KCa channels as therapeutic targets in episodic ataxia type-2

Abstract

Episodic ataxia type-2 (EA2) is an inherited movement disorder caused by mutations in the gene encoding the Ca(v)2.1alpha1 subunit of the P/Q-type voltage-gated calcium channel that result in an overall reduction in the P/Q-type calcium current. A consequence of these mutations is loss of precision of pacemaking in cerebellar Purkinje cells. This diminished precision reduces the information encoded by Purkinje cells and is thought to contribute to symptoms associated with this disorder. The loss of the precision of pacemaking in EA2 is the consequence of reduced activation of calcium-dependent potassium channels (K(Ca)) by the smaller calcium current and in vitro can be pharmacologically restored by K(Ca) activators. We used a well established mouse model of EA2, the tottering (tg/tg) mouse, to examine the potential therapeutic utility of one such Food and Drug Administration (FDA)-approved compound, chlorzoxazone (CHZ). Compared with wild-type Purkinje cells, we found the firing rate of tg/tg Purkinje cells in acutely prepared cerebellar slices to be very irregular. Bath application of CHZ successfully restored the precision of pacemaking in a dose-dependent manner. Oral administration of CHZ to tg/tg mice improved their baseline motor performance and reduced the severity, frequency, and duration of episodes of dyskinesia without producing any adverse effects. We propose the use of CHZ, which is currently FDA approved as a muscle relaxant, as a safe and novel treatment of EA2.

Figure 1.

CHZ decreases the irregularity of the intrinsic pacemaking of tg/tg Purkinje cells. A, Raw traces from extracellular recordings from wild-type (+/+; wt) and tg/tg Purkinje cells, with their interspike interval autocorrelograms below. Note the irregular firing of the tg/tg cell. B, CHZ dose dependently decreased the variation of the interspike interval in a tg/tg Purkinje cell whose spontaneous activity was extracellularly recorded. C, Average and individual values (circles) of the coefficient of variation of the interspike intervals in +/+ and tg/tg Purkinje cells. Note the higher values and the wider distribution of coefficient of variation in the mutant cells. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA, followed by Bonferroni's correction); n = 24 cells for tg/tg mice and n = 19 cells for +/+ mice. D, Average and individual (circles) firing rates of the +/+ and tg/tg Purkinje cells shown in C. n.s., Not significant. E, CHZ increased the magnitude of the positive deflection of the spikes recorded extracellularly in tg/tg Purkinje cells. The expanded figure shows an averaged trace of the activity of seven cells, in control and after adding CHZ at 60 μm. F, Average magnitude of the positive deflection in control and after adding CHZ in increasing concentrations. *p < 0.05 (one-way ANOVA, followed by Bonferroni's correction).

Figure 2.

CHZ decreases the irregularity pacemaking through SK channels. A, The precision of pacemaking of a wild-type cerebellar Purkinje cell was monitored by following coefficient of variation of its interspike intervals. Addition of low concentration of cadmium to partially block voltage-gated calcium channels made the firing of the cell irregular. Iberiotoxin at 100 nm was then added to block BK channels. Despite the blockade of BK channels, 30 μm CHZ restored the precision of pacemaking to control conditions. The average coefficient of variation of interspike intervals (B) and firing rate (C) of five Purkinje cells following the experimental procedure described in A. *p < 0.05 (one-way ANOVA, followed by Bonferroni's correction). n.s., Not significant. D, The pacemaking of a Purkinje cell was made irregular with addition of cadmium, and the selective BK channel activator NS1619 was bath applied at a concentration of 30 μm. Although NS1619 was ineffective in restoring the precision of pacemaking, subsequent addition of 30 μm CHZ returned the coefficient of variation of interspike intervals to pre-cadmium levels. The average coefficient of variation of interspike intervals (E) and firing rate (F) of all the Purkinje cells examined following the experimental procedure described in D. *p < 0.05; n.s., Not significant; Ctrl, control; NS, NS1619.

Figure 3.

Oral administration of CHZ improves motor performance in tg/tg mice. A, The performance of tg/tg (n = 10; orange symbols) and +/+ (n = 11; black symbols) mice was evaluated daily using an accelerating rotarod paradigm. CHZ treatment is denoted with the vertical bar. CHZ improved the motor performance of tg/tg but not wild-type (wt) mice. B, Maximum speed achieved before CHZ treatment (average of 5 d), during treatment (CHZ, average of 2 weeks), and after terminating treatment (Post, average of the last 5 d) for +/+ and tg/tg mice. ***p < 0.001 (one-way ANOVA, followed by Bonferroni's correction). n.s., Not significant.

Figure 4.

CHZ treatment reduces the frequency and severity of the stress-induced episodes of dyskinesia in tg/tg mice. A, The attacks of dyskinesia triggered in tg/tg mice as a consequence of the stress associated with the daily rotarod sessions were quantified in 10 min intervals for each session. A score of 0 denotes normal behavior, whereas a score of 5 corresponds to severe dyskinesia. During the treatment with CHZ, the average score was significantly reduced. B, The average severity of dyskinesia before, during, and after treatment with CHZ in the tg/tg mice shown in A. C, The average probability that the rotarod paradigm resulted in an episode of dyskinesia regardless of the severity of the attack. **p < 0.01, ***p < 0.001 (one-way ANOVA, followed by Bonferroni's correction). n.s., Not significant. D, The average severity of all attacks of dyskinesia before, during, and after treatment with CHZ. E, The duration of the attacks of dyskinesia shown in D. ***p < 0.001. n.s., Not significant. F, The average probability that the rotarod paradigm resulted in severe episodes of dyskinesia (dyskinesia score ≥3.5). ***p < 0.001. n.s., Not significant. G, The average of only severe attacks revealed a significant shortening of the duration of such episodes during the treatment with CHZ. The graph depicts the progression in severity from before the rotarod session up to 120 min after. H, Average duration of severe episodes of dyskinesia. ***p < 0.001. n.s., Not significant. I, Summary results on the efficacy of CHZ in increasing the frequency of sessions in which tg/tg mice were free of symptoms (no attack) and correspondingly increasing the frequency of sessions in which they had an episode of dyskinesia with a score <3.5 (mild) or one with a score ≥3.5 (severe).