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Keywords:

  • ATP-binding cassette transporter;
  • CFTR;
  • chloride ion channel;
  • open-channel blockade;
  • epithelial ion transport;
  • loop diuretics;
  • NKCC;
  • bumetanide;
  • furosemide;
  • glibenclamide

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Background and Purpose

Loop diuretics are widely used to inhibit the Na+, K+, 2Cl co-transporter, but they also inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel. Here, we investigated the mechanism of CFTR inhibition by loop diuretics and explored the effects of chemical structure on channel blockade.

Experimental Approach

Using the patch-clamp technique, we tested the effects of bumetanide, furosemide, piretanide and xipamide on recombinant wild-type human CFTR.

Key Results

When added to the intracellular solution, loop diuretics inhibited CFTR Cl currents with potency approaching that of glibenclamide, a widely used CFTR blocker with some structural similarity to loop diuretics. To begin to study the kinetics of channel blockade, we examined the time dependence of macroscopic current inhibition following a hyperpolarizing voltage step. Like glibenclamide, piretanide blockade of CFTR was time and voltage dependent. By contrast, furosemide blockade was voltage dependent, but time independent. Consistent with these data, furosemide blocked individual CFTR Cl channels with ‘very fast’ speed and drug-induced blocking events overlapped brief channel closures, whereas piretanide inhibited individual channels with ‘intermediate’ speed and drug-induced blocking events were distinct from channel closures.

Conclusions and Implications

Structure–activity analysis of the loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that loop diuretics are open-channel blockers of CFTR with distinct kinetics, affected by molecular dimensions and lipophilicity.


Abbreviations
BHK cells

baby hamster kidney cells

CF

cystic fibrosis

CFTR

cystic fibrosis transmembrane conductance regulator

FRT cells

Fischer rat thyroid cells

i

single-channel current amplitude

IClapical

apical membrane Cl current

M

transmembrane segment

MSD

membrane-spanning domain

NKCC

Na+, K+, 2Cl co-transporter

NMDG

N-methyl-D-glucamine

Po

open probability

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

During transepithelial Cl secretion, the Na+, K+, 2Cl co-transporter isoform 1 (NKCC1; SLC12A2) (Delpire et al., 1994; Haas and Forbush, 2000; Hebert et al., 2004) actively accumulates Cl within epithelial cells across the basolateral membrane, whereas the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) Cl channel (Riordan et al., 1989; Holland et al., 2003; Gadsby et al., 2006) mediates the passive exit of Cl across the apical membrane (receptor and channel nomenclature follows Alexander et al., 2013). High-affinity inhibitors of NKCC1 and CFTR are therefore valuable tools to identify these transport proteins, investigate transepithelial Cl transport mechanisms and treat disease (Russell, 2000; Sheppard, 2004). Loop diuretics, such as bumetanide and furosemide, are widely used to inhibit NKCC1 (Haas and Forbush, 2000). However, they are not selective blockers (Cabantchik and Greger, 1992). Interestingly, Reddy and Quinton (1999) demonstrated that bumetanide and furosemide inhibit salt reabsorption in native human sweat duct epithelia, a tissue with modest NKCC1 activity. These authors interpreted their data to suggest that loop diuretics inhibit salt reabsorption by blocking CFTR. Consistent with this idea, Venglarik (1997) reported that bumetanide and furosemide inhibit the single-channel activity of CFTR.

