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First published online 27 June 2006
doi: 10.1242/jcs.03030

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Molecular determinants for differential membrane trafficking of PMCA1 and PMCA2 in mammalian hair cells

M'hamed Grati^1,*, Nisha Aggarwal¹, Emanuel E. Strehler² and Robert J. Wenthold^1,

¹ Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, USA
² Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA

View larger version (34K): [in a new window]   Fig. 1. PMCA1 and PMCA2 alternative splicing. (A) PMCA secondary structure. The positions of the phospholipid binding region (G) and the location of the deafwaddler mouse Gly283Ser mutation (dfw G>S) in PMCA2 are shown. PM, plasma membrane. (B,C) Several PMCA1 and PMCA2 variants are encoded by differentially spliced mRNA. PMCA2 isoforms w, y, x and z are generated by alternative splicing at the A-splice site located in the first cytoplasmic loop. Isoforms differing in their C-termini are generated by alternative splicing at the C-splice site. (B) Aa sequences of PMCA2 exons 1, 2 and 3 of the A-splice site, and of the a-, b- and c-tails generated by alternative splicing at the C-splice site. (C) Aa sequence of the constitutively spliced exon in the PMCA1 A-splice site, and of the a-, b-, c-, d- and e-tails generated by alternative splicing at the C-splice site. Residues Glu-Thr-Ser-Leu (ETSL, underlined in B and C) in the most C-terminal sequence of the b-tail form a PDZ-domain-interacting motif. Aa numbers are according to GenBank PMCA2wb sequence (1243 aa, GenBank accession number P11506), PMCA1xd sequence (1258 aa, GenBank accession number P11505). (D) Aa sequence alignment of PMCA1 and PMCA2 b-tails.

View larger version (62K): [in a new window]   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.

View larger version (79K): [in a new window]   Fig. 3. Alternative A-site and C-site splicing affect apical and basolateral localization of PMCA2 isoforms in HCs. (A-F) Explant HCs were transfected with various EGFP-tagged PCMA2 isoform constructs and examined by immunofluorescence. An apical surface-scan of the stereocilia hair bundle (scanning plane 2 drawn in the HC schematic in G; actin labeled in red) and transverse view of the basolateral plasma membrane (plane 1 drawn in G) of a transfected HC expressing a transgene encoding for each of the EGFP-tagged PMCA2 isoforms (green) are shown in green and red, and merged channels. Arrowheads indicate strong light-scattering signal from the 1-µm-diameter gold bullets collected at 530-550 nm. Bars, 5 µm. Constructs are illustrated on the right. (G) Basolateral cross-sectional scan taken around the midpoint of the transfected HC is also shown on the right of the upper panel (scanning plane 3). (H) Summary of our data. a, a-tail; b, b-tail; AP, apical PM localization; BL, basolateral PM localization.

View larger version (70K): [in a new window]   Fig. 4. PMCA1xb basolateral localization in HCs requires the b-tail. (A) Secondary structure of PMC1xb with a single insert (13 aa) in the A-splice site (1, yellow) and the b-tail (b, purple). (B,C) Surface scans (plane 2 in D) and orthogonal views (plane 1 in D) of stacks of confocal images of HCs transfected with wild-type EGFP-tagged PMCA1xb (B) and PMCA1xb deleted of the b-tail aa sequence (C); green (GFP), red (actin), yellow (merged). Small panels show high-resolution cross-sectional (plane 3 drawn in D) scans in merged channels. n, number of examined transfected hair cells. Bars, 5 µm.

View larger version (59K): [in a new window]   Fig. 5. The PDZ motif and tyrosine-based motif are not important for PMCA2xb targeting in HCs. (A-D) Surface scans (plane 2 in E) and orthogonal views (plane 1 in E) of stacks of confocal images of HCs transfected with mutated PMCA2xb in separate and merged channels. Small panels on the right show cross-sectional high-resolution scan (plane 3 in E) of the basolateral PM taken around the midpoint of the transfected HC in merged channel. Aa sequences of the b-tail; mutations are in red. The Tyr-based Tyr-Glu-Gly-Leu motif at position 1121-1124 (YEGL), is not a basolateral targeting motif for PMCA2 (B,C). The C-terminal PDZ-domain-interacting Glu-Thr-Ser-Leu motif at position 1209-1212 (ETSL), does not seem to influence the basolateral targeting of the pump (A,C). However, the deletion of residues 1153-1166 in the double mutated Tyr1121Ala isoform and the deletion of the ETSL motif alters the vectorial basolateral targeting of the protein and causes it to localize to the apical pole as well (D). (F) Summary of our data. b, b-tail; AP, apical PM localization; BL, basolateral PM localization. Bars, 5 µm.

View larger version (82K): [in a new window]   Fig. 6. Identification of a region encompassing the basolateral signal in the b-tail. (A) Schematic representation of the wild-type PMCA2xb isoform (wt-2xb) used as a basic isoform for the determination of the basolateral-targeting motif present in the b-tail. (B-G) Apical surface-scan of the hair bundle (plane 2 in H, actin in red) and an orthogonal view of the basolateral plasma membrane (plane 1 in H) of a transfected hair cell expressing a transgene encoding EGFP-tagged (in green) PMCA2xb isoform (wt-2xb) and derived mutated isoforms, are presented in separated green and red, and merged channels. Small panels on the right show high-resolution basolateral cross-sectional scans (plane 3 in H). For each mutated variant, the corresponding mutation is shown in red on the b-tail aa sequence. The aa sequence Leu-Ile-Asp-Asp-Thr-Asp-Leu (LIDDTDL) at position 1153-1159 encompasses the crucial residues for the basolateral targeting of PMCA2xb (F). (I) Summary of our observations. 2xb, wild-type PMCA2xb isoform; b, b-tail; AP, apical PM localization; BL, basolateral PM localization. Bars, 5 µm.

