Western blot analysis shows plasma membrane calcium ATPase (PMCA) isoform expression in the adult mouse retina. Total protein lysates (40 μg protein per lane) from adult mouse retinae were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels, blotted onto polyvinylidene difluoride membrane, and processed for Western blotting by using PMCA isoform-specific antibodies NR1 (PMCA1), NR2 (PMCA2), NR3 (PMCA3), and NR4/JA9 (PMCA4). The pan-PMCA antibody 5F10 was used as a control to detect the sum of all PMCAs (All PMCAs). Molecular mass standards are indicated in kilodaltons on the sides, and the bands are marked by arrows.

Confocal fluorescence photomicrograph of a vertical frozen section through mouse retina labeled for plasma membrane calcium ATPase (PMCA)1plus secondary antibody (A) or with the secondary antibody only (B). A: PMCA1 immunofluorescence is prominent in the outer plexiform layer (OPL). Moderate PMCA1 signal is also observed in bipolar cell perikarya in the upper part of the inner nuclear layer (INL) and in the inner plexiform layer (IPL). ONL, outer nuclear layer; GCL, ganglion cell layer. Scale bar = 20 μm in B (applies to A,B).

Confocal fluorescence labeling indicates plasma membrane calcium ATPase 1 (PMCA1) expression in photoreceptor terminals. A: PMCA1 is strongly expressed in the outer plexiform layer (OPL), where it labels a horizontal band of ellipsoid structures corresponding to photoreceptor terminals. Moderate label is also present in perikarya of bipolar cells in the inner nuclear layer (INL). B: SV2 immunolabels photoreceptor synaptic terminals. C: Superposition of PMCA1 and SV2 immunofluorescence indicates substantial colocalization between the two antibodies in photoreceptor terminals. D–I: Higher power confocal fluorescence photomicrographs of immunoreactivity for PMCA1 and calbindin show colabeling of horizontal cells but not of photoreceptors, amacrine, or ganglion cells. D: PMCA1 is prominently expressed in photoreceptor synaptic terminals. The labeling is moderate in bipolar cell bodies in the distal INL and weak in horizontal cells (arrowhead). E: Calbindin antibody strongly labels horizontal cells. Prominent somas with dendritic knobs, extending into photoreceptor terminals (arrowheads), are observed. The antibody also labels a population of amacrine cells located in the proximal INL (arrow). F: Superposition of images from D and E. PMCA1 is expressed in photoreceptor terminals but is excluded from calbindin-immunopositive amacrine cells. G–I: High-power fluorescence photomicrograph of the same section as shown in D–F with magnification focused on the OPL. Each dendritic knob emanating from horizontal cells is associated with exactly one photoreceptor terminal immunolabeled by PMCA1. J–L: Section through macaque monkey retina immunostained for PMCA1 and calbindin. J: PMCA1 immunostained the two types of synaptic terminals in the OPL. Note that synaptic terminals are labeled uniformly. K: Calbindin selectively stains a population of monkey cones. L: PMCA1 signal is expressed in rod spherules (arrowheads) as well as in cone pedicles (arrows). Scale bars = 15 μm in C (applies to A–C), 10 μm in F (applies to D–F), 2.5 μm in I (applies to G–I), 20 μm in L (applies to J–L).

Plasma membrane calcium ATPase 1 (PMCA1) is expressed in cone but not rod bipolar cells. A–C: Double labeling for PMCA1 (A) and protein kinase C (PKC; B). PKC immunolabeled dendritic trees, perikarya, and synaptic terminals of rod bipolar cells. C: Little colocalization is observed between the PMCA1-immunopositive cells and PKC-immunopositive rod bipolar cells. D–F: Double labeling for β-galactosidase in the lacZ transgenic mouse. D: PMCA1 signal in the lacZ mutant is higher than in the wild-type control (Fig. 4A). E: β-Galactosidase antibody immunolabels a large population of cells with synaptic terminals in sublamina b of the inner plexiform layer (IPL). F: Significant colocalization is observed between PMCA1 and lacZ immunopositive perikarya in the INL. The lacZ-immunopositive terminals in the proximal IPL were typically unlabeled by PMCA1 (arrowheads). G–I: Double-labeling for vesicular glutamate transporter (VGLUT1) and PKC. G: VGLUT1 is expressed in glutamatergic synaptic terminals localized to both sublamina a (sub a) and sublamina b (sub b) of the IPL. H: Labeling for PKC. I: Colocalization of VGLUT1 and PKC signals in terminals of rod bipolar cells. The PMCA1-immunopositive puncta in sublamina b, unlabeled by the PKC antibody, correspond to synaptic terminals of ON cone bipolar cells. The PMCA1-immunopositive puncta in sub-lamina a, unlabeled by the PKC antibody, correspond to OFF cone bipolar cells terminals. Scale bars = 20 μm in C (applies to A–C) and I (applies to G–I), 25 μm in F (applies to D–F).

