Kourghi M et al. (2017), Divalent Cations Regulate the Ion Conductance P...

XB-ART-54241

Int J Mol Sci 2017 Nov 03;1811:. doi: 10.3390/ijms18112323.

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Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins.

Kourghi M , Nourmohammadi S , Pei JV , Qiu J , McGaughey S , Tyerman SD , Byrt CS , Yool AJ .

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Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophilamelanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction.

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Species referenced: Xenopus
Genes referenced: aqp1 aqp4 aqp5 tbl1x
GO keywords: ion channel activity

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	Figure 1. Confirmation of the expression of AQP1, AQP4, AQP5, PIP2;7, PIP2;2, PIP2;1, and BIB channels in Xenopus oocyte membranes. (A) Osmotic swelling responses as a function of time in 50% hypotonic saline for aquaporins (AQP)-expressing oocytes as compared with non-AQP-expressing control oocytes. Data are mean Â± SEM; n = 6 per treatment group. (B) Box plots of swelling rates for the data shown in (A). ANOVA and post hoc Bonferroni test; ** (p < 0.01) as compared with control; n = 6 per group. (C) Confocal image of immuno-labeled oocytes confirming BIB protein expression in the oocyte plasma membrane, as described previously [11].
	Figure 2. Effects of Mg2+ and Ca2+ divalent cations on ionic current responses in oocytes expressing different classes of AQPs. (A) Superimposed currents as a function of time measured by voltage clamp (steps from â110 to +60 mV, from a holding potential of â40 mV) at maximal activation in divalent-free saline (left), after application of Mg2+ (middle), and after application of Ca2+ (right). Control and PIP2;7-expressing oocytes lacked appreciable conductances. (B) Current-voltage relationships for the traces illustrated in (A). (C) Na+ concentrations in oocytes expressing PIP2;2 as compared with control oocytes after incubation in Frog Ringers containing 96 mM NaCl for three days. Data are from four replicates; each replicate contained five oocytes. (D) Box plot summary of compiled data for the conductances. Free Ca2+ concentrations are given in Î¼M. ** (p < 0.0001); * (p < 0.001); ** (p < 0.01); * (p < 0.05); NS (not significant); using ANOVA with post-hoc Bonferroni tests. n values are in italics below the x-axis.
	Figure 3. Effects of Ba2+ on ionic currents in oocytes expressing different AQPs. (A) Superimposed current traces as a function of time measured by voltage clamp at maximal activation in divalent-free saline (upper row) and after perfusion with bath saline containing 1 mM BaCl2 (lower). (B) Current-voltage relationships in Ba2+ for traces presented in (A). (C) Box plot summary of compiled data for AQP1, PIP2;1, PIP2;2 and BIB channels before and after 1 mM Ba2+ application. (D) Histogram showing the relative outward rectification values (I+60/Iâ80) as mean Â± SEM. Ratios were calculated as amplitude of outward (+60 mV) to inward (â80 mV) currents. ** (p < 0.0001); (p < 0.001); * (p < 0.01); NS (not significant); using ANOVA with post-hoc Bonferroni tests. n values are near the x-axis.