View larger version (80K): [in a new window]   Fig. 7. A Leu-Ile motif is required for the basolateral targeting of PMCA2xb. (A,B) Alignment of the basolateral targeting motifs of (A) E-cadherin and (B) CD147 with the crucial aa region for the basolateral targeting of PMCA2. Aa in red indicate the targeting motif of E-cadherin Leu587-[Leu/Ile588] and its homolog in PMCA2 Leu1153-Ile1154 (A), and the crucial residue Leu252 of CD147 and its homolog in PMCA2, Leu1165 (B). Aa in green indicate acidic aa residues. (C) Alignment of the distal C-terminal aa sequences of the b-tail of PMCA1, PMCA2, PMCA3, PMCA 4 from rat and from human PMCA4. Aa in red indicate the Leu[Leu/Ile] motif, aa in green indicate acidic residues, green shading indicates the conserved basolateral targeting motif found in all these PMCAs. Note the conservation of Leu1153-[Leu/Ile1154] and the downstream acidic residues in all four PMCAs. However, Leu1165, which is homologous to the residue crucial for targeting of CD147, is not conserved in all PMCAs. (D-I) Apical surface-scan of the hair bundle (plane 2 in J, actin in red) and an orthogonal view of the basolateral plasma membrane (plane 1 in J) of a transfected hair cell expressing a transgene encoding the EGFP-tagged PMCA2xb isoform (green) (wt-2xb; see Fig. 6A) and derived mutated isoforms, presented in separated green and red, and merged channels. High-resolution basolateral cross-sectional scans (plane 3 in J) are also shown in merged channels. Mutations are shown in red on the aa sequence of the b-tail. Substitutions Leu1153Ala and Ile1154Ala alter the basolateral targeting of PMCA2xb, whereas Phe1152Ala does not. The acidic cluster Asp-Asp-Thr-Asp at position 1155-1158 is not essential for the basolateral targeting of PMCA2xb. (K) Summary of our data. aa, amino acid; b, b-tail; AP, apical PM localization; BL, basolateral PM localization. Bars, 5 µm.

View larger version (158K): [in a new window]   Fig. 8. The basolateral targeting of PMCA2 is not mediated by adaptin AP1B, and the targeting signal is transposable to other PM proteins. (A-C) High-resolution surface confocal scans of transfected polarized LLC-PK1 cells, taken around the junctional area (upper panels) and orthogonal views of stacks of confocal images (lower panels). (A) PMCA2xb (green) is exclusively basolaterally targeted in LLC-PK1. (B) IL-chain (Tac) is delivered predominantly apically in LLC-PK1 cells, with a visible basolateral component. Under the influence of the b-tail, the chimeric protein Tac, fused to the b-tail (Tac-2b), is predominantly delivered to the basolateral PM (C). The mutations in the b-tail Leu1153Ala and Ile1154Ala, inactivate the signal in the b-tail and the product of construct Tac-2b-LI1153-1154AA is targeted predominantly apically (D), similarly to the Tac alone. Bars, 10 µm.

View larger version (75K): [in a new window]   Fig. 9. The size of the insert of the A-splice site determines the apical targeting of PMCA2. (A) Schematic representation of the wild-type PMCA2wa isoform (wt-2wa) used to study the targeting properties of isoforms with various insert sizes. Aa sequence shows the three consecutive exons forming the w-insert of the A-splice site. (B-H) Confocal scans of the hair bundles (plane 2 in I; actin in red) of HCs expressing mutated variants of PMCA2wa (green) are shown in green and merged channels. High-resolution cross-sectional scans of the basolateral PM (plane 3 in I) are shown in the green channel. Mutations generated for each variant are shown in red on the aa sequence of the w-insert. (J) Summary of our results. Only isoforms with inserts of at least 31 aa in the A-splice site are targeted exclusively apically. a, a-tail; AP, apical PM localization; BL, basolateral PM localization. Bars, 5 µm.

View larger version (68K): [in a new window]   Fig. 10. PMCA2wa apical targeting is not altered by deafwaddler Gly283Ser mutation and is independent of its phospholipid activation site (G domain). (A,B) High-resolution confocal scans of the hair bundles (plane 2 in C, actin in red) and cross-sectional scans of the basolateral PM (plane 3 in C) of HCs expressing mutated variants of PMCA2wa (green) are shown in merged channels. (A) Deafwaddler mouse mutant (Dfw; Gly283Ser) and (B) del12[380-91] mutations are represented in red in the aa sequence of the second cytosolic loop of PMCA2wa, the phospholipids-binding site is underlined (B). Both mutated variants are targeted exclusively apically. TM, Transmembrane domain; n, number of examined transfected hair cells. Bars, 5 µm.

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