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Fig. 2. PMCA1 and PMCA2 in rat inner ear. (A) RT-PCR detection of PMCA1 and PMCA2 isoforms in rat inner ear. Amplification of the A-splice regions of PMCA1 and PMCA2 showed the presence of a unique 519 bp band corresponding to the x-variant (top left), and of two distinct 762 bp and 627 bp bands corresponding to w- and z-variants, respectively (bottom left). RT-PCR product sampling after 15-25 rounds of amplification showed that the w-variant is the dominant form. RT-PCR of the exons encoding the C-terminal tail of PMCA1 and PMCA2 showed the presence of a single 409 bp and 543 bp band, respectively, corresponding to the isoforms encoding the b- and the a-tail, respectively (top right and bottom right, respectively). Thus, only two isoforms of PMCA2, PMCA2wa and PMCA2za, and a single isoform of PMCA1, are detectable in the rat inner ear. +, PCR product after RT; –, PCR product without RT. (B,C) Immunolocalization of PMCA1 in organ of Corti hair cells from rat. Immunofluorescence on adult rat organ of Corti with pan-PMCA1 antibodies reveals abundant labeling in the basolateral PM of inner hair cells. (B) Orthogonal views and stereocilia surface scan of a stack of confocal images are shown in the green (PMCA1), red (actin) and in merged channels. High-resolution hair-bundle surface and transverse basolateral scans in merged channels are also shown in (C). (D,E) Immunolocalization of PMCA2 in rat inner ear hair cells. Immunofluorescence of adult rat inner ear cells with pan-PMCA2 antibodies revealed abundant labeling in the stereocilia (co-stained for actin) of outer HCs (D) and vestibular HCs (E). Hair-bundle surface and transverse basolateral scans are shown in green (PMCA2), red (actin) and merged channels. Bars, 5 µm.

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