The chemical structures of bumetanide and furosemide show some similarities to that of glibenclamide, the best-studied CFTR blocker (Li and Sheppard, 2009). Therefore, we were interested to learn how loop diuretics inhibit CFTR. Our specific aims were, firstly, to understand the mechanism of CFTR inhibition and, secondly, to investigate the relationship between the chemical structures of loop diuretics and CFTR inhibition. We selected for study bumetanide, furosemide, piretanide and xipamide (Figure 1). These loop diuretics show a high degree of structural similarity, all being benzenesulfonamides with three further substituents on the aryl ring (Figure 1 and Supplementary Information Figure S1). Bumetanide and piretanide are very closely related, only differing in the 3-amino substituent (butylamino vs. pyrrolidino). Furosemide and xipamide differ from bumetanide and piretanide in substitution pattern (1,2,4,5-tetra substituted vs. 1,3,4,5-tetra substituted) and identity.

figure

Figure 1. Chemical structures of loop diuretics. The structures of glibenclamide and the agents used in this study are shown. The 3-cis and 4-trans positions of the cyclohexyl ring of glibenclamide are indicated by the numbers 1 and 2 respectively. For further details, see the text.

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To investigate mechanisms of CFTR inhibition, we studied macroscopic and single-channel currents in excised inside-out membrane patches from cells expressing recombinant wild-type human CFTR. We found that loop diuretics are open-channel blockers that inhibit CFTR with similar potencies, but distinct kinetics; bumetanide and piretanide with ‘intermediate’ speed, and furosemide and xipamide with ‘very fast’ speed. Analysis of the chemical structures of loop diuretics suggests that the phenoxy group present in bumetanide and piretanide, but absent in furosemide and xipamide, might account for the different kinetics of channel block by locking loop diuretics within the intracellular vestibule of the CFTR pore. We conclude that differences in molecular dimensions and lipophilicity affect CFTR inhibition by loop diuretics.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Cells and cell culture

We used baby hamster kidney (BHK), Fischer rat thyroid (FRT) and mouse mammary epithelial (C127) cells stably expressing wild-type human CFTR (Marshall et al., 1994; Farinha et al., 2002; Zegarra-Moran et al., 2002), as described previously (Sheppard and Robinson, 1997; Schmidt et al., 2008). Cells were generous gifts from MD Amaral (University of Lisboa; BHK cells); LJV Galietta (Istituto Giannina Gaslini; FRT cells) and CR O'Riordan (Genzyme; C127 cells). C127 cells are the cell line of choice for single-channel studies of wild-type human CFTR because they express low levels of recombinant CFTR (e.g. Sheppard and Robinson, 1997). By contrast, BHK cells are preferred for measurements of macroscopic CFTR Cl currents because they express high levels of recombinant CFTR (e.g. Schmidt et al., 2008). Because they form polarized epithelia with a high transepithelial resistance and because they lack expression of endogenous CFTR, FRT cells are the cell line of choice to investigate transepithelial Cl transport mediated by recombinant CFTR (Sheppard et al., 1994). The single-channel behaviour of wild-type human CFTR in excised membrane patches from different mammalian cells is equivalent (Chen et al., 2009).

Patch-clamp experiments

CFTR Cl channels were recorded in excised inside-out membrane patches using an Axopatch 200A patch-clamp amplifier and pCLAMP software (both from MDS Analytical Technologies, Union City, CA, USA) (Sheppard and Robinson, 1997). The pipette (extracellular) solution contained (mM): 140 N-methyl-D-glucamine (NMDG), 140 aspartic acid, 5 CaCl2, 2 MgSO4 and 10 N-tris[Hydroxymethyl]methyl-2-aminoethanesulphonic acid (TES), adjusted to pH 7.3 with Tris ([Cl], 10 mM). The bath (intracellular) solution contained (mM): 140 NMDG, 3 MgCl2, 1 CsEGTA and 10 TES, adjusted to pH 7.3 with HCl ([Cl], 147 mM; free [Ca2+], <10−8 M) and was maintained at 37°C. CFTR was activated, channel rundown was prevented and the effects of loop diuretics were tested as described previously for CFTR blockade by niflumic acid (Scott-Ward et al., 2004).

