A mutation in CaV2.1 linked to a severe neurodevelopmental disorder impairs channel gating

Abstract

Ca²⁺ flux into axon terminals via P-/Q-type Ca_V2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of Ca_V2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in Ca_V2.1 leading to neuronal Ca²⁺ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in Ca_V2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat Ca_V2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an ∼25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of Ca_V2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca²⁺ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type Ca_V2.1 channels, partially restored Ca²⁺ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca²⁺ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in Ca_V2.1.

Figure 1.

The R1624P mutation causes a profound depolarizing shift in Ca_V2.1 activation. (A) Schematic representation of Ca_V2.1 with Venus fluorescent protein fused to the N terminus (V-Ca_V2.1). The R to P substitution at human residue 1,673 (rat residue 1,624) is indicated by the star. (B) Sequence comparison of the RIV S4 helices of human Ca_V2.1 (GenBank accession no. NM_000068) and rat Ca_V2.1 (GenBank accession no. NM_012918), rat Ca_V2.1 with the R to P substitution at the R5 position and Drosophila Ca_V2.1 (UniProtKB accession no. P91645). Basic residues in positions R0–R5 are shown in green (Ca_V2.1 RIV S4 lacks a basic residue in the nominal R0 position). Nonconserved residues are in blue. (C and D) Ca²⁺ current families recorded from tsA-201 cells expressing V-Ca_V2.1 (C) or V-Ca_V2.1 R1624P (D) with β₄ and α₂δ-1. Currents were elicited by 25-ms step depolarizations from −90 mV to the indicated test potentials; the repolarization voltage was −40 mV. (E) Comparison of V-Ca_V2.1 (●; n = 24) and V-Ca_V2.1 R1624P (○; n = 23) average peak I-V relationships. Currents were evoked at 0.1 Hz by test potentials ranging from −50 mV through +90 mV in 10-mV increments. Amplitudes were normalized by capacitance (pA/pF). I-V curves are plotted according to Eq. 1 with the following respective parameters for V-Ca_V2.1 and V-Ca_V2.1 R1624P: G_max = 690 ± 30 and 560 ± 70 pS/pF, V_1/2 = −0.9 ± 1.0 and 18.2 ± 1.6 mV, V_rev = 71.1 ± 2.6 and 77.7 ± 2.0 mV and k = 3.4 ± 0.8 and 6.2 ± 0.9 mV. (F) Normalized G-V relationships were fit with Eq. 2 with the following respective parameters for V-Ca_V2.1 and V-Ca_V2.1 R1624P: V_1/2 = 2.3 ± 1.2 and 27.8 ± 2.1 mV; k = 4.3 ± 0.8 and 8.2 ± 1.2 mV. Error bars represent ±SEM.

Figure 2.

The R1624P mutation causes a profound depolarizing shift in the activation of Ba²⁺ currents. (A and B) Ba²⁺ current families recorded from tsA-201 cells expressing V-Ca_V2.1 (A) or V-Ca_V2.1 R1624P (B) with β₄ and α₂δ-1. Currents were elicited by 25-ms-step depolarizations from −90 mV to the indicated potentials; the repolarization voltage was −40 mV. (C) Normalized G-V relationships were fit with Eq. 2 with the following respective parameters for V-Ca_V2.1 (●; n = 16) and V-Ca_V2.1 R1624P (○; n = 10): V_1/2 = −8.9 ± 2.3 and 20.8 ± 2.7 mV; k = 6.9 ± 0.7 and 9.7 ± 0.4 mV. (D) Comparison of activation kinetics from tsA-201 cells expressing either V-Ca_V2.1 (●; n = 16) or V-Ca_V2.1 R1624P (○; n = 10). For both channels, the activation phase was fit by Eq. 3. Only test potentials in which a substantial amount of current was present are shown (i.e., ranging from 0 to 30 mV for V-Ca_V2.1 and 10 to 30 mV for V-Ca_V2.1 R1624P). Significant differences by two-tailed, unpaired t test are indicated (*, P < 0.05). Error bars represent ± SEM.

Figure 3.

V-Ca_V2.1 R1624P deactivation kinetics. (A) Ba²⁺ tail currents were elicited by 60-ms repolarizations to test potentials ranging from −90 mV to +10 mV following a 25-ms depolarization from −90 mV to either +30 mV or +10 mV. (B and C) Representative Ba²⁺ tail currents recorded from tsA-201 cells expressing V-Ca_V2.1 (B) or V-Ca_V2.1 R1624P (C) following depolarization from either +10 mV for V-Ca_V2.1 or +30 mV for V-Ca_V2.1 R1624P to the indicated test potentials. (D) Comparison of deactivation kinetics from tsA-201 cells expressing either V-Ca_V2.1 (●; n = 10) or V-Ca_V2.1 R1624P (○; n = 7). For both channels, deactivation was fit by Eq. 4. Significant differences by two-tailed, unpaired t test are indicated (*, P < 0.05).

