Whole-Cell and Single-Channel Analysis of P-Type Calcium Currents in Cerebellar Purkinje Cells of Leaner Mutant Mice

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Abstract
The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tglamutation occurs in a gene encoding the voltage-activated calcium channel α1Asubunit. The α1Asubunit is highly expressed in the cerebellum and is thought to be the pore-forming subunit of P- and Q-type calcium channels. In this study we used both whole-cell and single-channel patch-clamp recordings to examine the functional consequences of the tglamutation on P-type calcium currents. High-voltage-activated (HVA) calcium currents were recorded from acutely dissociated cerebellar Purkinje cells of homozygous leaner (tgla/tgla) and age-matched wild-type (+/+) mice. In whole cell recordings, we observed a marked reduction of peak current density in tgla/tglaPurkinje cells (−35.0 ± 1.8 pA/pF) relative to that in +/+ (−103.1 ± 5.9 pA/pF). The reduced whole-cell current in tgla/tglacells was accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents were predominantly of the ω-agatoxin-IVA–sensitive P-type. Cell-attached patch-clamp recordings revealed no differences in single-channel conductance between the two genotypes and confirmed the presence of three distinct conductance levels (9, 13–14, and 17–18 pS) in cerebellar Purkinje cells. Analysis of patch open-probability (NPo) revealed a threefold reduction in the open-probability of channels in tgla/tglapatches (0.04 ± 0.01) relative to that in +/+ (0.13 ± 0.02), which may account for the reduced whole-cell current in tgla/tglaPurkinje cells. These results suggest that the tglamutation can alter native P-type calcium channels at the single-channel level and that these alterations may contribute to the neuropathology of the leaner phenotype.