Ca²⁺ extrusion from rod and cone bipolar cells. A: Combined Nomarski and fluorescence photomicrograph of a rod bipolar cell dissociated from the mouse retina and immunostained for protein kinase C (PKC). The antibody labeled the cell body, the axon, and the synaptic terminal. B: [Ca²⁺]_i changes in a fura-2 loaded rod bipolar cell are shown as changes in the ratio between 340- and 380-nm wavelengths. The cell was superfused with 45 mM KCl (horizontal bar labeled KCl). At t = 2.3 min, the solution was switched (horizontal line labeled Wash) to a control 2.5 mM K⁺ -containing saline (solid line) or a test 2.5 mM K⁺ saline in which [Na⁺]_o was substituted by [Li⁺]_o (dotted line). [Ca²⁺]_i recovery to the baseline value after a depolarization-evoked increase in significantly prolonged in the absence of [Na⁺]_o C: A small proportion of bipolar cells were not labeled by the PKC antibody. These cells were classified as putative cone dipolar cells. D: [Ca²⁺]_i changes in a fura-2 loaded putative cone bipolar cell are shown as changes in the ratio between 340- and 380-nm wavelengths. The cell was depolarized (horizontal bar labeled KCl) with 45 mM KCl and then superfused (horizontal bar labeled Wash) with control saline (solid line) or a test saline in which [Na⁺]_o was substituted by [Li⁺]_o (dotted line). During washout with control saline, [Ca²⁺]_i recovered to the baseline value exponentially with two time constants that were very similar in control and Li⁺ saline. Scale bars = 5 μm in A, 5 μm in C.

Plasma membrane calcium ATPase 1 (PMCA1) is not localized to γ-aminobutyric acid-ergic neurons. Sections were labeled for PMCA1 and glutamic acid decarboxylase 65 (GAD-65). A: PMCA1 labeled the outer plexiform layer (OPL), inner plexiform layer (IPL), and the inner nuclear layer (INL). B: The same section as in A immunolabeled for GAD-65. The label is restricted to amacrine cell bodies in the INL (arrowheads) and to amacrine processes in the IPL. C: Superposition of A and B shows little apparent colocalization of PMCA1 and GAD-65 in the INL. Scale bar = 10 μm in C (applies to A–C).

Plasma membrane calcium ATPase 3 (PMCA3) immunoreactivity in the mouse retina. A: The PMCA3 immunofluorescence is prominent throughout the inner plexiform layer (IPL). Amacrine cell bodies in the inner nuclear layer (INL) and ganglion cell bodies in the granule cell layer (GCL) are also labeled. The optic nerve fiber layer is strongly labeled. Weaker immunofluorescence is observed in the outer plexiform layer (OPL). ONL, outer nuclear layer. B: Section stained with secondary antibody only. Scale bar = 20 μm.

Plasma membrane calcium ATPase 3 (PMCA3) and calbindin immunoreactivity demonstrate expression of PMCA3 in horizontal and amacrine cells. A: Section labeled for PMCA3. B: Same section shown in A labeled for calbindin. C: Horizontal cells, amacrine cells and displaced amacrine cells (arrowheads) are immunoreactive for both PMCA3 and calbindin. D–F. Same section as in A–C at higher magnification. OPL, outer plexiform layer; IPL, inner plexiform layer. D: PMCA3 is localized to horizontal cell bodies and processes and to amacrine cell bodies lining the proximal inner nuclear layer (INL). E: Same section as in D labeled for calbindin. F: Double-labeling for PMCA3 and calbindin. Both antigens are expressed in horizontal cells and in amacrine cells (arrowheads). Scale bar = 20 μm in C (applies to A–C), 15 μm in F (applies to A–F).

Colocalization of plasma membrane calcium ATPase 3 (PMCA3) and glutamic acid decarboxylase 65 (GAD-65) expression. A: PMCA3 immunoreactivity is expressed in both plexiform layers (inner, IPL; outer, OPL), the inner nuclear layer (INL), and the granule cell layer. B: Same section shown in A labeled for GAD-65. GAD-65 immunofluorescence is found in perikarya of amacrine cells localized to the proximal INL (arrowheads). Prominent immunoreactivity can be seen in the IPL. C: PMCA3 and GAD-65 colocalize in amacrine cell bodies in the INL. D–I: Same section shown at high magnification for INL (D–F) and IPL (G–I) labeled for PMCA3 (D and G) and GAD-65 (E,F). D: PMCA3 is expressed in a subset of perikarya at the proximal edge of the INL. E: GAD-65 labels perikarya in the proximal INL. F: PMCA3 is colocalized with GAD-65 in a subset of GAD-65–immunoreactive neurons in the INL (arrowheads in D,E). G: High-power photomicrograph of a PMCA3-immunolabeled retina focused on the IPL. PMCA3 antibody labeled distinct puncta within the IPL (arrows). H: Same section as in G labeled for GAD-65. GAD-65 immunolabeled distinct puncta in the IPL (arrows). I: PMCA3 and GAD-65 immunofluorescence colocalized in many puncta (arrows in G and H). Scale bars = 20 μm in C (applies to A–C), 7.5 μm in F (applies to D–F), 1.5 μm in I (applies to G–I).

