cells) and 3.3 ± 1.0 mV and 48.9 ± 18.9 pA pF⁻¹, respectively, for α1A_R192Q (n = 6 cells). Smooth curves correspond to a fit by the following Boltzmann equation: I_(V) =G_(V)(V – V_rev)/(1 + exp[(V_1/2 – V)/k]), where I_(V) is the peak current amplitude at potential V, V_rev is the apparent reversal potential, G_(V) is the conductance for the peak current, V_1/2 is the half-maximal voltage for activation and k is the slope factor of the curve. C, normalized voltage dependence of activation derived from data presented in B. For each cell, V_rev was measured by extrapolation from the I-V relationship and G_(V) was calculated by dividing I_(V) by (V – V_rev). V_1/2 was 21.1 ± 1.1 mV for WT and 16.7 ± 1.8 mV for R192Q. This difference was significant (P = 0.04).

The R192Q mutation reduces the extent of receptor-mediated inhibition

Dopamine receptors and modulation of dopaminergic neurotransmission might be involved in the pathophysiology of migraine (Peroutka, 1997; Fanciullacci et al. 2000). To determine whether G-protein-dependent modulation of α1A is affected by the R192Q mutation, inhibition by the D2 dopamine receptor was examined. D2 receptors couple to pertussis toxin (PTX)-sensitive Gi/Go G-proteins to inhibit Ca²⁺ channels (Lledo et al. 1992; Brown & Seabrook, 1995; Wolfe & Morris, 1999).

Exposure to 1 μm of the D2 receptor agonist quinpirole (Quin) produced a rapid (within 1-2 s) and substantial inhibition of the WT α1A current (Fig. 2A). This inhibition was abolished by incubation with PTX (500 ng ml⁻¹, overnight), confirming that inhibition by D2 receptors is also mediated by Gi/Go-type G-proteins in tsA-201 cells (Fig. 2C). Quin-inhibited currents displayed slowed activation kinetics and a current-voltage relationship that was shifted to positive potentials (see Fig. 2E and Fig. 4B), and were facilitated by prepulses to +100 mV (see Fig. 4). These characteristics suggest that inhibition of α1A Ca²⁺ channels by activated D2 receptors occurred through a membrane-delimited pathway and was, at least in part, voltage dependent (see Discussion). The R192Q mutation induced a significant reduction in the magnitude of inhibition (Fig. 2B and E). Thus, in α1A_WT-expressing cells, Quin reduced the Ca²⁺ current by 62.9 ± 0.9 % (n = 27) and by only 47.9 ± 1.8 % (n = 35, P < 0.001) in α1A_R192Q-expressing cells (Fig. 2C). The difference in the extent of inhibition was not due to a difference in Ca²⁺ current densities between α1A_WT and α1A_R192Q because, as mentioned above, Ca²⁺ current densities were comparable between the two channels and there was no correlation between inhibition and current density (Fig. 2D). This last observation suggests that D2 receptors were expressed at a saturating level.

Figure 2. Comparative receptor-mediated inhibition of α1A_WT and α1A_R192Q Ca²⁺ channels.

A, left, the amplitude of α1A_WTI_Ca, which were evoked at 1 Hz by a step depolarization from −90 to +30 mV, is plotted versus time. Rapid application of 1 μm quinpirole (Quin, indicated by horizontal bar) induced a rapid inhibition of I_Ca. Right, whole-cell I_Ca recorded at the times indicated. Scale: 200 pA, 10 ms. B, inhibition of α1A_R192Q; conditions otherwise as in A. C, averaged inhibition produced by application of 1 μm Quin. ***Significant at P < 0.001. Voltage protocol as in A. In some experiments, cells were incubated overnight with 500 ng ml⁻¹ pertussis toxin (PTX). Number of cells is indicated in parentheses. D, the magnitude of inhibition is not correlated with current density. Inhibition is plotted against initial current density for 32 α1A_WT-expressing cells (filled circles) and 42 α1A_R192Q-expressing cells (open circles). The dashed lines are linear regressions. E, voltage dependence of current inhibition. Average I-V relationships before (filled circles) and during (open circles) exposure to 1 μm Quin in 6 α1A_WT cells (left) and 11 α1A_R192Q cells (right). The steady-state holding potential was −90 mV; test potentials are indicated. Maximum voltage errors were 5.1 ± 1.4 mV for α1A_WT and 2.5 ± 1.9 mV for α1A_R192Q.

