Differential cerebellar GABAA receptor expression in mice with mutations in CaV2.1 (P/Q-type) calcium channels

Abstract

Ataxia is the predominant clinical manifestation of cerebellar dysfunction. Mutations in the human CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels, underlie several neurological disorders, including Episodic Ataxia type 2 and Familial Hemiplegic Migraine type 1 (FHM1). Several mouse mutants exist that harbor mutations in the orthologous Cacna1a gene. The spontaneous Cacna1a mutants Rolling Nagoya (tg(rol)), Tottering (tg) and Leaner (tg(ln)) mice exhibit behavioral motor phenotypes, including ataxia. Transgenic knock-in (KI) mouse strains with the human FHM1 R192Q and S218L missense mutations have been generated. R192Q KI mice are non-ataxic, whereas S218L KI mice display a complex behavioral phenotype that includes cerebellar ataxia. Given the dependence of γ-aminobutyric acid type A (GABAA) receptor subunit functioning on localized calcium currents, and the functional link between GABAergic inhibition and ataxia, we hypothesized that cerebellar GABAA receptor expression is differentially affected in Cacna1a mutants and contributes to the ataxic phenotype. Herein we quantified functional GABAA receptors and pharmacologically dissociated cerebellar GABAA receptors in several Cacna1a mutants. We did not identify differences in the expression of GABAA receptor subunits or in the number of functional GABAA receptors in the non-ataxic R192Q KI strain. In contrast, tg(rol) mice had a ∼15% decrease in the number of functional GABAA receptors, whereas S218L KI mice showed a ∼29% increase. Our data suggest that differential changes in cerebellar GABAA receptor expression profile may contribute to the neurological phenotype of cerebellar ataxia and that targeting GABAA receptors might represent a feasible complementary strategy to treat cerebellar ataxia.

Keywords: Cacna1a; Familial Hemiplegic Migraine; cerebellar ataxia; gamma aminobutyric acid receptor type A; pharmacology.

Figure 1

(A) Representative immunoblot for GABA_AR subunit expression in the cerebellum. (B) Quantification revealed no quantitative differences between overall cerebellar GABA_AR subunit protein levels in FHM1 R192Q KI cerebellum, compared with age-matched wild-type controls. (C) Representative autoradiograms of [³H] ligand binding to horizontal whole-brain sections of wild-type (left) and R192Q KI (right) mice. Non-specific binding was assessed by competitive binding of non-labeled ligands on wild-type cerebellar sections. (D) Autoradiogram densitometry of three separate experiments did not reveal any statistically significant differences between [³H] GABA_AR ligand binding between wild-type and FHM1 R192Q KI cerebella. (E) [³H] Muscimol binding to control and R192Q KI cerebellar membrane homogenates was similar; no differences in B_max and K_D were identified (cf. Table 1). Inset shows Scatchard plot. (F) Similarly, [³H] Ro15-4513 binding was not affected by the R192Q mutation in Cacna1a. Inset shows Scatchard plot. Numerical values for binding parameters to wild-type and R192Q KI cerebellar homogenates are provided in Table 2.

Figure 2

(A) The number of [³H] muscimol binding sites was reduced by 14% in the cerebellum of tg^rol mice, compared with wild-type (n=4; P<0.05). Binding affinity was similar between wild-type and tg^rol cerebella (cf. Table 2). (B) Data was transformed using the Rosenthal method and is presented as a Scatchard plot, showing an increase in B_max (x-axis intercept) with similar K_D (−1 × slope). (C) Representative immunobands for the GABA_AR δ subunit and β-actin. Semi-quantitative analysis revealed a 42.9 ± 4.4% reduction of the GABA_AR δ subunit in tg^rol cerebellum compared with wild-type (n=3, P<0.01).

Figure 3

(A) In contrast to loss-of-function Cacna1a mutants, [³H] muscimol binding was increased in S218L KI cerebellar membranes compared with wild-type (n=4, P<0.05), with no apparent change in K_D. (B) Scatchard plot of the Rosenthal transformation of [³H] muscimol binding to S218L KI cerebellar membranes and wild-type control homogenate. Numerical values for binding parameters to wild-type and S218L KI cerebellar homogenates are provided in Table 2.