Chow PH et al. (2021), Inhibition of the Aquaporin-1 Cation Conductanc...

XB-ART-59005

Front Pharmacol 2021 Oct 01;12:794791. doi: 10.3389/fphar.2021.794791.

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Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling.

Chow PH , Cox CD , Pei JV , Anabaraonye N , Nourmohammadi S , Henderson SW , Martinac B , Abdulmalik O , Yool AJ .

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In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 aqp4 piezo1 scd
GO keywords: ion channel activity [+]

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	FIGURE 1. Electrophysiological recordings illustrating the effects of furan derivatives on the 8CPT-cGMP-activated AQP1 ion conductance. Representative sets of traces recorded by two-electrode voltage clamp of AQP1-expressing oocytes showing the initial conductance; the response induced by the first application of membrane-permeable 8CPT-cGMP; and the response to a second application of cGMP after 2 h of incubation in isotonic saline containing (A) equivalent DMSO vehicle; and 0.5 mM of (B) 5-PMFC; (C) VZHE006; (D) 5-HMF; (E) 5-NMFC; (F) 5-CMFC.
	FIGURE 2. Trend plots showing the amplitude of the ionic currents, before (initial) and after the first activation by SNP (1st), the recovery after 2 h incubation with the indicated compound at 0.5 mM (post-incub), and the response reactivated by a second SNP application (2nd). Consistent recovery was seen after vehicle, 5-NMFC, or 5-CMFC but not after incubation with 5-PMFC or VZHE006 indicating ion channel inhibition. The n values are as shown; each line represents a series of recordings from one oocyte.
	FIGURE 3. Compiled data showing the effects of furan derivatives on the amplitude of the cGMP-dependent AQP1 ion conductance activated by SNP. (A) Compiled box plot data showing statistically significant inhibition of the AQP1 ion conductance by 5-PMFC or VZHE006, but not vehicle, 5-NMFC or 5-CMFC. n values were four each for all but 5-NMFC which was 5. Statistical significance was determine by paired Students t test; *p < 0.05; NS not significant. (B) Space filling structures of the compounds tested; the three on the left act as AQP1 inhibitors. (C) In silico docking model illustrating the predicted site for the most favorable interaction of 5-PMFC with AQP1, located near the central pore vestibule of the tetrameric channel in the loop D gating domain, which is seen as a curved purple strand connecting transmembrane helices (left panel). Higher magnification view of the predicted hydrogen bond interaction between 5-PMFC and Ser167 (right panel).
	FIGURE 4. Dose-dependent inhibition of AQP1 ion channel conductance by 5-PMFC. (A) Box plot compilation of SNP-activated conductance values measured after 1–2 h incubation with the indicated concentration of 5-PMFC in isotonic saline. Conductances were calculated from slopes of current-voltage (I–V) plots measured at 8 min after application of 3 mM extracellular SNP used to stimulate intracellular cGMP. No current activation by SNP was observed in non-AQP controls with or without 5-PMFC incubation, or in AQP4-expressing oocytes. n values in italics are above the x-axis. Significant differences were found between AQP1 at 0 µM 5-PMFC () and all other treatment groups shown, except AQP1 at 50 µM PMFC (#) which was not significantly different from AQP1 0 µM 5-PMFC. Statistical significance was assessed by one-way ANOVA and Šidák’s multiple comparisons test; p < 0.01; # not significant. (B) Current-voltage plots of responses recorded in series for the same AQP1-expressing oocyte, showing initial low conductance, activation by SNP (‘cGMP 1st′), recovery during 2 h incubation in 500 µM 5-PMFC (incub PMFC), and block of the second response to SNP (‘cGMP 2nd′). Reversal potentials shifted from near −20 mV in the initial and cGMP (1st) conditions to approximately −50 mV after 5-PMFC treatment (in incubation and cGMP (2nd) conditions), consistent with inhibition of a non-selective cation conductance, and not native oocyte K+ channels. (C) Traces illustrating data shown in the I-V plots in (B).