Figure 4. Voltage dependence of prepulse facilitation for I_Ca recorded from α1A_WT (left) and α1A_R192Q (right).

A, representative I_Ca recorded during inhibition by 1 μm Quin before (-PP) and after (+PP) a 25 ms depolarizing prepulse to +100 mV. Test depolarizations to the indicated potentials were given 10 ms before and 10 ms after the prepulse. Currents are superposed to facilitate comparison. Scale: 200 pA, 10 ms. B, I-V relationships: for each cell, the I_Ca recorded before and after the prepulse were normalized to the maximal current amplitude before the prepulse and then averaged. The inset histogram represents mean current amplitude facilitation versus test potential (*P < 0.05; **P < 0.01). These data are from 9 α1A_WT-expressing cells and 13 α1A_R192Q-expressing cells. I_Ca in α1A_WT-expressing cells were inhibited by 62.4 ± 2.4 % and those in α1A_R192Q-expressing cells were inhibited by 46.7 ± 3.6 %. This difference was significant (P = 0.004).

Ca²⁺ current inhibition was measured during exposure to a wide range of agonist concentrations (0.1 nm to 1 μm). Dose-response curves were obtained by application of increasing, cumulative concentrations of Quin. Increasing concentrations of agonist caused a progressive inhibition of Ca²⁺ currents (Fig. 3A). Figure 3B shows the steady-state dose-response curves for α1A_WT- and α1A_R192Q-expressing cells. This plot shows that the R192Q mutation reduced inhibition for all concentrations tested. Experimental data were fitted with a logistic equation (see legend to Fig. 3) to determine the EC₅₀ and maximum inhibition. Thus, maximum inhibition was significantly reduced (P = 0.015) from 51.9 ± 1.9 % (n = 5) for α1A_WT to 38.2 ± 3.5 % (n = 8) for α1A_R192Q. There was no change in agonist potency. The logEC₅₀ was −9.1 ± 0.1 (n = 5) for α1A_WT and −8.7 ± 0.2 (n = 8) for α1A_R192Q. Absolute values of maximum inhibition obtained with cumulative application of Quin were slightly lower than those obtained by acute application of a single, saturating concentration (1 μm). However, the R192Q mutation reduced inhibition by the same proportion (26 versus 24 %, respectively).

Figure 3. Concentration-response analysis.

A, representative dose-response experiment in a control cell expressing α1A_WT. Currents were evoked by step depolarizations from −90 to +30 mV at 10 s intervals. Shown are selected I_Ca recorded before (Control) and during exposure to increasing concentrations of Quin. Scale: 500 pA, 10 ms. B, dose- response relationships in α1A_WT- and α1A_R192Q-expressing cells. Error bars represent ± s.e.m. for 5 α1A_WT and 8 α1A_R192Q cells. Experimental points were fitted with the following dose-response equation: I_Ca (% of initial) =A₁+[(A₂ – A₁)/1 + 10^{(logEC50 – Quin) Hill slope)}], where A₂ is the top asymptote (fixed to 100 %) and A₁ is the bottom asymptote, corresponding to the percentage of I_Ca that could not be inhibited. Maximum inhibition was 51.9 ± 1.9 % for α1A_WT and 38.2 ± 3.5 % for α1A_R192Q. LogEC₅₀ was −9.1 ± 0.1 for α1A_WT and −8.7 ± 0.2 for α1A_R192Q. The Hill slope was −1.17 ± 0.07 for α1A_WT and −1.10 ± 0.03 for α1A_R192Q.

The R192Q mutation reduces prepulse facilitation

One key feature of voltage-dependent inhibition is its transient relief by a depolarizing prepulse. This facilitation is coupled to the dissociation of Gβγ subunits as channels undergo conformational changes in response to depolarization. Figure 4A shows WT (left panel) or R192Q (right panel) Ca²⁺ currents recorded during inhibition by 1 μm Quin and elicited by 25 ms test pulses to the indicated potentials 10 ms before and 10 ms after a 25 ms prepulse to +100 mV. For each cell, Ca²⁺ currents recorded before and after the prepulse were normalized to the maximal current amplitude recorded before the prepulse. Normalized current-voltage relationships are shown in Fig. 4B. An alternative representation of the data, which reports the direct ratio of Ca²⁺ current amplitude before and after the prepulse (without normalization), is shown in the inset histogram. Before agonist application, no basal facilitation was observed. For example, at +30 mV, the mean facilitation ratio was 0.92 ± 0.01 (n = 10) for WT and 0.94 ± 0.01 (n = 11) for R192Q channels. In the presence of agonist, the amplitudes of WT and R192Q Ca²⁺ currents were significantly facilitated at test potentials ranging from +10 to +40 mV. However, a clear difference in the magnitude of facilitation was observed. Both channels exhibited maximum facilitation at +20 mV, which was 1.80 ± 0.08 (n = 9) for WT and only 1.45 ± 0.08 for R192Q channels (n = 13; P = 0.01). The biggest reduction in facilitation was observed at +30 mV. At this potential, facilitation was reduced from 1.64 ± 0.08 to 1.24 ± 0.07 (P = 0.002). A significant difference in the facilitation ratio was also measured at +40 mV, with mean values of 1.30 ± 0.06 for WT and 1.09 ± 0.04 for R192Q channels (P = 0.008).

