Altered calcium homeostasis in cerebellar Purkinje cells of leaner mutant mice.

The leaner (tg(la)) mouse mutation occurs in the gene encoding the voltage-activated Ca(2+) channel alpha(1A) subunit, the pore-forming subunit of P/Q-type Ca(2+) channels. This mutation results in dramatic reductions in P-type Ca(2+) channel function in cerebellar Purkinje neurons of tg(la)/tg(la) mice that could affect intracellular Ca(2+) signaling. We combined whole cell patch-clamp electrophysiology with fura-2 microfluorimetry to examine aspects of Ca(2+) homeostasis in acutely dissociated tg(la)/tg(la) Purkinje cells. There was no difference between resting somatic Ca(2+) concentrations in tg(la)/tg(la) cells and in wild-type (+/+) cells. However, by quantifying the relationship between intracellular Ca(2+) elevations and depolarization-induced Ca(2+) influx, we detected marked alterations in rapid calcium buffering between the two genotypes. Calcium buffering values (ratio of bound/free ions) were significantly reduced in tg(la)/tg(la) (584 +/- 52) Purkinje cells relative to +/+ (1,221 +/- 80) cells. By blocking the endoplasmic reticulum (ER) Ca(2+)-ATPases with thapsigargin, we observed that the ER had a profound impact on rapid Ca(2+) buffering that was also differential between tg(la)/tg(la) and +/+ Purkinje cells. Diminished Ca(2+) uptake by the ER apparently contributes to the reduced buffering ability of mutant cells. This report constitutes one of the few instances in which the ER has been implicated in rapid Ca(2+) buffering. Concomitant with this reduced buffering, in situ hybridization with calbindin D28k and parvalbumin antisense oligonucleotides revealed significant reductions in mRNA levels for these Ca(2+)-binding proteins (CaBPs) in tg(la)/tg(la) Purkinje cells. All of these results suggest that alterations of Ca(2+) homeostasis in tg(la)/tg(la) mouse Purkinje cells may serve as a mechanism whereby reduced P-type Ca(2+) channel function contributes to the mutant phenotype.