Carpentier GA et al. (2016), Identification of key residues involved in Si t...

XB-ART-54252

J Gen Physiol 2016 Sep 01;1483:239-51. doi: 10.1085/jgp.201611598.

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Identification of key residues involved in Si transport by the aquaglyceroporins.

Carpentier GA , Garneau AP , Marcoux AA , Noël M , Frenette-Cotton R , Isenring P .

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We recently demonstrated that the aquaglyceroporins (AQGPs) could act as potent transporters for orthosilicic acid (H4SiO4). Although interesting, this finding raised the question of whether water and H4SiO4, the transportable form of Si, permeate AQGPs by interacting with the same region of the pore, especially in view of the difference in molecular radius between the two substrates. Here, our goal was to identify residues that endow the AQGPs with the ability to facilitate Si diffusion by examining the transport characteristics of mutants in which residues were interchanged between a water-permeable but Si-impermeable channel (aquaporin 1 [AQP1]) and a Si-permeable but water-impermeable channel (AQP10). Our results indicate that the composition of the arginine filter (XX/R), known to include three residues that play an important role in water transport, may also be involved in Si selectivity. Interchanging the identities of the nonarginine residues within this filter causes Si transport to increase by approximately sevenfold in AQP1 and to decrease by approximately threefold in AQP10, whereas water transport and channel expression remain unaffected. Our results further indicate that two additional residues in the AQP arginine filter may be involved in substrate selectivity: replacing one of the residues has a profound effect on water permeability, and replacing the other has a profound effect on Si permeability. This study has thus led to the identification of residues that could play a key role in Si transport by the AQGPs and shown that substrate selectivity is likely ensured by more than one checkpoint within or near the pore.

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Species referenced: Xenopus
Genes referenced: aqp1 aqp10 aqp11 aqp12a ren