The kinetics of facilitation and slow activation are not affected by the R192Q mutation

The time constants for the onset and decay of facilitation, which reflect the apparent dissociation and association rates of the G-protein, respectively, can be measured by varying the parameters of a standard two-pulse protocol (Elmslie et al. 1990). Figure 5 illustrates the decay and development of prepulse facilitation in experiments performed using the protocols illustrated. The decay of facilitation was monitored by plotting the ratio of current amplitudes measured before and after the prepulse as a function of the variable interval (Δt) between the conditioning prepulse and the second test pulse (Fig. 5A). For each cell, the decay of facilitation was fitted by a single exponential to obtain a time constant (τ-decay). Facilitation decayed with an averaged time constant of 49.7 ± 4.4 ms for α1A_WT(n = 7) and 48.9 ± 3.9 ms for α1A_R192Q(n = 7). To monitor the onset of facilitation, the ratio of current amplitudes measured before and after the prepulse was plotted as a function of the conditioning pulse duration (Fig. 5B). Facilitation developed with an averaged time constant (τ-onset) of 4.2 ± 0.6 ms for α1A_WT(n = 7) and 4.1 ± 0.5 ms for α1A_R192Q(n = 8). These results show that when measured at extreme voltages (+100 and −90 mV, respectively), time constants for facilitation and re-inhibition were identical for WT and R192Q channels.

Figure 5. Facilitation decays and develops with a similar time course for α1A_WT and α1A_R192Q.

A, decay of facilitation. Top, representative experiment with an α1A_WT-expressing cell during inhibition of I_Ca by 1 μm Quin. The voltage protocol is illustrated above the traces. Step depolarizations were from −90 to +30 mV. Scale: 200 pA, 50 ms. The interval between the first test pulse, P1, and the prepulse (PP) was 10 ms; the interval between PP and the second test pulse, P2, (Δt) varied from 10 to 290 ms. Successive episodes of this voltage protocol were delivered at 10 s intervals. Bottom, current amplitude facilitation is plotted as a function of Δt. Symbols represent averaged data from 7 α1A_WT-expressing cells and 7 α1A_R192Q-expressing cells. For each cell, facilitation decay was fitted by a single exponential function to obtain a time constant (τ-decay). Averaged values of τ-decay are summarized in the inset histogram. τ-decay was 49.7 ± 4.4 ms for α1A_WT and 48.9 ± 3.9 ms for α1A_R192Q. In this set of experiments, inhibition was 62.3 ± 1.7 % for α1A_WT and 51.6 ± 3.7 % for α1A_R192Q. This difference was significant (P = 0.02). B, development of facilitation. Scale: 200 pA, 25 ms. The prepulse duration varied from 0 to 30 ms. The P1-PP and PP-P2 intervals were fixed to 10 ms. The time constant of facilitation onset (τ-onset) was 4.2 ± 0.6 ms for α1A_WT and 4.1 ± 0.5 ms for α1A_R192Q. In this set of experiments, inhibition was 61.6 ± 2.0 % for α1A_WT and 49.3 ± 3.4 % for α1A_R192Q. This difference was significant (P = 0.003). Legend otherwise as in A.

