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

Abstract

The leaner (tgla) mutation in mice results in severe ataxia and an overt neurodegeneration of the cerebellum. Positional cloning has revealed that the tgla mutation occurs in a gene encoding the voltage-activated calcium channel alpha1A subunit. The alpha1A subunit 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 tgla mutation 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/tgla Purkinje cells (-35.0 +/- 1.8 pA/pF) relative to that in +/+ (-103.1 +/- 5.9 pA/pF). The reduced whole-cell current in tgla/tgla cells was accompanied by little to no alteration in the voltage dependence of channel gating. In both genotypes, HVA currents were predominantly of the omega-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/tgla patches (0.04 +/- 0.01) relative to that in +/+ (0.13 +/- 0.02), which may account for the reduced whole-cell current in tgla/tgla Purkinje cells. These results suggest that the tgla mutation 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.

Familial hemiplegic migraine mutations increase Ca 2+ influx through single human Ca V 2.1 channels and decrease maximal Ca V 2.1 current density in neurons

“…Tg la mutants also showed a slower velocity and frequent failure of propagation of CSD and a much larger reduction of excitatory with respect to inhibitory neurotransmitter release. It has been shown that tg and tg la mutations lead to a decreased P͞Q current density in both native cerebellar neurons and in heterologous expression systems, and that the tg la mutation shifts the activation curve of Ca V 2.1 channels to depolarized voltages and reduces their singlechannel open probability (26)(27)(28). These data support the conclusion that a reduced Ca 2ϩ entry through Ca V 2.1 channels reduces neuronal cortical network excitability and makes the cortex more resistant to CSD.…”

Spinocerebellar ataxia type 6 knockin mice develop a progressive neuronal dysfunction with age-dependent accumulation of mutant Ca V 2.1 channels

“…Peak current amplitude during the test pulse was normalized and plotted against conditioning pulse voltage to generate the curves in C. phenotype in the relatively short life span of the mouse (17,(20)(21)(22). The absence of motor phenotype in heterozygous or homozygous Sca6 30Q mice also appears to support the notion that SCA6 is not a simple channelopathy because previously described loss-of-function Cacna1a mutants often show early-onset motor phenotypes associated with distinct alterations of Ca 2ϩ channel properties (23,24).…”

Spinocerebellar Ataxia Type 6 Mutation Alters P-type Calcium Channel Function

“…In addition to SCA6, the ␣1A channel is linked to tottering (tg) and leaner (tg la ) mice. The tg la mice have a severe progressive ataxia, and this mutated channel was reported to exhibit a considerably reduced Ca 2ϩ influx into Purkinje cells (27)(28)(29), whereas tg, which expresses mild phenotype, showed a smaller functional alteration (29). Accordingly, the reduced Ca 2ϩ influx by these mutations may account for their ataxic phenotype.…”