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	Figure 1. Hydropathy plot models and transport characteristics of wild-type AQP1 and AQP10. (A) Model of AQP1. Each symbol corresponds to a single residue within a transmembrane domain (red), a connecting segment (pink), the XX/R filter (yellow), or the NPA motifs (black). Residues above the transmembrane domains face the extracellular side of the membrane. The cartoon was drawn with the program PLOT based on the hydropathy model of Murata et al. (2000). (B) Model of AQP10. Each symbol corresponds to a single residue in a transmembrane domain (blue), a connecting segment (pale blue), the XX/R filter (brown), or the NPA motifs (black). The cartoon was drawn with the program PLOT based on sequence alignments of transmembrane domains with AQP1. (C) Multiple alignment analysis of AQP family members with Clustal Omega. The residue segment used for each of the channels corresponds to the one that is flanked by green lines in A and B; the N and C termini were excluded from the analysis given that they are poorly conserved among the isoforms. AQP11 and AQP12 were also excluded given that they share much lower homologies with the other family members. Gray boxes correspond to transmembrane segments based on alignments with AQP1, and colored residues correspond to those that were interchanged between AQP1 and AQP10 in this study.
	Figure 2. Location of residues that were substituted in AQP1 and AQP10. The cartons were drawn as described in Fig. 1 using the same color code. (A and B) Chimeras. (C and D) Point substitutions.
	Figure 3. Si influx in oocytes expressing AQP1, expressing AQP10, or injected with water. (A) Oocytes incubated in medium B2 for 90 min at room temperature were assayed for Si content. Data are expressed as means ± SE of 10 oocytes among 25–26 experiments, using the asterisk to indicate that they are significantly different statistically (, P < 0.05) compared with oocytes injected with water. (B) Water permeability in oocytes expressing AQP1, expressing AQP10, or injected with water. Oocytes incubated in plain water were assayed for volume measurements as described in Materials and methods. Data are expressed as mean Pf ± SE of three to five oocytes among five to six experiments, using the asterisk to indicate that they are significantly different statistically (, P < 0.05) compared with oocytes injected with water.
	Figure 4. Transport characteristics and membrane expression of AQP11-10-1 and AQP1010-1-10. (A) Si influx. Oocytes incubated in medium B2 for 90 min at room temperature were assayed for Si content. They were from 10 oocytes among three to four experiments. According to this presentation, a mean of 1 indicates no n-fold differences (Δ) in Si influx. (B) Water permeability. Oocytes incubated in plain water were assayed for volume measurements as described in Materials and methods. They were from three to five oocytes among three experiments. (A and B) Data are expressed as n-fold differences ± SE between AQP11-10-1 and AQP1 (left bar) or between AQP1010-1-10 and AQP10 (right bar) after background subtraction and normalization to channel expression levels. (C) AQP expression at the cell surface (EXPcs). Oocytes incubated in medium B2 for 90 min were lysed for Western blot analyses using specific anti-AQP Abs, and the signals obtained were quantified through densitometry. Data are expressed as n-fold differences ± SE between AQP11-10-1 and AQP1 (left bar) or between AQP1010-1-10 and AQP10 (right bar) after background subtraction. They were from three experiments. (A–C) The asterisk is used to indicate that the mean is significantly different statistically (*, P < 0.05) compared with the wild-type channel.
	Figure 5. Transport characteristics and membrane expression of AQP1LGRND→IFATY and AQP10IFATY→LGRND. Experimental conditions were as described in Fig. 4. Data expression is also as described in Fig. 4. (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three experiments. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (A and B) The asterisk is used to indicate that the mean is significantly different statistically (*, P < 0.05) compared with the wild-type channel. (C) AQP expression at the cell surface (EXPcs). Data are expressed as means ± SE among three to four experiments.
	Figure 6. Transport characteristics and membrane expression of AQP1L84C and AQP10C90L. Experimental conditions were as described in Fig. 4. Data expression is also as described in Fig. 4. (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three to six experiments. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (B) The asterisk is used to indicate that the mean is significantly different statistically (*, P < 0.05) compared with the wild-type channel. (C) AQP expression at the cell surface (EXPcs). Data are expressed as means ± SE among three to four experiments.
	Figure 7. Transport characteristics and membrane expression of AQP1Y186N and AQP10N208Y. Experimental conditions were as described in Fig. 4. Data expression is also as described in Fig. 4. (A) Si influx. Data are expressed as means ± SE of 10 oocytes among three to four experiments. The asterisk is used to indicate that the mean is significantly different statistically (*, P < 0.05) compared with the wild-type channel. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among three to four experiments. (C) AQP expression at the cell surface (EXPcs). Data are expressed as means ± SE among three experiments.
	Figure 8. Transport characteristics and membrane expression of AQP1F56G/H180G and AQP10G62F/G202H. Experimental conditions were as described in Fig. 4. Data expression is also as described in Fig. 4. (A) Si influx. Data are expressed as means ± SE of 10 oocytes among eight to nine experiments. The asterisk is used to indicate that the mean is significantly different statistically (*, P < 0.05) compared with the wild-type channel. (B) Water permeability. Data are expressed as means ± SE of three to five oocytes among four to five experiments. (C) AQP expression at the cell surface (EXPcs). Data are expressed as means ± SE among three to four experiments.
	Figure 9. Illustrative experiments. (A) Water transport. After incubation in plain water, an oocyte expressing AQP1 and an oocyte expressing AQP10 were microphotographed at different time points for cell volume measurements. (B) Expression of wild-type and mutant AQPs by Western blot analyses. Samples correspond to cell surface proteins detected with an anti-AQP1 Ab (left) or an anti-AQP10 Ab (right). In each panel, water-injected oocytes were used as negative controls. (C) Immunofluorescence experiments. Cryosections postfixed in paraformaldehyde were obtained from AQP-expressing oocytes after a 3-d incubation in medium B1 at 18°C. Wild-type and mutant AQPs were detected with an anti-AQP1 Ab (left) or an anti-AQP10 Ab (right). For each Ab, water-injected oocytes were used as negative controls. Micrographs were taken under confocal microscopy and are shown in the panel for some of the AQPs: (top left) water, (second left) AQP11-10-1, (third left) AQP1LGRND→IFATY, (bottom left) AQP1L84C, (top right) water, (second right) wild-type AQP10, (third right) AQP10N208Y, and (bottom right) AQP10G62F/G202H. Note that each of the micrographs is ~300 μm in actual width.
	Figure 10. Crystal structure of human AQP1. The images were screen-printed from an open access database available at http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1FX8. The yellow circles correspond to carbon atoms within individual residues. The model was originally determined by Ren et al. (2000) at 3.70-Å resolution through electron crystallography of ice-embedded 2-D crystals.

References [+] :

Beitz, Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons. 2006, Pubmed, Xenbase