John T. Penniston

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 Ca2+, which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca2+ that enters transduction channels is extruded by the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ 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 Ca2+-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca2+ pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca2+pumping in bundles.

Protein kinase C phosphorylates the 'a' forms of plasma membrane Ca2+ pump isoforms 2 and 3 and prevents binding of calmodulin

Phosphorylation by protein kinase C of the “a” and “b” variants of plasma membrane Ca2+pump isoforms 2 and 3 was studied. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the “a” forms were phosphorylated easily with PKC, isoform 2b was phosphorylated only a little, and isoform 3b was not phosphorylated at all. Phosphorylation of isoforms 2a and 3a did not affect their basal activity, but prevented the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicated that phosphorylation prevented activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca2+ levels in those cells where isoforms 2a and 3a are expressed.

CALMODULIN REGULATION OF THE Ca2+ PUMP OF ERYTHROCYTE MEMBRANES*

The Ca2+ pumping ATPase of plasma membranes is an important mechanism by which the intracellular concentration of Ca2+ is controlled. In mature mammalian erythrocytes, and in squid giant axon this ATP requiring Ca2+ pump is the main mechanism by which Ca2+ is removed from the cell. Only the (Ca2-M

Protein Kinase C Phosphorylates Plasma Membrane Ca2+Pump Isoform 4a at Its Calmodulin Binding Domain

+)-ATPase from erythrocytes has been studied extensively, but Ca2+ requiring ATPases with similar properties have been found in plasma membranes from brain,3 luteal c e k 4 ad ip~cytes ,~ neutrophils,6 heart, and pancreatic islet membranes7 Calmodulin, in one of its many roles, interacts with this Ca2+ pump; we will discuss whether this interaction is likely to affect the properties of the pump in vivo, and how calmodulin may regulate the pump. Because of their possible importance in this regulation, it is necessary briefly to discuss the evidence for states of the pump with high and low affinity for Ca2. High and low Ca2+ affinity forms of the Ca2+ ATPase from erythrocyte membranes were reported at an early point in the study of this a~tivity,~, but physiological significance for the low Caz+ affinity form was dismissed as probably being due to denaturation of the ATPase.. The question was reopened by Quist and Roufogalis who showed that the high affinity form could be eliminated by extraction of a factor from the membranes and regenerated by restoration of the extract.I3 They interpreted these results on the basis of two different (Ca+-M

PURIFICATION AND RECONSTRUCTION OF THE CALCIUM, MAGNESIUM ATPASE OF THE ERYTHROCYTE MEMBRANE*

+]-ATPases, while acknowledging the possibility of two types of Caz+ sites as an alternative explanation. Scharff succeeded in preparing erythrocyte membranes in either the low or the high affinity state, depending on the method of preparation, and interpreted this as corresponding to two different states of the Caz+ pump.4 In his subsequent study on a reversible shift between two states of this ATPase he reiterated the position that the two affinities represent two states of the same enzyme. The factor that causes this change in affinity is the same factor that was discussed above as stimulating the ATPase, namely calmodulin.6 A11 of these studies of Ca2+ affinity were done on whole erythrocyte membranes; they were subject to uncertainties about the number of Ca2+ and M&+ ATPases present and to interference due to the presence of other enzymes in the membrane. The present study on the purified Ca pumping ATPase avoids these uncertainties. The work leading up to the purification of the erythrocyte membrane Ca2+ pump to homogeneity has been presented e l s e ~ h e r e . ~ . ~ We do not propose to review that material again here, but rather to present data concerning the

Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin

Phosphorylation by protein kinase C of isoform 4a of the human plasma membrane Ca2+ pump (hPMCA4a) was studied using the COS cell expression system. Phosphorylation of several truncated mutants of hPMCA4a indicated that a single phosphorylation site lies in a region between residues 1113 and 1125. This region is within the calmodulin binding domain and contains a single phosphorylatable residue, serine 1115. Converting this serine to an alanine diminished phosphorylation greatly. Phosphorylation, done in the absence of calmodulin, did not affect subsequent calmodulin binding, but previous binding of calmodulin did inhibit phosphorylation. Moreover, no significant shift in the calmodulin response curve of hPMCA4a was observed when phosphorylation was mimicked by converting serine 1115 to an acidic residue. The calmodulin binding domain of hPMCA4a is much longer than other calmodulin binding domains and has been suggested to consist of two binding lobes interrupted by a short nonbinding region. The findings of this study indicate that serine 1115 is the residue phosphorylated by protein kinase C, and that it lies within the nonbinding region of the calmodulin binding domain.

Plasma Membrane Ca2+ Pumpsa

The ( Ca2+ + Mg) -ATPase of the erythrocyte plasma membranes was discovered by Schatzman and Vincenzi in 1969, and was suggested to be the enzyme responsible for the ejection of CaZ+ from erythrocytes.2 The enzyme can be solubilized from the membrane environment with the help of detergents, particularly Triton X-100.3-5 In both ghosts and solubilized preparations it has been possible to establish that acidic phospholipids possess an activating effect.5* By the use of detergent-solubilized, impure preparations, the A T p a ~ e , ~ and the Ca2+-pumping activity,?, 8 have been reconstituted in vesicular phospholipid membrane systems. The characterization of the enzyme in molecular terms would of course require its purification in a functionally competent state, and this has proven to be an extremely difficult task. This is due ( 1 ) to the fact that the enzyme is contained in the membrane in exceedingly small amounts: (2) to its lability in the solubilized ~ t a t e , ~ and (3) to the fact that, in purification attempts, it co-elutes with Band 3, one of the most abundant protein components of the erythrocyte membrane.3 The purification attempts published in the past have produced fractions with specific activities 20 to 155 times higher than the original membrane material.3~ 4, As judged from the protein patterns in SDS polyacrylamide gel electrophoresis, however, the preparations were in all cases highly heterogenous, or no data on purity was given. that calmodulin stimulates the (Ca2+ + MgZ+)-ATPase in erythrocyte ghosts, and the related finding that the ATPase solubilized by Triton X-100 is still activated by ca1m0dulin.l~ Studies with radioactively labeled calmodulin have shown that it binds to the ATPase with high affinity (Kd about 5 nM) . This interaction of calmodulin with the ATPase suggested that calmodulin-containing affinity columns might be useful in purification of (Ca2+ + Mg2+) -ATPase from impure detergent-solubilized preparations. The first results obtained have been very

Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids.

Isoform 3f of the plasma membrane Ca2+ pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K d of 15 nm) than the corresponding peptide of isoform 2b (K d value was 0.2 nm). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.

Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column.

Plasma membrane Ca2+ pumps are P-type ATPases and therefore are members of the same superfamily as the Na+,K+ ATFase and the sarcoplasmic reticulum Ca2+ pump. Like them, this pump moves a biologically essential ion up its concentration gradient at the expense of hydrolysis of ATP. The pump is important in shaping the Ca2+ signal that controls the biological responses of cells. When cells are simulated by agonists, Ca2+ flows into the cytosol through channels from either intracellular stores or outside the cell. Activation of these channels determines the rising phase of the Ca2+ signal, but the falling phase is controlled by the two Ca2+ pumps, those of the plasma membrane and the sarco/endoplasmic reticulum, and in some cells by the Na+/Ca2+ exchanger.