Abstract
The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.
Highlights
The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants
We report the raising of these antibodies, the determination of their specificities, and their use to localize the alternatively spliced variants in the cerebellum, frontal cortex, and hippocampal region of rat brain
Nomenclature—In naming the different isoforms of the pump we use names such as rPMCA1a, where the letter r refers to the rat species, PMCA stands for plasma membrane calcium pump, the number 1 refers to the product of the first gene, and the last letter refers to the alternate splicing
Summary
The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. The specific presence of the proteins of the a and b variants of the different isoforms is interesting, since these variants are products of the alternate splicing at site C in the region where most of the regulatory properties of the pump are located [5, 6]. In this paper we present our work on the production of highly sensitive variant-specific polyclonal antibodies that recognize the alternatively spliced variants (called a and b) of the different isoforms of the plasma membrane calcium pump. We report the raising of these antibodies, the determination of their specificities, and their use to localize the alternatively spliced variants in the cerebellum, frontal cortex, and hippocampal region of rat brain. Our evidence indicates that specific proteolysis of isoforms 3b and 4b removes a portion of the carboxyl terminus, leaving only 2b with a fulllength carboxyl terminus
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