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The four basic isoforms of the plasma membrane Ca 2؉ pump and the two C-terminally truncated spliced variants PMCA4CII(4a) and 3CII(3a) were transiently overexpressed in Chinese hamster ovary cells together with aequorin targeted to the cytosol, the endoplasmic reticulum, and the mitochondria. As PMCA3CII (3a) had not yet been cloned and studied, it was cloned for this study, partially purified, and characterized. At variance with the corresponding truncated variant of PMCA4, which had been studied previously, PMCA3CII(3a) had very high calmodulin affinity. All four basic pump variants influenced the homeostasis of Ca 2؉ in the native intracellular environment. The level of [Ca 2؉ ] in the endoplasmic reticulum and the height of the [Ca 2؉ ] transients generated in the cytosol and in the mitochondria by the emptying of the endoplasmic reticulum store by inositol 1,4,5-trisphosphate were all reduced by the overexpression of the pumps. The effects were much greater with the neuron-specific PMCA2 and PMCA3 than with the ubiquitously expressed isoforms 1 and 4. Unexpectedly, the truncated PMCA3 and PMCA4 were as effective as the full-length variants in influencing the homeostasis of Ca 2؉ in the cytosol and the organelles. In particular, PMCA4CII(4a) was as effective as PMCA4CI(4b), even if its affinity for calmodulin is much lower. The results indicate that the availability of calmodulin may not be critical for the modulation of PMCA pumps in vivo.

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca 2؉ release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca 2؉ pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca 2؉ homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca 2؉ revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca 2؉ transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca 2؉ derangements of different degrees account for the specific cellular phenotypes of the two disorders.