Cell-specific expression of plasma membrane calcium ATPase isoforms in retinal neurons.

Ca2+ functions as a critical and ubiquitous second messenger by regulating a variety of key intracellular processes (Berridge et al., 2000). The concentration of free intracellular Ca2+ ions, [Ca2+]i, is set by the balance between Ca2+ entry into the cytoplasm and Ca2+ removal by means of buffering proteins, uptake into intracellular organelles and extrusion across the plasma membrane. Together with Na+/Ca2+ exchangers (NCXs), the plasma membrane calcium ATPases (PMCAs) are the principal mechanism for Ca2+ extrusion from most vertebrate cells. The mammalian PMCA family is encoded by four genes whose protein products have been designated PMCA1–4 (reviewed in Guerini and Carafoli, 1998; Strehler and Zacharias, 2001). Expression studies in heterologous cell systems have revealed that the members of the family differ significantly in their affinities for Ca2+ and calmodulin and are linked differently to multiprotein complexes containing PDZ domain proteins (Enyedi et al., 1994; DeMarco and Strehler, 2001; Caride et al., 2001b). PMCA isoforms also are differentially regulated by intracellular messengers such as protein kinases A and C, proteases, and acidic phospholipids (reviewed by Monteith and Roufogalis, 1995; Penniston and Enyedi, 1998; Strehler and Zacharias, 2001). However, immunolocalization of PMCA isoforms to different neuron subtypes within a tissue has hitherto been seldom carried out (de Talamoni et al., 1993; Dumont et al., 2001). Moreover, the functional significance of differential localization of the PMCA isoforms to central nervous system (CNS) tissues and to different cell types is poorly understood. The vertebrate retina consists of cell types with different light response properties. Some classes of retinal neurons signal with graded potentials (photoreceptors, horizontal and bipolar cells), others communicate by means of action potentials (ganglion cells), and still others use both graded and action potentials (amacrine cells; Werblin and Dowling, 1969; Kolb, 1995; Bieda and Copenhagen, 1999). Spiking induces transient elevations in [Ca2+]i compared with the sustained elevations in nonspiking, graded potential neurons. Bipolar, amacrine, and ganglion cells can be further divided into ON and OFF subclasses, which respond to light with depolarization and hyperpolarization, respectively (Werblin and Dowling, 1969). These differences in light-evoked responses presumably place very different demands on the calcium regulation systems of different classes of retinal neuron. In this study, we examine the hypothesis that functionally highly diverse populations of retinal neuron rely on different Ca2+ extrusion mechanisms. By using electrophysiological methods and optical imaging techniques, Ca2+ extrusion from photoreceptor terminals in tiger salamander and tree shrew was shown to be exclusively mediated by a PMCA-like mechanism (Krizaj and Copenhagen, 1998; Morgans et al., 1998). In contrast, a Na+/K+,Ca2+ exchanger mechanism mediates Ca2+ extrusion from photoreceptor outer segments, suggesting that Ca2+ clearance is highly compartmentalized in different regions of the same cell (Miller et al., 1994; Krizaj and Copenhagen, 1998). The localization of PMCAs to synaptic terminals of rods and cones was confirmed by immunohistochemistry by using a pan-specific PMCA antibody (Morgans et al., 1998; Krizaj and Copenhagen, 1998). However, it is not known which particular PMCA isoforms are expressed in photoreceptors. The mechanisms of Ca2+ extrusion from bipolar cells are currently unclear. In one study, Ca2+ efflux from synaptic terminals of teleost bipolar cells was shown to be mediated exclusively by PMCA-like extrusion mechanisms (Zenisek and Matthews, 2000). However, Kobayashi and Tachibana (1995) found evidence for Na+/Ca2+ exchanger-mediated Ca2+ extrusion from these same type of fish bipolar cells. No information is available about Ca2+ extrusion systems used in mammalian bipolar cells. PMCA-mediated Ca2+ efflux also contributes to Ca2+ extrusion from third-order retinal neurons (Gleason et al., 1994, 1995). In cultured chick amacrine cells, PMCAs contribute from ~0 to ~50% of total Ca2+ clearance (Gleason et al., 1995). A Na+/Ca2+ exchange mechanism constitutes the remaining component of Ca2+ extrusion. Na+/Ca2+ exchange also may be prominent in fish ganglion cells (Bindokas et al., 1994). However, no PMCA-dependent Ca2+ extrusion has yet been detected in retinal ganglion cells. From the above, it is clear that much still needs to be learned about Ca2+ extrusion mechanisms from neurons in the mammalian retina. Moreover, the PMCA isoforms associated with Ca2+ extrusion from specific retinal cell classes have yet to be identified. The aim of the current study was to map and compare the distribution and cellular localization of all PMCA isoforms in a mammalian retina. Particular attention was paid to photoreceptors and bipolar cells, cell types that are the most likely to rely on PMCA-mediated extrusion of Ca2+ (Kobayashi and Tachibana, 1995; Krizaj and Copenhagen, 1998; Zenisek and Matthews, 2000). We report here that PMCA isoforms are differentially distributed within different classes of retinal neuron and to different subcellular locations within neurons. Specifically, we found that photoreceptors and cone bipolar cells possess PMCA1, rod bipolar cells express PMCA2, whereas the spiking amacrine and ganglion cells express both PMCA2 and PMCA3. PMCA4 was present in synaptic structures of several retinal neurons. We propose that differential expression of PMCA isoforms in different classes of retinal neurons is likely of fundamental importance for the synaptic communication of the visual signal.