Figure 4.

Open-state inactivation of Ca_V2.1 is accelerated by the R1624P mutation. (A and B) Representative Ba²⁺ currents recorded from tsA-201 cells expressing V-Ca_V2.1 (A) or V-Ca_V2.1 R1624P (B) near the peak of the I-V relationship for either channel. Currents were elicited by 500-ms depolarizations from −90 mV to +10 mV (A) and +30 mV (B). In both A and B, half-times of decay are indicated by the red dot. (C) Summary of half-times of current decay for V-Ca_V2.1 (●; n = 10) and V-Ca_V2.1 R1624P (○; n = 13). Means and medians are indicated by the dashed and solid black lines of the boxes, respectively. Boxes represent the 25th/75th percentiles. Bars represent the 5th/95th percentiles. A significant difference by two-tailed, unpaired t test is indicated (P < 0.001).

Figure 5.

GV-58 promotes Ca²⁺ flux via both Ca_V2.1 and Ca_V2.1 R1624P. (A) Ca²⁺ currents recorded before (1) and during (2) application of 12.5 µM GV-58 to a tsA-201 cell expressing V-Ca_V2.1, β₄, and α₂δ-1. (B) Time course of step current amplitude before (black filled circles) and during (red filled circles) GV-58 application. Currents were evoked by the protocol illustrated in A at 0.1 Hz. Numbers correspond to traces shown in A. (C) Comparison of τ_deact of V-Ca_V2.1 tail currents evoked by repolarization from +10 mV to −40 mV in the absence (black filled circles) or presence (red filled circles) of GV-58 (n = 8). (D) Normalized G-V relationships before and during application of GV-58. Currents were evoked at 0.1 Hz by test potentials ranging from −50 mV to +90 mV in 10-mV increments. G-V curves are plotted according to Eq. 2 with the respective parameters for control and GV-58: V_1/2 = 1.7 ± 1.3 and −5.7 ± 2.8 mV and k = 4.8 ± 0.4 and 5.6 ± 0.8 mV. (E) Ca²⁺ currents recorded before (1) and during (2) application of 12.5 µM GV-58 to a tsA-201 cell expressing V-Ca_V2.1 R1624P, β₄, and α₂δ-1. (F) The corresponding time course. (G) Comparison of V-Ca_V2.1 R1624P deactivation upon repolarization from +10 mV to −40 mV in the absence (open circles) or presence (red circles) of GV-58 (n = 7). (H) Normalized G-V relationships for V-Ca_V2.1 R1624P before and during application of GV-58. The respective G-V fit parameters for control and GV-58 were V_1/2 = 27.9 ± 2.2 and 10.8 ± 1.9 mV and k = 8.3 ± 0.4 and 8.6 ± 0.5 mV. For reference, the G-V curve for wild-type V-Ca_V2.1 in the absence of GV-58 is shown as a dashed black line. As in Figure 3, tail currents were fit by Eq. 4. Means and medians in C and G are indicated by the dashed and solid lines of the boxes, respectively. Boxes represent the 25th/75th percentiles. Bars represent the 5th/95th percentiles. In D and H, error bars represent ±SEM. Significant differences by two-tailed, paired t test are indicated.

Figure 6.

GV-58 increases Ca²⁺ flux via V-Ca_V2.1 and V-Ca_V2.1 R1624P in response to an action potential–like waveform. (A and D) Ca²⁺ currents recorded before (1) and during (2) application of 12.5 µM GV-58 to tsA-201 cells expressing either V-Ca_V2.1 (A) or V-Ca_V2.1 R1624P (D). In both cases, Ca²⁺ currents were evoked by an action potential–like waveform similar to that used by Bahamonde et al. (2015). Specifically, this stimulus consisted of a 1-ms rising phase from −80 mV to +30 mV followed by a 1-ms decline back to −80 mV (illustrated in A and D, top). (B and C) Comparison of current amplitudes (B) and total charge flux (C) for cells expressing V-Ca_V2.1 in the absence (gray box) or presence (black box) of GV-58 (n = 8). (E and F) Comparison of current amplitudes (E) and total charge flux (F) for cells expressing V-Ca_V2.1 R1624P in the absence (gray box) or presence (black box) of GV-58 (n = 7). Means and medians are indicated by the dashed and solid lines of the boxes, respectively. Boxes represent the 25th/75th percentiles. Bars represent the 5th/95th percentiles. Significant differences by two-tailed, paired t test are indicated.