	FIGURE 5. Osmotic water permeability of AQP1-expressing oocytes is not altered by treatment with the furan derivatives. (A) Mean swelling responses of AQP1 expressing oocytes in 50% hypotonic saline were not affected after 2 h of preincubation in the furan derivatives (2 mM). Data are mean ± SEM; n values are as shown in the key. (B) Compiled box plot data showing comparable swelling rates measured in the same oocytes for the first (S1) assay before and second (S2) assay after 2-h incubation in saline with indicated treatments. Statistical significance was assessed by ANOVA and post hoc paired Student T tests; NS not significant. (C) The plot of S1 vs. S2 swelling rates for individual oocytes shows a linear relationship (slope values near 1.0) in all treatment conditions, indicating no effect on water channel activity, and no change in oocyte membrane integrity or levels of AQP1 channel expression during pharmacological incubation or repeated assays. “Vehic” is vehicle control (equivalent DMSO).
	FIGURE 6. The Piezo1 ion channel conductance is not sensitive to block by 5-HMF. (A) Representative traces from cell-attached recordings of human Piezo1 currents (upper row) measured in HEK293T cells stably transfected with Piezo1, shown for cells in the control treatment (LEFT), after 2 mM 5-HMF for 60 min (CENTER), or after 100 µM GdCl3 applied in the bath for ≥5 min before patching (RIGHT). Square wave pressure pulses (lower row) were applied to the patch pipette using a high-speed pressure clamp. (B) Amplitudes of peak currents recorded from cell attached patches from HEK293T cells in control, 5-HMF and Gd3+ treatment groups as specified in panel A. is p < 0.01; NS is not significant; n values are above the x-axis. (C) Pressure-sensitive activation of Piezo1 currents in control, 5-HMF and Gd3+ treatment groups. Statistical significance was assessed by Kruskal-Wallis ANOVA and post hoc Dunn’s multiple comparisons tests; p < 0.01; NS not significant.
	FIGURE 7. Effects of selected furan compounds on percent block of AQP1 ion conductance amplitudes, percent structural modification of hemoglobin (Hb), and percent inhibition of sickling in SCD red blood cells exposed to low oxygen. Tests of AQP1 block were done with agents at 0.5 mM each; tests for hemoglobin modification and sickling risk were done at 2 mM each. Data for Hb modification and red blood cell sickling were based on results presented in published tables (Xu et al., 2017).
	FIGURE 8. Combined application of agents that inhibit AQP1 ion channels (AqB011) and that modify hemoglobin (5-NMFC) reduce the rate of sickling of human SCD cells in hypoxic conditions, approaching the protection seen with 5-PMFC alone. (A) Data for percent sickling were standardized to the mean maximum sickling (at 45 min) measured in the vehicle controls in the same experimental group; data within treatments were averaged for all experiments to determine mean and SEM values, and plotted as a function of time in full nitrogen starting at time zero. Estimated t1/2 values (figure legend) show the times needed in minutes to reach 50% of the net maximal sickling level, set as the level observed for vehicle-treated cells at 45 min. Data were compiled from three separate experiments with 2 to 3 replicate samples each. n values are as shown in the dot plot below. (B) Dot plot summary of raw data for the percentages of sickled cells at 20 min hypoxia compiled from three independent experiments. Concentrations of agents used were 2.5 mM for 5-NMFC and 5-PMFC, 80 µM for AqB011, and equivalent DMSO for the vehicle control. Significant differences were determined by ordinary one-way ANOVA with Šidác’s multiple comparisons test. **p < 0.01; NS is not significant.
	Supplemental Figure 1. Dot plots showing initial cGMP-activated conductance values for individual AQP1-expressing oocytes, and the effects of incubation with vehicle or furan compounds on the amplitude of the second cGMP-induced current activation responses. Horizontal lines show median values. (Corresponding box plots for the same data are shown in Figure 3).
	Supplemental Figure 2. Additional positions detected by in silico modeling as candidate sites of interaction of 5-PMFC across the intracellular face of the AQP1 channel tetramer, with calculated values for the predicted theoretical energies of interaction of the ligand with the channel at the various positions. (See Methods for details.)

References [+] :

Abdulmalik, 5-hydroxymethyl-2-furfural modifies intracellular sickle haemoglobin and inhibits sickling of red blood cells. 2005, Pubmed