To confirm this conclusion, we attempted to measure slow activation and deactivation at intermediate voltages, parameters that are also believed to reflect the time-dependent dissociation and re-association of the Gβγ subunit, respectively. Ca²⁺ current kinetics exhibited fast and slow components of activation during agonist exposure, reflecting the kinetics of uninhibited and inhibited channels, respectively. In order to minimize inactivation, short (25 ms) pulses were used in the two-pulse protocol (Fig. 4 and Fig. 5), which may, however, not be suitable for providing a good estimation of the time constant of slow activation. The use of longer pulses was also prohibitive as they dramatically increased inactivation. Instead of β1b, therefore, we used β2a subunit, which slows inactivation (Patil et al. 1998). The basal activation kinetics of WT and R192Q α1A were identical when co-expressed with β1b and were not affected when β2a was co-expressed instead of β1b (data not shown). The R192Q mutation did not affect the time course of inactivation of the Ca²⁺ channel α1 subunit co-expressed with either β1b or β2a (Fig. 6A). The extent of inhibition of WT channels and the effect of the R192Q mutation were also unchanged by the β2a subunit (Fig. 6B). Thus, in the presence of β2a, α1A_WT and α1A_R192Q were inhibited by 60.3 ± 1.6 % (n = 8) and 46.1 ± 2.3 % (n = 9), respectively, values that are similar to those obtained with β1b (62.9 ± 0.9 % for α1A_WT and 47.9 ± 1.8 % for α1A_R192Q). Figure 6C shows Ca²⁺ currents recorded in a cell expressing α1A_WT and β2a, using the protocol illustrated at the top of the trace, with a test pulse duration of 150 ms. Slow activation was measured from control minus Quin-inhibited difference currents in order to minimize contamination of the time constant measurement by current inactivation (Elmslie & Jones, 1994). However, even with the β2a subunit, measurement of the deactivation time constant following the prepulse was still obstructed by inactivation. Slow activation could be resolved reliably for test potentials from +20 to +40 mV. For this range of potentials, no significant difference in slow activation was observed between the WT and the R192Q channels (Fig. 6D).

Figure 6. Slow activation measured from the difference current is identical for WT and R192Q α1A co-expressed with β2a.

A, time course of inactivation. Currents were evoked by step depolarization from −90 to +30 mV and normalized to maximum amplitude. WT (left) or R192Q (right) α1A subunits were co-transfected with either β1b or β2a. Scale: 500 ms. For WT channels, the mean fast inactivation time constant was 246 ± 23 ms (n = 15) in the presence of β1b and 590 ± 67 ms (n = 6) in the presence of β2a. These values were similar for R192Q channels (226 ± 15 ms, n = 11, and 510 ± 74 ms, n = 6, respectively). B, averaged inhibition produced by application of 1 μm Quin for WT and R192Q α1A subunits co-expressed with β1b or β2a. Values for β1b are from Fig. 2C. C, I_Ca recorded from a cell co-transfected with α1A_WT and β2a using the protocol illustrated on top of the trace with a prepulse to +100 mV. The difference current (labelled Cont-Quin) was obtained by subtracting the Quin-inhibited current from the control current and the time constant of slow activation was determined by fitting the difference current with a single exponential. Scale: 200 pA, 50 ms. The averaged time constants of slow activation for WT and R192Q channels are summarized in D. Time constants measured at +20, +30 and +40 mV were 78.1 ± 17.8, 48.2 ± 8.2 and 20.1 ± 2.7 ms, respectively, for WT (n = 8 cells), and 80.8 ± 13.9, 41.1 ± 7.2 and 19.1 ± 2.3 ms, respectively, for R192Q channels (n = 9 cells). Inhibition was 60.3 ± 1.6 % for α1A_WT and 46.1 ± 2.3 % for α1A_R192Q (P = 0.006). Numbers in parentheses in B and D are number of cells.

Taken together, these results indicate that the kinetics of facilitation and slow activation are similar for WT and R192Q channels, suggesting indirectly that the mutation does not affect the binding affinity between α1A and Gβγ. Thus, we reason that the effects of the R192Q mutation occur after the Gβγ subunit has bound to the channel.

Discussion

Our results show that the FHM mutation R192Q significantly reduces the P/Q-type Ca²⁺ channel inhibition produced by PTX-sensitive Gi/o-type G-proteins through the activation of dopamine D2 receptors. These findings are likely to be relevant to other neurotransmitter receptors that are coupled to the same type of G-proteins.

