THE CALCIUM-PUMP OF THE PLASMA-MEMBRANE - MEMBRANE TARGETING, CALCIUM-BINDING SITES, TISSUE-SPECIFIC ISOFORM EXPRESSION

The two Ca2+ pumps of higher eucaryotes are strictly targeted to diffe rent membrane systems: the plasma membrane (PMCA) and the sarco(endo)p lasmic reticulum (SERCA). Chimeric constructs of the two pumps express ed in COS-7 cells have revealed a strong signal for endoplasmic reticu lum retention in the N-terminal cytosolic portion of the SERCA pump: t he signal is contained in a stretch of 28 amino acids that follows the N-terminus. A second, but masked, endoplasmic reticulum retention sig nal is contained in a cytosolic C-tenminal sequence immediately preced ing the calmodulin-binding domain of the Ca2+ pump. Selective mutation s on the SERCA pump have led to the conclusion that 5 conserved residu e membrane domains (TM) 4, 5, and 6 form the Ca2+ channel through the pump protein. A comparative sequence inspection has failed to reveal a ny of these residues in TM5 of the PMCA pump. Mutation of the conserve d residue in TM4 and of two in TM6 abolished the ability of the pump t o form the Ca2+-dependent phosphoenzyme. However, one of the mutations (N979, TM6) also caused retention of the PMCA pump in the reticulum, suggesting structural alterations. Of the four basic isoforms of the p ump, two (1, 4) are ubiquitously expressed, two (2, 3) are essentially brain specific. Isoform 2 has the highest calmodulin affinity. Primar y cultures of cerebellar granule cells from newborn rats did not expre ss isoforms 2 and 3 at plating time. Incubation of the cells in depola rizing concentrations of KCl, which promote Ca2+ influx, promoted the expression of isoforms 2 and 3, and of a brain specific spliced varian t of isoform 1. Incubation of the cells in L-type Ca2+ channel blocker s abolished the upregulation of the pump genes.