Chauvigné F et al. (2015), The pH sensitivity of Aqp0 channels in tetraplo...

XB-ART-50163

FASEB J 2015 May 01;295:2172-84. doi: 10.1096/fj.14-267625.

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The pH sensitivity of Aqp0 channels in tetraploid and diploid teleosts.

Chauvigné F , Zapater C , Stavang JA , Taranger GL , Cerdà J , Finn RN .

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Water homeostasis and the structural integrity of the vertebrate lens is partially mediated by AQP0 channels. Emerging evidence indicates that external pH may be involved in channel gating. Here we show that a tetraploid teleost, the Atlantic salmon, retains 4 aqp0 genes (aqp0a1, -0a2, -0b1, and -0b2), which are highly, but not exclusively, expressed in the lens. Functional characterization reveals that, although each paralog permeates water efficiently, the permeability is respectively shifted to the neutral, alkaline, or acidic pH in Aqp0a1, -0a2, and -0b1, whereas that of Aqp0b2 is not regulated by external pH. Mutagenesis studies demonstrate that Ser(38), His(39), and His(40) residues in the extracellular transmembrane domain of α-helix 2 facing the water pore are critical for the pH modulation of water transport. To validate these findings, we show that both zebrafish Aqp0a and -0b are functional water channels with respective pH sensitivities toward alkaline or acid pH ranges and that an N-terminal allelic variant (Ser(19)) of Aqp0b exists that abolishes water transport in Xenopus laevis oocytes. The data suggest that the alkaline pH sensitivity is a conserved trait in teleost Aqp0 a-type channels, whereas mammalian AQP0 and some teleost Aqp0 b-type channels display an acidic pH permeation preference.

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Species referenced: Xenopus laevis
Genes referenced: aqp4 gnl3 mip rps18

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	Figure 1. Genomic organization of Atlantic salmon aqp0 genes. Schematic representation of aqp0a1, -0a2, -0b1, and -0b2 gene loci. Gray boxes indicate exons with coding regions only.
	Figure 2. Phylogenetic relationships of Aqp0 in Gnathostomata. Bayesian majority rule consensus tree of a codon alignment of vertebrate aqp0 orthologs resulting from 5 million Markov chain Monte Carlo generations and a burn-in of 25%. Posterior probabilities of the codon/amino acid analyses are shown at each node, where - indicates <50%. The tree is rooted with Inshore hagfish (Eptatretus burgeri) aqp4, with the scale bar indicating the rate of nucleotide substitution per site. Whole genome duplication events are shown as black squares at the relevant nodes.
	Figure 3. Tissue expression pattern of Atlantic salmon aqp0 genes. Tissue distribution of aqp0a1, -0a2, -0b1, and -0b2 transcripts determined by quantitative RT-PCR and using rps18 as reference gene. Data are means Â± sem (n = 5 fish). Significant differences (P < 0.05; P < 0.01; **P < 0.001) between paralogs in each tissue are indicated. The bracket indicates significant differences of the expression levels in the lens with respect the other tissues. NS, not significant.
	Figure 4. Structural analysis of tetrapod and teleost AQP0. A) Amino acid alignment of Atlantic salmon and representative teleost Aqp0 sequences in relation to bovine AQP0. Fully conserved residues are boxed in dark gray, whereas the His residue involved in pH sensitivity is shaded in magenta. The zebrafish allelic variant at position Gly19 is circled in red. Conserved Asn-Pro-Ala (NPA) motifs (pale red arrows) are highlighted with Î±-helical regions shown for TMDs 1â6 (light blue arrows), hemihelices 3 (yellow arrow), 7 (green arrow), and 9 (orange arrow), intra- and extracellular loops (AâE; pink lines) with the N (NT) and C (CT) termini (palid orange lines). Taxa as follows: Bt, Bos taurus; Fh, Fundulus heteroclitus; Ss, Salmo salar; Dr, Danio rerio. BâD) Extracellular (upper) and lateral (lower) views of bovine AQP0 (B) and Atlantic salmon Aqp0a2 (C) and Aqp0b1 (D) are rendered with MacPymol.
	Figure 5. Functional characterization of Atlantic salmon Aqp0 paralogs. Osmotic water permeability (Pf) of X. laevis oocytes injected with water (W) or cRNA encoding Aqp0a1, -0a2, -0b1, and -0b2 and exposed to different pHs before and during the swelling assays. The Pf was calculated using an estimated surface area of 9Ã the geometric area. Data are the mean Â± sem (n = 12 oocytes per treatment) of 3 independent experiments. Significant differences (P < 0.05; P < 0.01; **P < 0.001) for each aquaporin at the 3 pHs are indicated. The bracket indicates significant differences with respect water-injected oocytes. NS, not significant.
	Figure 6. Both zebrafish aqp0a and -0b paralogs encode functional water channels. Osmotic water permeability (Pf) of X. laevis oocytes injected with water or cRNA encoding zebrafish wild-type Aqp0a or -0b or the Aqp0b-G19S mutant. The Pf was calculated using an estimated surface area of 9Ã the geometric area. Oocytes were exposed to different pH conditions before and during the swelling assays. Data are the mean Â± sem (n = 8â12 oocytes per treatment) of 4 independent experiments. Significant differences (P < 0.05; P < 0.01; **P < 0.001) for each construct at the 3 pHs are indicated. The bracket indicates significant differences with respect water-injected oocytes, NS, not significant.
	Figure 7. Role of Ser38 and His39-His40 on Atlantic salmon Aqp0a1 and -0b2 pH sensitivity. Osmotic water permeability (Pf) of X. laevis oocytes injected with water or cRNA encoding wild-type Aqp0a1 or Aqp0b2 or different mutants in which Ser39 was replaced by Pro in Aqp0a1 (Aqp0a1-S39P) or in which a single His in position 39 or 40 replaced the double His in Aqp0b2 (Aqp0b2âH40N and Aqp0b2-H39N, respectively). The Pf was calculated using an estimated surface area of 9Ã the geometric area. Oocytes were exposed to different pH before and during the swelling assays. Data are the mean Â± sem (n = 12 oocytes per construct) of a representative experiment. Significant differences (P < 0.05; P < 0.01; **P < 0.001) for each construct at the 3 pHs are indicated. The bracket indicates significant differences with respect water-injected oocytes. NS, not significant.