Effects of R192Q mutation on Ca²⁺ current properties and channel expression

Previous studies have investigated changes in the biophysical characteristics of P/Q-type Ca²⁺ channels induced by the introduction of the R192Q and other FHM mutations in α1A subunits. Kraus et al. (1998) introduced the mutations R192Q, T666M, V714A and I1819L into the rabbit α1A (BI-2) and investigated changes in channel properties by expression in Xenopus oocytes. They found that mutations T666M, V714A and I1819L shifted activation towards more negative potentials and modified the rate of recovery from inactivation. The R192Q mutation had no detectable effects in this study. Hans et al. (1999a) introduced the same mutations into the human α1A-2 subunit and investigated their functional consequences after expression in HEK293 cells using a combination of whole-cell and single-channel recording. All four mutations shifted current activation towards more negative potentials, induced a change in the density of functional channels in the membrane and affected their unitary current properties. More specifically, the R192Q mutation increased the whole-cell current density mainly by increasing the expression of functional Ca²⁺ channels, and to a lesser extent by increasing the open probability. In our study, we confirmed a slight (≈5 mV) but significant negative shift in the voltage dependence of channel activation induced by the R192Q mutation. However, we did not observe any significant effect of the mutation on Ca²⁺ current density. This discrepancy with a previous study in HEK293 cells (Hans et al. 1999a) might arise from differences in cell culture conditions (incubation temperature and duration after transfection), the types of β subunits expressed or a slight variation in the α1A-2 sequence (V⁷²⁶EA splice deletion). As reported previously (Kraus et al. 1998; Hans et al. 1999a), we also did not observe any obvious effects of the R192Q mutation on current inactivation with either the β1b or β2a subunit (Fig. 6A). Similarly, the time course of current activation was not affected. Thus, under our experimental conditions, the whole-cell effects of the R192Q mutation seem to be restricted to a slight negative shift in activation, allowing easier interpretation of the differences in G-protein modulation.

G-protein-mediated inhibition of α1A-2

It is well established that P/Q-type Ca²⁺ channels are inhibited by G-proteins. Kinetic slowing and the magnitude of prepulse facilitation depend upon the type of Gβ subtype that induces inhibition (Arnot et al. 2000). The presence of an alternatively spliced Val⁴²¹ residue located within one of the two Gβγ binding sites in the I-II linker was reported to increase μ-opioid receptor-induced inhibition of α1A in Xenopus oocytes (Bourinet et al. 1999). Despite the absence of a Val⁴²¹ residue in the α1A variant used here, marked inhibition (> 60 %) by dopamine D2 receptors was observed. This inhibition was completely blocked by PTX and was thus caused by Gi/o-type G-proteins. Consistent with a previous study (Bourinet et al. 1996), the extent of inhibition of α1A was independent of the co-expressed β subunit (β1b or β2a). We also found that the effect of the R192Q mutation on channel inhibition was independent of the β subunit expressed. D2 receptors were expressed at a saturating level in both α1A_WT- and α1A_R192Q-expressing cells (Fig. 2D), and the decay of prepulse facilitation, which is proportional to the concentration of Gβγ dimers, was not affected by the presence of the R192Q mutation (Fig. 5A). These observations rule out the possibility that the reduced G-protein inhibition of the mutant channel is due to a lower level of receptor-activated G-proteins.

D2 receptor-induced inhibition was both voltage dependent and voltage independent, as prepulse facilitation failed to completely reverse the inhibition. In 10 cells expressing WT channels, voltage-dependent and voltage-independent inhibition contributed to 38.1 ± 5.2 and 50.4 ± 2.9 %, respectively, of the total inhibition. The residual component (≈10 %) was due to prepulse-induced inactivation. Interestingly, these relative contributions of voltage-dependent and voltage-independent inhibition were not affected by the R192Q mutation (37.5 ± 7.2 and 51.8 ± 7.5 %, respectively; n = 11). Both components appear to be mediated by Gi/o-type G-proteins, as PTX completely blocked this inhibition (Fig. 2C). Since we did not investigate in detail the origin of the voltage-independent inhibition, it is not clear whether the two components represent different signalling pathways (i.e. Gαi/o versus Gβγ; Kinoshita et al. 2001) or whether there is only partial reversal of a single inhibitory mechanism (i.e. Gβγ-induced inhibition). However, our results demonstrate clearly that the R192Q mutation induces a reduction in facilitation, which is characteristic of voltage-dependent inhibition, but also implies an effect of the mutation on the operationally voltage-independent inhibition, because the relative contribution of the two components was identical in WT and R192Q channels. This latter observation would rather favour the implication of a single functional mechanism.

How does the R192Q mutation reduce G-protein inhibition?

