A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis

Abstract

Familial hemiplegic migraine (FHM)-causing mutations in the gene encoding the P/Q Ca(2+) channel alpha(1A) subunit (CACNA1A) locate to the pore and voltage sensor regions and normally involve gain-of-channel function. We now report on a mutation identified in the first intracellular loop of CACNA1A (alpha(1A(A454T))) that does not cause FHM but is associated with the absence of sensorimotor symptoms in a migraine with aura pedigree. Alpha(1A(A454T)) channels showed weakened regulation of voltage-dependent steady-state inactivation by Ca(V)beta subunits. More interestingly, A454T mutation suppressed P/Q channel modulation by syntaxin 1A or SNAP-25 and decreased exocytosis. Our findings reveal the importance of I-II loop structural integrity in the functional interaction between P/Q channel and proteins of the vesicle-docking/fusion machinery, and that genetic variation in CACNA1A may be not only a cause but also a modifier of migraine phenotype.

Fig. 1.

Pedigree segregating the A454T mutation and protein location of the amino acid change. (A) Clinical and CACNA1A genetic characterization of a three-generation migraine with aura pedigree. Affected individuals are denoted by solid symbols (squares indicate male family members; circles, female family members; symbols with a slash, members who had died). The type of aura experienced is indicated below. (B) Location of the alanine-to-threonine mutation at position 454 (A454T) in the I-II intracellular loop of the P/Q channel α_1A subunit.

Fig. 2.

A454T lessens modulation of P/Q channel activity by Ca_Vβ subunits. Steady-state inactivation was studied with the voltage protocol shown (A). Typical traces obtained from cells expressing WT or A454T P/Q channels including either the β_2a (B) or the β₃ subunit (C). (D) Steady-state inactivation curves. WTβ_2a (○, n = 11); A454Tβ_2a (•, n = 14); WTβ₃ (△, n = 10), and A454Tβ₃ (▲, n = 9). Average k_inact for all four channel combinations were between –4.38 and –5.42 mV and did not differ statistically (ANOVA, P = 0.55).

Fig. 3.

A454T prevents modulation of P/Q channel activity by syntaxin 1A and SNAP-25. (A) Representative current traces from cells expressing WT (Left) or A454T (Right) P/Q channels in the presence of syntaxin 1A (to compare in the absence of syntaxin 1A see Fig. 2B). Western blot of plasma membrane proteins obtained from HEK 293 cells transfected with CFP-α_1A, Ca_Vβ_2a, and α₂δ P/Q channel subunits in the absence or presence of syntaxin 1A and probed with anti-GFP, syntaxin 1A, and β-actin antibodies. Heterologous expression of P/Q channel subunits does not induce the expression of endogenous syntaxin 1A. Methodological details can be found in SI Methods. (B and C) Steady-state inactivation curves. V_1/2,inact and k_inact values were (in mV): WT (○, n = 11) –3 ± 0.6 and –4.5 ± 0.5; WT + syntaxin 1A (, n = 22) –12.8 ± 0.5 and –5.1 ± 0.4; A454T (•, n = 14) –9.2 ± 0.5 and –4.6 ± 0.5; A454T + syntaxin 1A (, n = 15) –7.8 ± 0.5 and –4.8 ± 0.5. (D) Inhibition of WT and A454T P/Q channels alone (Left) or coexpressed with SNAP-25 (Right) evoked by a 20-ms test pulse to +20 mV following a 30-s prepulse to −80 mV (black trace) or −20 mV (green trace). (E) Average percentage I_Ca inhibition of WT and A454T P/Q channels in the absence or presence of SNAP-25. Ca²⁺ currents obtained as indicated in D were normalized to the current following the −80 mV prepulse. *P < 0.05 vs. WT. (F) Representative current traces from HEK 293 cells expressing WT P/Q channels coexpressed with WT (I-II_WT loop) or A454T (I-II_A545T loop) intracellular loops connecting domains I and II in the absence or presence of syntaxin 1A (stx 1A). Ca²⁺ currents were evoked by a 20-ms test pulse to +20 mV following a 30-s prepulse to −80 mV (black trace) or −20 mV (green trace). (G) Average percentage of WT P/Q I_Ca inhibition obtained under the experimental conditions shown in F (*P < 0.01).

Fig. 4.

Mutation A454T prevents P/Q channel regulation by MPC endogenous syntaxin 1A. (A) Inhibition of WT P/Q channels alone (Left, control) or coexpressed with BTX C (Right) in mouse pheochromocytoma (MPC 9/3L-AH) cells, evaluated as described in Fig. 3. (B) Average percentage I_Ca inhibition for WT P/Q channels expressed alone (control) or with BTX C in MPC 9/3L-AH cells. Ca²⁺ currents were normalized to the current following the −80-mV prepulse. *P < 0.01. (C) Average current evoked by every 10th pulse of a 200-Hz train of 2-ms depolarizations from −80 mV to +20 mV, normalized to the current evoked by the first pulse of the train, obtained from MPC 9/3L-AH cells expressing WT or A454T P/Q channels alone or coexpressed with BTX C, as indicated. (D) Western blot of cell lysates obtained from HEK 293 cells expressing syntaxin 1A alone or with botulinum toxin C (BTX C) and probed with anti-syntaxin 1A and α-tubulin antibodies. Full-length and cleaved syntaxin 1A are detected in the presence of BTX C. Methodological details can be found in SI Methods.

Fig. 5.

Mutation A454T decreases secretion efficiency. (A) Currents from two MPC 9/3L-AH cells expressing either WT (Upper) or A454T channels (Lower) in response to a train of five successive 200-ms depolarizing voltage steps to +20 mV delivered at 20 Hz. Arrows indicate the 0 current level. (B) Capacitance traces, plotted as a function of time, from the same cells showed in A. WT (○) and A454T (•) P/Q-expressing cells were stimulated at 20 Hz as described. (C) Amperometric recording from a MPC 9/3L-AH cell loaded with dopamine during the application of a local puff of a depolarizing high-K⁺ solution. Note the synchronous release of dopamine after the onset of the stimulus. (Inset) Higher time-scale resolution of the amperometric spike indicated with an asterisk. (D) Averaged data for Ca²⁺ influx normalized by the whole-cell capacitance [Q_Ca density (pC/pF)] elicited by the first depolarizing pulse, the first two depolarizing pulses, and all five depolarizing pulses (as indicated) under three different intracellular Ca²⁺-buffering conditions in WT (EGTA 0.1 mM, n = 8; EGTA 1 mM, n = 21; EGTA 5 mM, n = 7) and A454T MPC 9/3L-AH transfected cells (EGTA 0.1 mM, n = 10; EGTA 1 mM, n = 21; EGTA 5 mM, n = 7). (E) Exocytosis [ΔCm (fF)] normalized as a function of Ca²⁺ entry [Q_Ca density (pC/pF)] corresponding to the conditions described in D. *P < 0.05. (F) Averaged data for Ca²⁺ influx normalized by cell size [Q_Ca density (pC/pF)] elicited by the first depolarizing pulse, the first two depolarizing pulses, and all five depolarizing pulses (as indicated) under intermediate intracellular Ca²⁺-buffering conditions (1 mM EGTA) in MPC 9/3L-AH cells transfected with either the human WT (hWT, n = 12) or human A454T (hA454T, n = 7) P/Q channel. (G) Exocytosis [ΔCm (fF)] normalized as a function of Ca²⁺ entry [Q_Ca density (pC/pF)] corresponding to the conditions described in F. *P < 0.05.