Abstract
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.
Highlights
Aquaporins (AQPs) are a diverse family of channels for water and solutes, classified as major intrinsic proteins (MIPs) (Benga et al, 1986; Agre et al, 1993; Reizer et al, 1993)
Voltage clamp recordings showed that application of extracellular CPT-cGMP activated ionic conductance responses in human AQP1 wild type and R159A+R160A mutant expressing oocytes (Figure 1A)
The ionic conductance increased after application of CPT-cGMP in AQP1 wild type and R159A+R160A expressing oocytes, but in not non-AQP control oocytes
Summary
Aquaporins (AQPs) are a diverse family of channels for water and solutes, classified as major intrinsic proteins (MIPs) (Benga et al, 1986; Agre et al, 1993; Reizer et al, 1993). Some classes of aquaporin channels have been found shown to transport molecules other than water across the cell membrane, including glycerol, ammonia, urea, protons, as well as CO2 and O2 gases (Madeira et al, 2014; Kitchen et al, 2015), and ions (Yool, 2007; Yool and Campbell, 2012). Aquaporin ion channel functions have been described for multiple members of the MIP family. The precise roles of their ion channel activities in cell signaling and development remain to be determined
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