Plasma membrane calcium ATPase 3 (PMCA3) is not expressed in rod bipolar cell bodies and synaptic terminals. A: Outer plexiform layer (OPL) and inner nuclear layer (INL) regions double labeled for PMCA3 (green) and protein kinase C (PKC; red). PMCA3 is not expressed in perikarya of rod bipolar cells. B1–3: Same section, magnified view of the IPL. B1: Little colocalization between PMCA3 and PKC is seen at the level of rod bipolar cell terminals. B2: Same section as B1; PMCA3 signal in the IPL. B3: Same section as B1; PKC in the IPL immunolabels synaptic terminals of rod bipolar cells. Scale bars = 10 μm in A; 7.5 μm in B3 (applies to B1–3).

Plasma membrane calcium ATPase 2 (PMCA2) immunolocalization in the mouse retina. A: The PMCA2 antibody labeled the IPL with the most prominent signal shown in two strata in sublaminae a and b, respectively. Moderate immunoreactivity is seen in the outer plexiform layer (OPL) and in the proximal inner nuclear layer (INL). B: Section stained with secondary antibody only. Scale bar = 20 μm in B (applies to A,B).

Plasma membrane calcium ATPase 2 (PMCA2) is expressed in synaptic terminals of rod bipolar cells and in horizontal cells. A: Magnified view of the outer plexiform layer (OPL) labeled for PMCA2. PMCA2 is expressed diffusively throughout the OPL. B: Same section immunolabeled for calbindin. Calbindin immunofluorescence is found in horizontal cells. C: PMCA2 and calbindin colocalize in horizontal cell bodies and dendrites. D–F: Retinal section double labeled for PMCA2 and protein kinase C (PKC). D: PMCA2 signal in a low-power photomicrograph. E: Same section showing the PKC signal. F: Double labeling for PMCA2 and PKC. INL, inner nuclear layer. G–I. Same section as in D–F, high-power view focused on sublamina b of the inner plexiform layer (IPL). G: PMCA2 immu-noreactivity in sublamina b. H: PKC immunoreactivity in sublamina b. I: Colocalization with PMCA2 is seen in synaptic terminals of PKC-immunoreactive rod bipolar cell synaptic terminals (arrowheads in G). Scale bars = 10 μm in C (applies to A–C), 20 μm in F (applies to D–F), 8 μm in I (applies to G–I).

Plasma membrane calcium ATPase 4 (PMCA4) immunolocalization in the retina. A: PMCA4 immunostained both plexiform layers (outer, OPL; inner, IPL). Nonspecific signal is seen at the level of photoreceptor outer segments. INL, inner nuclear layer; ONL, outer nuclear layer. B: Section stained with secondary antibody only. Scale bar = 20 μm.

Plasma membrane calcium ATPase 4 (PMCA4) is localized to synaptic terminals. A: PMCA4 is prominently expressed in the outer plexiform layer (OPL) and the inner plexiform layer (IPL). B: Same section labeled for SV2. C: Combined images for the section shown in A and B show high degree of colocalization in both synaptic layers for PMCA4 and SV2 signals. ONL, outer nuclear layer; INL, inner nuclear layer. D–F: Higher magnification view of the section shown in A–C, focusing on the OPL. Note the colocalization of PMCA4 and SV2 in photoreceptor terminals. Scale bar = 20 μm in C (applies to A–C), 2 μm in F (applies to D–F).

Schematic localization of plasma membrane calcium AT-Pase (PMCA) isoforms in the mammalian retina. The distribution of PMCA isoforms is indicated by shading of neuronal classes associated with the appropriate isoform. PMCA was expressed in photoreceptors and cone bipolar cells. Weak signal was also observed in horizontal cells. PMCA2 was present in rod bipolar cells, amacrine cells, and ganglion cells. PMCA3 was highly expressed in amacrine and ganglion cells, and weakly in horizontal cells. PMCA4 was found to label synaptic elements in the outer plexiform layer (OPL) at the level of photoreceptor terminals, and in the inner plexiform layer (IPL) where synaptic terminals of bipolar and amacrine cells are found.