Plasma membrane Ca2+-ATPase isoform 2a is the PMCA of hair bundles.

Mechanoelectrical transduction channels of hair cells allow for the entry of appreciable amounts of Ca(2+), which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca(2+) that enters transduction channels is extruded by the plasma membrane Ca(2+)-ATPase (PMCA), a Ca(2+) pump that is highly concentrated in hair bundles and may be essential for normal hair cell function. Because PMCA isozymes and splice forms are regulated differentially and have distinct biochemical properties, we determined the identity of hair bundle PMCA in frog and rat hair cells. By screening a bullfrog saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones as well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immunoprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes and that PMCA2a is the only PMCA present in hair bundles. This complete segregation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular hair cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cells and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca(2+)-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca(2+) pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca(2+) pumping in bundles.

Deletion of amino acid residues 18-75 inactivates the plasma membrane Ca2+ pump.

A mutant of the plasma membrane Ca2+ pump hPMCA4b(d18-75)(ct120) containing a deletion of the N-terminal amino acid residues 18-75 and lacking the C-terminal 120 amino acid residues was expressed in COS-1 cells. The deletion in the N-terminal region did not significantly affect the level of expression of the Ca2+ pump. Tryptic digestion of the hPMCA4b(d18-75)(ct120) mutant resulted in the appearance of the same fragments obtained by proteolysis of the hPMCA4b(ct120) enzyme, suggesting that deletion of residues 18-75 neither impeded the insertion in the membrane nor extensively affected the folding of the mutant protein. The functional competence of the hPMCA4b(d18-75)(ct120) enzyme was examined by measuring the Ca2+ transport and the Ca2+ ATPase activity of COS-1 cell microsomes expressing the mutant protein. Both tests proved the mutant to be inactive. Under conditions in which hPMCA4b(ct120) becomes phosphorylated, hPMCA4b(d18-75)(ct120) was incapable of reacting with ATP and Ca2+ to form the phosphoenzyme. Taken together these results suggest that the segment of amino acids 18-75 is essential for the activity of the plasma membrane Ca2+ pump.

Molecular cloning of a plasma membrane calcium pump from human osteoblasts.

The osteoblast plays a critical role in bone formation, bone remodelling, bone matrix formation, and matrix calcification. To better understand the process of osteoblast-controlled bone formation, we determined the structure and isoform types of the plasma membrane calcium pump from normal human osteoblasts. A complementary DNA library from normal human osteoblasts was screened for plasma membrane calcium pump clones. Sequencing and analysis of cDNA clones revealed the presence of a 3986 base pair cDNA that encoded a 1220 amino acid protein that was similar to the human plasma membrane calcium pump isoform 1. Polyadenylated RNA from human osteoblast cells contains bands of RNA approximately 5050 and 6750 bases long. Reverse transcription of polyadenylated RNA from human osteoblasts followed by amplification of the RNA-DNA duplex with calcium pump isoform-specific primers revealed the presence of isoforms 1 and 2 of the calcium pump. Isoform 4 was not detected. We conclude that normal adult human osteoblasts contain a plasma membrane calcium pump that is similar to the human plasma membrane calcium pump isoform 1. It is likely that this pump plays an important role in the cell biology of the human osteoblast.

Vitamin D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine.

The basolateral membrane of the enterocyte was previously shown to contain an adenosine triphosphate-dependent calcium pump. Using immunological procedures, the localization of the Ca2+ pump in chick intestine, and the effect of dietary variables on the concentration of the pump, were studied. A monoclonal antibody produced against the human erythrocyte calcium pump was shown to cross-react with a chick intestinal Ca2+ pump epitope. The most intense staining of intestinal tissue, as determined immunohistochemically, occurred at the basolateral membrane of the duodenum, jejunum, ileum, and colon, with minor staining elsewhere. By the Western blotting procedure, vitamin D repletion of vitamin D-deficient chicks was shown to significantly increase the concentration of the Ca2+ pump epitope of duodenal, jejunal, and ileal mucosa by a factor of 2-3. Chicks were also fed diets deficient in calcium or phosphorus, a situation known to result in the stimulation of the synthesis of calbindin-D28k and an enhancement of the efficiency of Ca2+ absorption. Adaptation of the chicks to these deficient diets was verified by an increase in intestinal levels of calbindin-D28k, and is now shown to increase the Ca2+ pump epitope. From these immunological studies, it seems apparent that dietary variables that enhance intestinal Ca2+ absorption also increase the amount of the intestinal basolateral Ca2+ pump.

Multiple divergent mRNAs code for a single human calmodulin.

The isolation of a novel complementary DNA (cDNA) clone coding for human calmodulin (CaM) is reported. Although it encodes a protein indistinguishable from the only known higher vertebrate calmodulin, its nucleotide sequence varies extensively from that of two previously reported human CaM cDNAs (Wawrzynczak and Perham, 1984; SenGupta et al., 1987). Only 82 and 81% identity, respectively, is found between the newly isolated and the two known human mRNAs in their coding regions. No striking homology is present in their noncoding regions. Codon usage in the three CaM mRNAs is also surprisingly divergent. A 2.3-kilobase mRNA corresponding to the newly isolated clone is expressed to varying extents in several human tissues, together with an approximately 0.8-kilobase mRNA species presumably arising from alternative polyadenylation of the same primary transcript. The results indicate that the human genome contains at least three divergent CaM genes that are under selective pressure to encode an identical protein while maintaining maximally divergent nucleotide sequences. Partial characterization of a genomic clone specifying the 3' portion of the newly identified CaM mRNA shows that this gene contains introns at identical positions as the previously characterized bona fide vertebrate CaM genes. Evolutionary implications of the presence of a CaM multigene family are discussed.