For single-channel studies, we used membrane patches containing ≤3 active channels. To determine channel number, we used the maximum number of simultaneous channel openings observed during the course of an experiment (Cai et al., 2006). To investigate the voltage and external Cl concentration dependence of channel block, we used voltage-ramp protocols to acquire current–voltage (I–V) relationships (Cai et al., 2003). To explore the time dependence of blockade, we bathed membrane patches in symmetrical Cl-rich solutions and stepped voltage first from 0 to −100 mV and then from −100 to +100 mV before returning to 0 mV; the duration of each voltage step was 250 ms. We averaged multiple current records (5–30) acquired in the absence and presence of drugs before subtracting basal currents with no active channels recorded in the absence of PKA (75 nM) and ATP (1 mM) to isolate macroscopic CFTR Cl currents.

We recorded, filtered and digitized data as described previously [macroscopic (Scott-Ward et al., 2004); single-channel (Sheppard and Robinson, 1997)]. Time-course data were analysed and the relationship between drug concentration and CFTR inhibition was fitted to the Hill equation as described (Scott-Ward et al., 2004). To investigate the time dependence of CFTR inhibition following a hyperpolarizing voltage step, exponential functions were fit to current relaxations; capacitance transients were excluded from these fits. To measure single-channel current amplitude (i), Gaussian distributions were fit to current amplitude histograms. For open probability (Po) and kinetic analyses, lists of open and closed times were created and dwell-time histograms were constructed, fitted and statistically tested as previously described (Winter et al., 1994; Sheppard and Robinson, 1997). Only membrane patches that contained a single-active CFTR Cl channel were used for kinetic analyses.

Ussing chamber studies

We measured CFTR-mediated apical membrane Cl current (IClapical) in FRT epithelia after permeabilizing the basolateral membrane with nystatin as described previously (Li et al., 2004).

Data analysis

Results are expressed as means ± SEM of n observations. To compare sets of data, we performed either an anova or Student's t-test using SigmaStatTM (Systat Software Inc., Richmond, CA, USA). Differences were considered statistically significant when P < 0.05.

Materials

Unless specified, chemicals were purchased from the Sigma-Aldrich Company Ltd. (Gillingham, UK). Xipamide and piretanide were generous gifts of Dishman Europe Ltd. (London, UK) and Sanofi-Aventis Deutschland GmbH (Frankfurt am Main, Germany) respectively. Genistein was obtained from LC Laboratories (Woburn, MA, USA), CFTRinh-172 from Calbiochem (Merck Chemicals Ltd., Nottingham, UK) and PKA purified from bovine heart was from Promega UK (Southampton, UK) or Calbiochem.

ATP was dissolved in intracellular solution, forskolin in methanol and all other reagents in DMSO. Stock solutions were stored at −20°C, with the exception of ATP, which was prepared directly before each experiment. Immediately before use, stock solutions were diluted to achieve final concentrations and, where necessary, the pH of the intracellular solution was readjusted to pH 7.3 to avoid pH-dependent changes in CFTR function (Chen et al., 2009). DMSO did not affect CFTR activity (Sheppard and Robinson, 1997).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

Loop diuretics inhibit CFTR Cl currents

In this study, we investigated how the loop diuretics, bumetanide, furosemide, piretanide and xipamide, inhibit CFTR. Inspection of their chemical structures identifies a number of features, which could influence channel block (Figure 1; Li and Sheppard, 2009). All are large, organic anions, each possesses a benzenesulfonamide moiety, and with the exception of xipamide, each possesses a carboxyl group. Because the chemical structures of loop diuretics show some similarities to that of glibenclamide, the best-studied CFTR blocker (Figure 1), we compared the effects of loop diuretics with that of glibenclamide.

