Functional roles of γ2, γ3and γ4, three new Ca2+channel subunits, in P/Q‐type Ca2+channel expressed inXenopusoocytes

Stargazin or γ2, a protein selectively expressed in the brain with homology to the skeletal muscle L-type Ca2+ channel γ1 subunit (Bosse et al. 1990; Pragnell et al. 1991) was recently discovered as the product of the gene mutated by insertion of a retrotransposon in the stargazer mouse model of petit mal epilepsy and ataxia (Letts et al. 1998). In skeletal muscle, γ1 can be co-immunoprecipitated from muscle extracts with the pore-forming L-type Ca2+ channel subunit α1S (Flockerzi et al. 1986a, b; Sieber et al. 1987). The γ1 subunit is a 32 kDa, 222 amino acid, glycosylated transmembrane protein with 10 cysteines that are supposed to form disulfide bridges of major importance for the secondary structure of the native protein (Bosse et al. 1990; Pragnell et al. 1991). Co-expression of γ1 with L-type (α1C or CaV1.2) or P/Q-type (α1A or CaV2.1) Ca2+ channels in heterologous systems has evidenced minor modulatory function including slight changes in the activation and inactivation properties and modification in the peak current amplitude of these Ca2+ channels (Mori et al. 1991; Wei et al. 1991; Singer Lahat et al. 1994; Sipos et al. 1995; Eberst et al. 1997; Ren & Hall, 1997; Freise et al. 2000). The stargazin protein displays 38 % similarity (25 % identity) with γ1 and shares the same gene organization and protein secondary structure including four putative transmembrane segments (Letts et al. 1998; Klugbauer et al. 2000). The stargazer mouse has recessively inherited epilepsy and ataxia characterized by spike wave seizures, characteristic of petit mal or absence epilepsy (Chen et al. 1999). Cerebellar disorders have also been noted, including, most prominently, abnormal migration and maturation and reduced brain-derived neurotrophic factor production of cerebellar granule cells. These features are coincindent with the start of ataxia (Qiao et al. 1996, 1998). Immature or reduced synaptic transmission at parallel fibre-Purkinje cell synapses, Golgi cell-granule cell synapses and mossy fibre-granule cell synapses has also been noticed (Chen et al. 1999; Hashimoto et al. 1999), suggesting a crucial role of the γ2/stargazin protein for normal synaptic transmission. The phenotypic similarities between stargazer mice and the other neurological mutant mice, tottering and lethargic, which have defects in genes encoding the α1A and β4 subunits of the P/Q-type Ca2+ channels, together with the homology of gene and protein structures with the γ1 subunit, have led to the hypothesis that the γ2 subunit is indeed a Ca2+ channel subunit and suggest a channel dysfunction as the basis of the disease. Three new members of this family (γ3, γ4 and γ5) have now been isolated in mice and humans (Black & Lennon, 1999; Klugbauer et al. 2000). Co-expression studies of these four γ subunits with the L-type (α1C) or P/Q-type (α1A) Ca2+ channels did not change the kinetics and voltage dependence of activation of the channel (Klugbauer et al. 2000). Analysis of the effects of these γ subunits on channel inactivation has produced contradictory results, and shifts in the steady-state inactivation curves in both the depolarized and the hyperpolarized direction have been reported, depending on the α1 and β subunits (α1A, α1C, β1 or β2) used, as well as the cation used as charge carrier (Ba2+versus Ca2+, Klugbauer et al. 2000). These data suggest that the subunit composition of the channel might be an important determinant of the effects of the γ subunits on channels properties, and therefore on the physiopathological consequences of its absence in the stargazer mice. In this work we have analysed the effects of human γ2, γ3 and γ4 subunits and the modulatory roles of the β1, β2, β3 and β4 subunits on P/Q-type Ca2+ channel properties.