References [+] :

Agre, Aquaporin water channels--from atomic structure to clinical medicine. 2002, Pubmed

	Figure 1. Genomic organization of Atlantic salmon aqp0 genes. Schematic representation of aqp0a1, -0a2, -0b1, and -0b2 gene loci. Gray boxes indicate exons with coding regions only.
	Figure 2. Phylogenetic relationships of Aqp0 in Gnathostomata. Bayesian majority rule consensus tree of a codon alignment of vertebrate aqp0 orthologs resulting from 5 million Markov chain Monte Carlo generations and a burn-in of 25%. Posterior probabilities of the codon/amino acid analyses are shown at each node, where - indicates <50%. The tree is rooted with Inshore hagfish (Eptatretus burgeri) aqp4, with the scale bar indicating the rate of nucleotide substitution per site. Whole genome duplication events are shown as black squares at the relevant nodes.
	Figure 3. Tissue expression pattern of Atlantic salmon aqp0 genes. Tissue distribution of aqp0a1, -0a2, -0b1, and -0b2 transcripts determined by quantitative RT-PCR and using rps18 as reference gene. Data are means Â± sem (n = 5 fish). Significant differences (P < 0.05; P < 0.01; **P < 0.001) between paralogs in each tissue are indicated. The bracket indicates significant differences of the expression levels in the lens with respect the other tissues. NS, not significant.
	Figure 4. Structural analysis of tetrapod and teleost AQP0. A) Amino acid alignment of Atlantic salmon and representative teleost Aqp0 sequences in relation to bovine AQP0. Fully conserved residues are boxed in dark gray, whereas the His residue involved in pH sensitivity is shaded in magenta. The zebrafish allelic variant at position Gly19 is circled in red. Conserved Asn-Pro-Ala (NPA) motifs (pale red arrows) are highlighted with Î±-helical regions shown for TMDs 1â6 (light blue arrows), hemihelices 3 (yellow arrow), 7 (green arrow), and 9 (orange arrow), intra- and extracellular loops (AâE; pink lines) with the N (NT) and C (CT) termini (palid orange lines). Taxa as follows: Bt, Bos taurus; Fh, Fundulus heteroclitus; Ss, Salmo salar; Dr, Danio rerio. BâD) Extracellular (upper) and lateral (lower) views of bovine AQP0 (B) and Atlantic salmon Aqp0a2 (C) and Aqp0b1 (D) are rendered with MacPymol.
	Figure 5. Functional characterization of Atlantic salmon Aqp0 paralogs. Osmotic water permeability (Pf) of X. laevis oocytes injected with water (W) or cRNA encoding Aqp0a1, -0a2, -0b1, and -0b2 and exposed to different pHs before and during the swelling assays. The Pf was calculated using an estimated surface area of 9Ã the geometric area. Data are the mean Â± sem (n = 12 oocytes per treatment) of 3 independent experiments. Significant differences (P < 0.05; P < 0.01; **P < 0.001) for each aquaporin at the 3 pHs are indicated. The bracket indicates significant differences with respect water-injected oocytes. NS, not significant.
	Figure 6. Both zebrafish aqp0a and -0b paralogs encode functional water channels. Osmotic water permeability (Pf) of X. laevis oocytes injected with water or cRNA encoding zebrafish wild-type Aqp0a or -0b or the Aqp0b-G19S mutant. The Pf was calculated using an estimated surface area of 9Ã the geometric area. Oocytes were exposed to different pH conditions before and during the swelling assays. Data are the mean Â± sem (n = 8â12 oocytes per treatment) of 4 independent experiments. Significant differences (P < 0.05; P < 0.01; **P < 0.001) for each construct at the 3 pHs are indicated. The bracket indicates significant differences with respect water-injected oocytes, NS, not significant.
	Figure 7. Role of Ser38 and His39-His40 on Atlantic salmon Aqp0a1 and -0b2 pH sensitivity. Osmotic water permeability (Pf) of X. laevis oocytes injected with water or cRNA encoding wild-type Aqp0a1 or Aqp0b2 or different mutants in which Ser39 was replaced by Pro in Aqp0a1 (Aqp0a1-S39P) or in which a single His in position 39 or 40 replaced the double His in Aqp0b2 (Aqp0b2âH40N and Aqp0b2-H39N, respectively). The Pf was calculated using an estimated surface area of 9Ã the geometric area. Oocytes were exposed to different pH before and during the swelling assays. Data are the mean Â± sem (n = 12 oocytes per construct) of a representative experiment. Significant differences (P < 0.05; P < 0.01; **P < 0.001) for each construct at the 3 pHs are indicated. The bracket indicates significant differences with respect water-injected oocytes. NS, not significant.