To investigate loop diuretic inhibition of CFTR, we studied CFTR Cl currents in excised inside-out membrane patches from C127 cells expressing wild-type human CFTR. Following CFTR activation, we added increasing concentrations of loop diuretics (10–250 μM) to the intracellular solution in the continuous presence of PKA (75 nM) and ATP (0.3 mM) and monitored current inhibition (Figure 2). For each loop diuretic, the relationship between drug concentration and current inhibition at −50 mV was well fitted by the Hill equation [furosemide: drug concentration causing half-maximal inhibition (Ki) = 71.24 ± 15.08 μM, Hill coefficient (n) = −1.07 ± 0.19; xipamide: Ki = 45.03 ± 3.51 μM, n = −0.78 ± 0.07; bumetanide: Ki = 55.58 ± 10.68 μM, n = −1.04 ± 0.10; piretanide: Ki = 57.73 ± 17.62 μM, n = −1.00 ± 0.07; n = 5 for all values; Figure 2]. When tested at lower concentrations, neither furosemide nor piretanide inhibited significantly CFTR [furosemide (1 μM), IDrug/IControl = 92.7 ± 2.6%; piretanide (1 μM), IDrug/IControl = 96.3 ± 1.8%; n = 6 for both; P > 0.1]. Channel blockade by loop diuretics was readily reversible (Figure 2A and C; data not shown). Two conclusions can be drawn from these data: firstly, loop diuretics interact with single site(s) on CFTR; and secondly, they inhibit CFTR with similar potency, which approaches that of glibenclamide (Ki = 36 ± 3 μM, n = −1.1 ± 0.1; n = 6; Sheppard and Robinson, 1997, see also Schultz et al., 1996).

figure

Figure 2. Loop diuretics inhibit CFTR. (A, C) Time courses of CFTR Cl current in excised inside-out membrane patches from C127 cells expressing wild-type human CFTR. During the periods indicated by the bars, ATP (0.3 mM), PKA (75 nM) and either furosemide (furo; 25–250 μM) or piretanide (piret; 20–250 μM) were present in the intracellular solution. Unless otherwise indicated, in this and subsequent figures, voltage was −50 mV and there was a large Cl concentration gradient across the membrane patch ([Cl]internal = 147 mM; [Cl]external = 10 mM). For the purpose of illustration, the time courses have been inverted so that upward deflections represent inward current. (B, D) Hill plots of CFTR inhibition by loop diuretics. Data are means ± SEM (n = 5). The continuous lines are the fits of first-order regression to the data (furosemide, r2 = 0.98; xipamide, r2 = 0.99; bumetanide, r2 = 0.99; piretanide, r2 = 1.00).

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Time dependence following a hyperpolarizing voltage step distinguishes channel blockade by furosemide and piretanide

In preliminary studies (see the Supporting Information Results), we applied noise analysis to macroscopic currents (e.g. Venglarik et al., 1996; Gong et al., 2002; Scott-Ward et al., 2004). In the presence of furosemide and xipamide, but not bumetanide and piretanide, a loop diuretic-induced Lorentzian component was observed (Supporting Information Figure S2; Supporting Information Table S1). These data suggest that the kinetics of CFTR inhibition by bumetanide and piretanide are slower than those of furosemide and xipamide. To explore this idea further, we investigated the time dependence of macroscopic current inhibition because CFTR blockade by some agents (e.g. glibenclamide), but not by others (e.g. tolbutamide), leads to current relaxation following a hyperpolarizing voltage step (Zhou et al., 2002; Zhang et al., 2004a; Cui et al., 2012). We selected for study furosemide and piretanide.

Figure 3A, D and G–I demonstrates that CFTR inhibition by furosemide (100 μM) is time independent following a hyperpolarizing voltage step. Over the duration of a 250 ms voltage step from 0 to −100 mV in the presence of furosemide (100 μM), there was no change in the magnitude of channel blockade (Figure 3A and D). The initial fraction of current remaining was similar to that at steady state, and hence, the current ratio for furosemide was close to 1 (Figure 3G–I). By contrast, CFTR inhibition by piretanide (100 μM) was time dependent following a hyperpolarizing voltage step like that of glibenclamide (50 μM) (Figure 3B, C and E–I). During a 250 ms voltage step from 0 to −100 mV in the presence of either blocker, current relaxed to a steady-state value; relaxation was smooth for piretanide, but sharp for glibenclamide (Figure 3B, C, E and F). For both piretanide and glibenclamide, the initial fraction of current remaining was ∼30% greater than that at steady state, and hence, the current ratio for both blockers was about 0.6 (Figure 3G–I).

