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.

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.

Protein expression of plasma membrane Ca(2+)-ATPases (PMCAs) and the putative Golgi secretory pathway Ca(2+)-ATPase (SPCA) was examined in rat mammary tissue. As lactation started, PMCA protein expression increased dramatically, and this increased expression paralleled milk production. Mammary PMCA was primarily PMCA2b but was approximately 4,000 daltons larger than expected. RT-PCR showed that the primary mammary PMCA2b transcript was alternatively spliced, at splice site A, to include an additional 135 bp, resulting in the insertion of 45 amino acids. This splice form is designated 2bw. PMCA2bw is secreted into milk, associated with the milk fat globule membrane. Therefore, PMCA2bw is located on the apical membrane of the secretory cell. Smaller amounts of PMCA1b and 4b protein were found in mammary tissue. PMCA4b was the major PMCA expressed in developing tissue, and its level declined as lactation started. PMCA1b expression increased moderately during lactation. SPCA protein expression increased 1 wk before parturition and increased further as lactation proceeded. The abundance and cell location of PMCA2b suggest that it is important for macro-Ca(2+) homeostasis in lactating tissue. The pattern of expression and abundance of SPCA suggest that it is a candidate for the Golgi Ca(2+)-ATPase.

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.

To understand how the plasma membrane Ca 2؉ pump (PMCA) behaves under changing Ca 2؉ concentrations, it is necessary to obtain information about the Ca 2؉ dependence of the rate constants for calmodulin activation (k act ) and for inactivation by calmodulin removal (k inact ). Here we studied these constants for isoforms 2b and 4b. We measured the ATPase activity of these isoforms expressed in Sf9 cells. For both PMCA4b and 2b, k act increased with Ca 2؉ along a sigmoidal curve. At all Ca 2؉ concentrations, 2b showed a faster reaction with calmodulin than 4b but a slower off rate. On the basis of the measured rate constants, we simulated mathematically the behavior of these pumps upon repetitive changes in Ca 2؉ concentration and also tested these simulations experimentally; PMCA was activated by 500 nM Ca 2؉ and then exposed to 50 nM Ca 2؉ for 10 to 150 s, and then Ca 2؉ was increased again to 500 nM. During the second exposure to 500 nM Ca 2؉ , the activity reached steady state faster than during the first exposure at 500 nM Ca 2؉ . This memory effect is longer for PMCA2b than for 4b. In a separate experiment, a calmodulin-binding peptide from myosin light chain kinase, which has no direct interaction with the pump, was added during the second exposure to 500 nM Ca 2؉ . The peptide inhibited the activity of PMCA2b when the exposure to 50 nM Ca 2؉ was 150 s but had little or no effect when this exposure was only 15 s. This suggests that the memory effect is due to calmodulin remaining bound to the enzyme during the period at low Ca 2؉ . The memory effect observed in PMCA2b and 4b will allow cells expressing either of them to remove Ca 2؉ more quickly in subsequent spikes after an initial activating spike.There are only two mechanisms that actively extrude Ca 2ϩ from the cytosol to the extracellular space: the Na ϩ /Ca 2ϩ exchanger and the plasma membrane Ca 2ϩ pump (PMCA). 1 From comparison of the sequence of PMCA with the x-ray structure of the homologous sarco/endoplasmic reticulum Ca 2ϩ -ATPase(1), it is accepted that its basic structure contains 10 transmembrane domains and two large cytosolic loops. The larger cytosolic loop contains the sites for ATP and formation of the phosphorylated intermediate. In addition, after the last transmembrane domain, PMCAs have a cytoplasmic C-terminal region that includes the calmodulin-binding domain. Although there is strong evidence supporting the formation of oligomers of PMCA when this protein has been solubilized with detergents and purified to a high concentration (2), there is no evidence that these oligomers form when the pump is in the plasma membrane. There are four genes encoding PMCAs; the resulting isoforms are accordingly named PMCA 1, 2, 3, and 4. The diversity of PMCA isoforms is greatly enhanced by the existence of two alternative splicing sites, denoted A and C. No functional differences have yet been found for the alternative splices at site A. Splicing site C is located in the middle of the calmodulinbinding domain in the C-terminal region of the molec...