	Figure 4. Effects of Cd2+ on ion current responses in oocytes expressing different classes of AQPs. (A) Superimposed current traces under voltage clamp recorded at maximal activation (if present) in divalent-free saline (left) and after perfusion with bath saline containing 1 mM CdCl2 (right). (B) Current-voltage relationships for data presented in (A). (C) Rates of onset of block after perfusion of bath saline containing 1 mM Cd2+, as measured using repeated voltage steps to +40 mV. (D) Box plot summary of compiled data for control, AQP4, AQP5, AQP1, PIP2;1, PIP2;2 and BIB expressing oocytes before and after Cd2+ application. n values are below the x-axis. (E) Histogram showing relative outward rectification values (mean Â± SEM), calculated as the ratio of outward to inward currents at +60 and â80 mV (I+60/I-80). ** (p < 0.0001); * (p < 0.005); NS (not significant); using ANOVA with post-hoc Bonferroni tests; n values are shown above the histogram bars.
	Figure 5. Effects of sequential application of Ca2+ followed by Ba2+ on ionic current responses in oocytes expressing different classes of AQPs. (A) Superimposed current traces under voltage clamp showing the maximal responses of AtPIP2;2, AtPIP2;1, HsAQP1 and DmBIB expressing oocytes recorded in divalent-free saline (left), after perfusion of saline containing the indicated amount of free Ca2+ (middle), and after application of Ba2+ in the continuing presence of the same concentration of Ca2+ (right). (B) Current-voltage relationships for traces shown in (A). (C) Summary box plot of conductance values for AtPIP2;2, AtPIP2;1, HsAQP1 and DmBIB expressing oocytes in different divalent cation salines. * (p < 0.001); (p < 0.01); * (p < 0.05); NS (not significant); using ANOVA with post hoc Bonferroni tests; n values are below the x-axis.
	Figure 6. Effects of the arylsulfonamide compound AqB011 on ionic current responses in oocytes expressing different classes of AQPs. (A) Responses were recorded for control (DMSO vehicle), and AtPIP2;1, AtPIP2;2, and HsAQP1 expressing oocytes before (left) and after 2 h incubation (right) in AqB011 at the indicated doses. HsAQP1 was blocked by 20 Î¼M AqB011; AtPIP2;1 and AtPIP2;2 currents were not affected by 100 Î¼M AqB011. (B) Box plot summary of the conductance levels before and after treatment with AqB011. **** (p < 0.0001); NS (not significant); using ANOVA with post hoc Bonferroni tests; n values are below the x-axis.
	Figure 7. Amino acid sequence alignment of HsAQP1, RnAQP4, RnAQP5, AtPIP2;1, AtPIP2;2, AtPIP2;7 and DmBIB. Sequences from the NCBI Protein Database were aligned using Clustal Omega software (Available online: http://www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 18 October 2017). HsAQP1 protein topology was from the Topology Data Bank of Transmembrane Proteins (TOPDB). Symbols: asterisk (*) identical residues across all sequences; colon (:) highly conserved residue; period (.) semi-conserved residue. Hyphens (-) show gaps; black vertical lines separate predicted domains, as labelled. Colors illustrate chemical properties: positive charged (magenta), negative charged (blue), polar (green), hydrophobic (red). Highlighted in yellow are NPA (Asn-Pro-Ala) signature motifs located in loops B and E, conserved in most aquaporins. The carboxy terminus of DmBIB was truncated for this figure.

