Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles

Abstract

Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport.

Figure 1.

Location of splice-site A in the amino acid sequence and predicted structure of PMCA2. A, Alignment of amino acid sequences of rabbit muscle SERCA with rat PMCA2 splice variants in the vicinity of splice-site A. SERCA lacks sequences corresponding to this splicing region. B, Scheme of the PMCA protein and diagram of splicing at sites A and C in frog and rat PMCA2. The PMCA is shown at the top as a gray bar; the 10 membrane-spanning domains are indicated by black boxes and numbered from 1 to 10. N, N terminus; C, C terminus. Colored boxes denote the positions where alternative splicing alters PMCA2 at sites A and C. Splicing at site A affects the first cytosolic loop between membrane-spanning domains 2 and 3; splicing at site C alters the C-terminal sequence of the pump protein. The colored boxes below the PMCA scheme indicate the amino acid insertions encoded by separate exons in different PMCA2 splice variants (labeled on the left). The colors of the boxes correspond to the colored bars above the sequence in A. Note that a unique sequence insertion in the 2v splice form of frog PMCA2 (indicated by an additional dark green box) interrupts the 2w sequence. This sequence insertion does not exist in rat (or human) PMCA2 and hence is not shown in A; frog PMCA2v is nevertheless targeted to hair bundles. C, Side view of the SERCA structure (1SU4). PMCA2 identities are red; nonidentical but aligned amino acids are blue. Gray areas do not align between the two sequences. The location of the actuator domain, N terminus (orange), and splice-site A (yellow) are indicated. The shaded box corresponds to the approximate location of the membrane bilayer. Note the proximity of the N terminus to splice-site A. The structure was generated with Cn3D version 4.1.

Figure 2.

R2w and R2z specifically recognize PMCA2w/a and PMCA2z/a in transfected COS-7 cells. A, B, Immunoprecipitation from COS-7 cells. A, Lysates from cells expressing 2w or 2z variants of PMCA2a or negative control plasmid (pJPA5) were incubated with R2w or F2a. The pan-PMCA antibody 5F10 was used to detect immunoprecipitated PMCA; in hair bundles, 5F10 precipitates bands of 150–170 kDa (Yamoah et al., 1998). R2w precipitated a band of ∼170 kDa only from cells expressing PMCA2w/a. F2a precipitated 170 kDa bands from cells expressing PMCA2w/a or PMCA2z/a. B, Lysates from cells expressing C8 epitope-tagged PMCA2z or pJPA5 were incubated with R2z or anti-C8. 5F10 detection was used as in A. R2z and anti-C8 precipitated a band of ∼170 kDa from transfected cells. C, Immunocytochemistry in COS-7 cells. R2w detects cells expressing PMCA2w/a but not PMCA2z/a. NR2 demonstrated that each PMCA2 variant was expressed in transfected cells. IP, Immunoprecipitation; Detect, detection; Ab, antibody.

Figure 3.

PMCA2w is the predominant PMCA2 isoform of rat inner ear. A, Sequential immunoprecipitation (IP) scheme. P, Immunoprecipitate; S, supernatant. B–D, Sequential immunoprecipitations with PMCA2 antibodies. B, 5F10 detection of PMCA isoforms sequentially immunoprecipitated with PMCA2-selective antibodies from 36 ear equivalents of purified rat hair bundles. Nearly all bundle PMCA (∼170 kDa) were precipitated with R2w. F/T, PMCA not precipitated by any of the antibodies. C, Sequential immunoprecipitation from six ear equivalents of residual maculae. D, Sequential immunoprecipitation negative control; agarose from the bundle isolation procedure was used as the starting material. E, Location of primers used for nested PCR relative to splice-site A. F, Ethidium bromide-stained gel (inverted grayscale image) separating rat cochlear RT-PCR products. The sizes of products expected for PMCA2 splice forms and the sizes (in base pairs) of molecular weight markers (MW) are indicated.

Figure 4.

Localization of PMCA2w in rat hair bundles by immunofluorescence. A–D, Confocal cross sections through the P21 rat organ of Corti using R2w to detect PMCA2w (A; D, green), 5F10 to detect all PMCA isoforms (B; D, blue), and phalloidin to detect actin (C; D, red). Note the strong 5F10 immunoreactivity in basolateral membranes of inner hair cells (B, arrowhead). E–H, Utricular hair bundles were strongly labeled by R2w, as was the pericuticular necklace (H, arrow); the z-projection of two adjacent slices is shown. I–L, No R2w labeling of the lateral membranes of utricular hair cells; the z-projection of two adjacent slices is shown. M, En face view of a cochlea whole mount, with a line indicating the location of the x–z section for N–Q. OHC, Outer hair cells; OPC, outer pillar cells; IPC, inner pillar cells; IHC, inner hair cells. N–Q, The x–z section of cochlear whole mount shown in M. The color scheme in Q is the same as in M. PMCA2w immunoreactivity is strong in bundles of outer hair cells but absent from the cell bodies of outer hair cells (O, P, arrows), inner hair cells, and other cells of the organ of Corti. R, Lateral view of rat ampulla labeled with R2w. Scale bars: (in A) A–D, R, 10 μm; (in E) E–L, 5 μm; M, 91 × 91 μm; N–Q, 110 × 21 μm.

Figure 5.

Targeting of PMCA2w/a and PMCA2w/b transfected in rat hair cells. A, Bundle-specific PMCA2w/a expression in outer hair cells labeled with anti-C8 for epitope-tagged PMCA2w/a (green), GFP-actin (blue), and phalloidin to detect actin (red). Arrows indicate cortical actin from the GFP-actin signal in a transfected cell; note the absence of the C8-PMCA2w/a signal in the corresponding panel. The white color of the hair bundle in the last panel indicates overlap of PMCA2w/a, GFP-actin, and phalloidin signals. B, Hair-bundle targeting of PMCA2w/a in a utricular hair cell. C, Hair-bundle targeting of PMCA2w/b in a cochlear outer hair cell. D, Hair-bundle targeting of PMCA2w/b in a utricular hair cell. Scale bars, 5 μm.

Figure 6.

Targeting of PMCA2z/a in transfected rat hair cells. A, B, PMCA2z/a-C8 expression in the membrane of a cochlear outer hair cell at the level of the bundle (A) and near the nucleus (B). The magenta color of the hair bundle indicates lack of PMCA2z/a; the strong expression of PMCA2z/a on the basolateral membrane is apparent in B (green). C, D, PMCA2z/a-C8 on the membrane of a utricular hair cell, visualized at the level of the bundle (C) and near the nucleus (D). Green, Epitope-tagged PMCA2w/a; blue, GFP-actin; red, phalloidin to detect actin. Scale bars, 2 μm.

Figure 7.

Expression of GFP-PMCA2w/a in rat hair cells. Representative hair cells transfected with GFP-PMCA2w/a alone (A–H) or with GFP-PMCA2w/a and C8-actin (I–L). A–D, Vestibular hair cell transfected with GFP-PMCA2w/a (green) and labeled with phalloidin to detect actin (red). A, x–z projection. B–D, Individual sections at levels indicated in A. E–H, Cochlear outer hair cell transfected as in A–D. I–L, Cochlear outer hair cell transfected with GFP-PMCA2w/a (green) and C8-actin (blue); actin was detected with phalloidin (red). Scale bars: A, E, I, 5 μm; (in B, F, J) B–D, F–H, J–L, 5 μm.