Kourghi M et al. (2018), Identification of Loop D Domain Amino Acids in ...

XB-ART-54893

Front Chem 2018 Apr 27;6:142. doi: 10.3389/fchem.2018.00142.

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Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011.

Kourghi M , De Ieso ML , Nourmohammadi S , Pei JV , Yool AJ .

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Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1) channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 μM) and AqB011 (IC50 14 μM). In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5), but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A), and mutants with proline substituted for single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), or glycine (G165P) were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50 μM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the role of the loop D as a gating domain for AQP1 ion channels, and identify the likely site of interaction of AqB011 in the proximal loop D sequence.

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Species referenced: Xenopus laevis
Genes referenced: aqp1

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	Figure 1. Human AQP1 ionic conductances activated by cGMP differ in sensitivity to the inhibitor AqB011 in wild type and R159A+R160A expressing oocytes. (A) Electrophysiology traces showing currents recorded in control non-AQP oocytes, and in hAQP1 wild type and R159A+R160A expressing oocytes. The current traces are shown prior to stimulation (initial), after the first maximal response to CPT-cGMP (1st cGMP), and after the second maximal response (2nd cGMP) following a 2 h incubation with 50 Î¼M AqB011 or vehicle (DMSO). (B) Trend plots show the ionic conductance amplitudes for individual oocytes through each series of treatments for AQP1 wild type, mutant, and non AQP-expressing control oocytes, measured before stimulation (initial), after the first CPT-cGMP (1st cGMP), after 2-h recovery in cGMP-free saline containing vehicle or 50 Î¼M AqB011 (â€œincubatâ€), and after the second CPT-cGMP (2nd cGMP). (C) Compiled box plot data illustrate statistically significant block of AQP1 wild type but not R159A+R160 ion conductances following incubation in 50 Î¼M AqB011. n values are above the x-axis. Boxes show 50% of data points; error bars show the full range; horizontal bars show median values. ****p < 0.0001.
	Figure 2. Rates of activation of ion conductance responses to CPT-cGMP in oocytes expressing AQP1 wild type or R159A+R160A channels. (A) Ion current responses were monitored after application of CPT-cGMP using repeated series of brief steps to +40, 0, and âˆ’80 mV from a holding potential of âˆ’40 mV (10 per minute; 150 ms each). Traces are shown at 4 min intervals for clarity. Numbers indicate time in minutes post-application of CPT-cGMP. (B) The plot of steady state current amplitudes at +40 mV as a function of time after application of CPT-cGMP at time zero illustrates the difference in latency to activation in representative examples of AQP1 wild type and R159A+R160A expressing oocytes.
	Figure 3. Confirmation of expression of AQP1 wild type and AQP1 R159A+R160 mutant channels in oocyte plasma membranes by significantly increased osmotic water permeabilities as compared to non-AQP1 expressing controls. (A) Osmotic water permeabilities (mean Â± SEM) assessed by quantitative swelling assays for AQP1 wild type (open circles) and AQP1 R159A+R160 mutant (squares) compared with non-AQP1 expressing control oocytes (filled circles). Relative volumes as a function of time after introduction into 50% hypotonic saline at time zero were measured from video-imaged cross-sectional areas. (n = 6 per group). (B) Box plot data showing osmotic swelling rates were higher in oocytes expressing AQP1 wild type and AQP1 R159A+R160 mutants than non-AQP1 expressing controls (one way ANOVA; post-hoc Bonferroni tests). p < 0.05; ***p < 0.0001; n = 6 per group. Boxes show 50% of data points; error bars show the full range; horizontal bars show median values.
	Figure 4. Amino acid sequence alignment for the loop D and flanking domains of AQP1 channels from diverse classes of vertebrates (mammals, fish, and birds). Amino acid sequences downloaded from the National Center for Biotechnology Information (NCBI) Protein database (www.ncbi.nlm.nih.gov/protein) were aligned using the NCBI BlastP online application (blast.ncbi.nlm.nih.gov) for multiple sequences. Residues in black are identical with the query sequence Homo sapiens AQP1. Variations in sequence are highlighted in red.
	Figure 5. Conserved amino acids in the AQP1 loop D domain influence the ion conductance response, as assessed by proline mutagenesis. (A) Schematic summary of the level of conservation of loop D amino acids in AQP1 sequences (data shown in Figure 4) created with the online WebLogo tool (http://weblogo.berkeley.edu/logo.cgi). Letter sizes represent corresponding relative frequencies of occurrence at that position in the sequence set. (B) Box plot data showing the net conductance values (maximalâ€”initial) for hAQP1 wild type and proline substituted mutant channels. Position-specific effects of proline-scanning mutagenesis on ion conductance responses in human AQP1 suggested less conserved positions are more tolerant of proline substitution. Statistical significance was evaluated with ANOVA and post-hoc two-tailed Mann Whitney. p < 0.01; NS not significant, as compared with wild type; n values are above the x-axis. (C) Graph of mean volumes measured during optical swelling assays and standardized as a percentage of initial volume, for control oocytes and oocytes expressing wild type hAQP1 and proline-substituted mutants as a function of time after introduction of the oocyte into 50% hypotonic saline at time zero. (n values given in D). (D) Box plot histogram of swelling rates for hAQP1 wild type, proline mutant and control oocytes. Significant differences were determined by ANOVA (p < 0.0001) and post-hoc unpaired T-tests. indicates a significant difference from control, p < 0.0001. O indicates no significant difference from wild type, p > 0.05. A significant difference from wild type is indicated as # at p < 0.05, or ## for p < 0.0001.
	Figure 6. Confocal images of anti-AQP1 immunolabeled oocytes expressing wild type and proline substituted mutant channels confirmed protein expression in the oocyte plasma membrane. See Methods for details.
	Figure 7. Ion conductance responses of oocytes expressing human AQP1 wild type and G166P channels. (A) Currents recorded from wild type (left), G166P (middle), and non-AQP expressing control oocytes (right) by two-electrode voltage clamp before (initial) and after stimulation of intracellular cyclic GMP by application of the nitric oxide donor, sodium nitroprusside at a final concentration of 7.5 mM (after SNP). Perfusion of fresh bath saline without SNP (wash) promoted rapid recovery. (B) Current voltage relationships for the traces shown in (A). (C) Steady state current amplitudes at +40 mV monitored as a function of time after three sequential applications of SNP (2.5 mM each) at times indicated by arrows, and after perfusion of bath saline without SNP (wash) shown by the horizontal bar, for wild type (C1), G166P (C2), and control (C3) oocytes. Data are from the same oocytes as shown in (A).
	Figure 8. Schematic diagrams illustrating the separate pathways proposed to mediate water and ion transport in the AQP1 tetrameric channel, and the position of the mutations tested in the loop D domain by proline mutagenesis. (A) AQP1 channels assemble as homomeric tetramers in the membrane bilayer. Water pores (blue) are located in each subunit; cations are thought to permeate via the central pore at the four-fold axis of symmetry in the channel (rose). (B) Loop D is a cytoplasmic loop between the 4 and 5th transmembrane domains in each subunit; loops D in the tetramer surround the central pore. (C) Amino acid residues in loop D tested by mutation to proline. Structural data used to create the diagrams were downloaded from the NCBI Structure database (www.ncbi.nlm.nih.gov/structure/), for PDB ID 1IH5 human AQP1 (Ren et al., 2001); and PDB 1JN4 bovine AQP1 (Sui et al., 2001).

References [+] :

Agre, Aquaporin CHIP: the archetypal molecular water channel. 1993, Pubmed, Xenbase