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	Figure 1. Confirmation of the expression of AQP1, AQP4, AQP5, PIP2;7, PIP2;2, PIP2;1, and BIB channels in Xenopus oocyte membranes. (A) Osmotic swelling responses as a function of time in 50% hypotonic saline for aquaporins (AQP)-expressing oocytes as compared with non-AQP-expressing control oocytes. Data are mean Â± SEM; n = 6 per treatment group. (B) Box plots of swelling rates for the data shown in (A). ANOVA and post hoc Bonferroni test; ** (p < 0.01) as compared with control; n = 6 per group. (C) Confocal image of immuno-labeled oocytes confirming BIB protein expression in the oocyte plasma membrane, as described previously [11].
	Figure 2. Effects of Mg2+ and Ca2+ divalent cations on ionic current responses in oocytes expressing different classes of AQPs. (A) Superimposed currents as a function of time measured by voltage clamp (steps from â110 to +60 mV, from a holding potential of â40 mV) at maximal activation in divalent-free saline (left), after application of Mg2+ (middle), and after application of Ca2+ (right). Control and PIP2;7-expressing oocytes lacked appreciable conductances. (B) Current-voltage relationships for the traces illustrated in (A). (C) Na+ concentrations in oocytes expressing PIP2;2 as compared with control oocytes after incubation in Frog Ringers containing 96 mM NaCl for three days. Data are from four replicates; each replicate contained five oocytes. (D) Box plot summary of compiled data for the conductances. Free Ca2+ concentrations are given in Î¼M. ** (p < 0.0001); * (p < 0.001); ** (p < 0.01); * (p < 0.05); NS (not significant); using ANOVA with post-hoc Bonferroni tests. n values are in italics below the x-axis.
	Figure 3. Effects of Ba2+ on ionic currents in oocytes expressing different AQPs. (A) Superimposed current traces as a function of time measured by voltage clamp at maximal activation in divalent-free saline (upper row) and after perfusion with bath saline containing 1 mM BaCl2 (lower). (B) Current-voltage relationships in Ba2+ for traces presented in (A). (C) Box plot summary of compiled data for AQP1, PIP2;1, PIP2;2 and BIB channels before and after 1 mM Ba2+ application. (D) Histogram showing the relative outward rectification values (I+60/Iâ80) as mean Â± SEM. Ratios were calculated as amplitude of outward (+60 mV) to inward (â80 mV) currents. ** (p < 0.0001); (p < 0.001); * (p < 0.01); NS (not significant); using ANOVA with post-hoc Bonferroni tests. n values are near the x-axis.
	Figure 4. Effects of Cd2+ on ion current responses in oocytes expressing different classes of AQPs. (A) Superimposed current traces under voltage clamp recorded at maximal activation (if present) in divalent-free saline (left) and after perfusion with bath saline containing 1 mM CdCl2 (right). (B) Current-voltage relationships for data presented in (A). (C) Rates of onset of block after perfusion of bath saline containing 1 mM Cd2+, as measured using repeated voltage steps to +40 mV. (D) Box plot summary of compiled data for control, AQP4, AQP5, AQP1, PIP2;1, PIP2;2 and BIB expressing oocytes before and after Cd2+ application. n values are below the x-axis. (E) Histogram showing relative outward rectification values (mean Â± SEM), calculated as the ratio of outward to inward currents at +60 and â80 mV (I+60/I-80). ** (p < 0.0001); * (p < 0.005); NS (not significant); using ANOVA with post-hoc Bonferroni tests; n values are shown above the histogram bars.
	Figure 5. Effects of sequential application of Ca2+ followed by Ba2+ on ionic current responses in oocytes expressing different classes of AQPs. (A) Superimposed current traces under voltage clamp showing the maximal responses of AtPIP2;2, AtPIP2;1, HsAQP1 and DmBIB expressing oocytes recorded in divalent-free saline (left), after perfusion of saline containing the indicated amount of free Ca2+ (middle), and after application of Ba2+ in the continuing presence of the same concentration of Ca2+ (right). (B) Current-voltage relationships for traces shown in (A). (C) Summary box plot of conductance values for AtPIP2;2, AtPIP2;1, HsAQP1 and DmBIB expressing oocytes in different divalent cation salines. * (p < 0.001); (p < 0.01); * (p < 0.05); NS (not significant); using ANOVA with post hoc Bonferroni tests; n values are below the x-axis.
	Figure 6. Effects of the arylsulfonamide compound AqB011 on ionic current responses in oocytes expressing different classes of AQPs. (A) Responses were recorded for control (DMSO vehicle), and AtPIP2;1, AtPIP2;2, and HsAQP1 expressing oocytes before (left) and after 2 h incubation (right) in AqB011 at the indicated doses. HsAQP1 was blocked by 20 Î¼M AqB011; AtPIP2;1 and AtPIP2;2 currents were not affected by 100 Î¼M AqB011. (B) Box plot summary of the conductance levels before and after treatment with AqB011. **** (p < 0.0001); NS (not significant); using ANOVA with post hoc Bonferroni tests; n values are below the x-axis.
	Figure 7. Amino acid sequence alignment of HsAQP1, RnAQP4, RnAQP5, AtPIP2;1, AtPIP2;2, AtPIP2;7 and DmBIB. Sequences from the NCBI Protein Database were aligned using Clustal Omega software (Available online: http://www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 18 October 2017). HsAQP1 protein topology was from the Topology Data Bank of Transmembrane Proteins (TOPDB). Symbols: asterisk (*) identical residues across all sequences; colon (:) highly conserved residue; period (.) semi-conserved residue. Hyphens (-) show gaps; black vertical lines separate predicted domains, as labelled. Colors illustrate chemical properties: positive charged (magenta), negative charged (blue), polar (green), hydrophobic (red). Highlighted in yellow are NPA (Asn-Pro-Ala) signature motifs located in loops B and E, conserved in most aquaporins. The carboxy terminus of DmBIB was truncated for this figure.