Voltage-dependent inhibition exhibits characteristic gating shifts that are manifested as slowed activation kinetics, reduced inhibition at positive voltages and partial relief from inhibition by a conditioning prepulse (Bean, 1989; Elmslie et al. 1990). These effects have been incorporated into allosteric models in which the channels exhibit two functional gating states (Bean, 1989; Herlitze et al. 2001). In the willing state, channels activate rapidly at relatively negative membrane potentials. In the reluctant state, activation is slower and requires stronger depolarization. A prolonged first latency to single-channel opening after G-protein activation accounts for the alterations observed in whole-cell current (Patil et al. 1996) and G-proteins act to retard voltage-sensor activation and the transduction of voltage-sensor movement to channel opening (Jones et al. 1997). In a recent study (Zhong et al. 2001), the functional properties of two isoforms of rat brain N-type Ca²⁺ channels (Ca_V2.2a and Ca_V2.2b) that differ in their susceptibility to G-protein modulation were investigated. It was proposed that the reluctant state is independent of G-protein activation but depends upon the channel structure and the presence of a negatively charged amino acid at position 177 in segment IS3, which may interact electrostatically with a positive charge in the S4 segment and trap the voltage sensor in an inward, not-activated position. G-protein βγ subunits may impede the outward gating movement of the S4 voltage sensor and produce reluctant channels by a similar molecular mechanism (Zhong et al. 2001). We can speculate that the mutation R192Q, by neutralizing a positive charge in the IS4 segment, might modify electrostatic interactions and reduce the effectiveness of G-protein-induced charge trapping, resulting in attenuated inhibition and facilitation.

The onset and decay of prepulse facilitation during Gβγ-induced inhibition can be represented by a simple two-state model (Scheme 1; Currie & Fox, 1997; Zhou et al. 1997; Arnot et al. 2000).

Scheme 1.

When measured at extreme voltages (+100 and −90 mV), the time constants for facilitation and re-inhibition were identical for WT and R192Q channels. These measurements reflect at best the off-rate for the open channel and the on-rate for the closed channel. However, the presence of a voltage-independent inhibition in our experiments could be interpreted by the fact that some G-proteins remain bound at +100 mV, suggesting that the on-rate of the open channel could affect the measured time constant. At −90 mV, the time constant may be significantly affected by the off-rate from the closed channel because of the presence of a rapid component of activation during inhibition, indicating that all of the channels are not initially bound to G-proteins. These measurements thus only provide an estimation of apparent association and dissociation rates. A prediction of the willing-reluctant model (Elmslie et al. 1990; Boland & Bean, 1993; Colecraft et al. 2000) is that the dissociation of the Gβγ subunit at intermediate voltages is dominated by the closed channel off-rate. Our measurement of slow activation at potentials between +20 and +40 mV showed no difference between WT and R192Q channels. Since the R192Q mutation does not alter facilitation onset and decay or slow activation (see Fig. 5 and Fig. 6C and D), we can safely assume that the Gβγ-Ca²⁺ channel affinity is not affected and that the consequences of the mutation are restricted to allosteric modifications that occur after the Gβγ subunits bind to the channel.

Conclusion

This study shows that the R192Q mutation reduces the G-protein inhibition of P/Q-type Ca²⁺ channels, probably by altering the mechanisms by which binding of the Gβγ subunit induces changes in channel gating. Our results are likely to have significant physiological relevance. Altered G-protein inhibition and consequent reduced presynaptic inhibition may contribute to the promotion of a persistent state of hyperexcitability, which is believed to be the basis for susceptibility to migraine attacks (Ptacek, 1998; Welch, 1998). Cortical spreading depression (CSD) is a critical event in the initiation of migraine with aura, and migraine/pain probably results from a dysfunction of brainstem nuclei such as the trigeminal nucleus caudalis, involved in the nociceptive modulation of craniovascular afferents (Goadsby et al. 2002). Recent findings indicate that CSD stimulates the trigeminovascular system, evoking a series of cortical meningeal and brainstem events that are consistent with the development of headache (Bolay et al. 2002). P/Q-type Ca²⁺ channels are involved in the initiation and propagation of CSD (Ayata et al. 2000). It is thus tempting to speculate that hyperexcitability due to a lower susceptibility to G-protein inhibition contributes to migraine pathophysiology by favouring CSD. It is unlikely that all FHM mutations reduce G-protein inhibition, and an explanation of how mutations that are either gain- or loss-of-function produce a similar FHM phenotype is still missing. The development of knock-in mice expressing FHM mutations will allow a direct study of altered synaptic transmission by the mutant P/Q-type Ca²⁺ channels involved in human neurological disorders.