figure

Figure 3. Time dependence of CFTR inhibition following a voltage step. (A–C) CFTR Cl currents recorded with (black) and without (grey) furosemide (100 μM), piretanide (100 μM) or glibenclamide (glib; 50 μM) by stepping voltage first from 0 to −100 mV and then from −100 to +100 mV before returning to 0 mV; holding voltage was 0 mV and voltage steps were 250 ms in duration. The data were acquired using membrane patches excised from BHK cells expressing wild-type human CFTR bathed in symmetrical 147 mM Cl solutions. ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution. Dotted lines indicate zero current. (D–F) Higher resolution recordings of currents acquired at −100 mV in the presence of furosemide, piretanide and glibenclamide from the experiments shown in A–C. The grey continuous lines show the fit of exponential functions. (G–I) Fraction of current remaining at the start (initial) and end (steady state; SS) of voltage step and the ratio of the current remaining. Data are means + SEM (n = 4–6); *P < 0.05 versus furosemide; †P < 0.001 versus furosemide. (J–L) Voltage dependence of time constants fit to current relaxations in the presence of piretanide (100 μM) and glibenclamide (50 μM). The piretanide and glibenclamide data were fit with two- and three-component exponential functions respectively. Data are means ± SEM (piretanide, n = 3; glibenclamide, n = 5); *P < 0.05 versus glibenclamide data at −80 mV.

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Consistent with previous results (Zhang et al., 2004a; Cui et al., 2012), Figure 3C demonstrates that there was substantially less current relaxation when membrane voltage was stepped from −100 to +100 mV in the presence of glibenclamide (50 μM) to elicit channel unblocking. Figure 3B shows similar results for piretanide (100 μM) when voltage was stepped from −100 to +100 mV; relief of piretanide blockade at +100 mV was rapid in marked contrast to the slow development of block when voltage was stepped from 0 to −100 mV. However, Figure 3A demonstrates that for furosemide (100 μM), there was no marked asymmetry between the onset and relief of block following voltage steps; both were time independent.

To understand better the time dependence of block development for piretanide and glibenclamide following a hyperpolarizing voltage step, we fitted exponential functions to current relaxations at −80, −100 and −120 mV. Two time constants were required to fit the piretanide data and three the glibenclamide data (Figure 3J–L). For both piretanide and glibenclamide, the slow time constant was voltage independent, but the faster time constants were voltage dependent, slowing with membrane depolarization (Figure 3J–L). We conclude that furosemide and piretanide inhibit macroscopic CFTR Cl currents with distinct kinetics, with those of piretanide resembling glibenclamide blockade of CFTR (Zhang et al., 2004a).

Furosemide and piretanide inhibit single channels with distinct kinetics

To understand better how loop diuretics inhibit CFTR, we studied single channels. Figures 4 and 5 demonstrate that furosemide and piretanide markedly altered CFTR gating. The gating behaviour of wild-type human CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures and separated by longer closures between bursts (Figures 4A and 5A). Furosemide (100 μM)-induced blocking events were very brief, with the result that the frequency of flickery closures interrupting bursts of channel openings increased markedly (Figure 4A). To quantify channel block, we measured i and Po. Furosemide (100 μM) caused a small reduction in i (17%; P < 0.05), but a large decrease in Po (59%; P < 0.05) (Figure 4B and C).

figure

Figure 4. Furosemide block of individual CFTR Cl channels. (A) Representative recordings show the effects of furosemide (100 μM) on a single CFTR Cl channel in an excised inside-out membrane patch from a C127 cell expressing wild-type human CFTR. ATP (0.3 mM) and PKA (75 nM) were continuously present in the intracellular solution. Dotted lines indicate the closed-channel state and downward deflections channel openings. Beneath each prolonged 10 s recordings, 1 s portions indicated by the bars are shown on an expanded time scale. (B) Single-channel current amplitude histograms in the absence and presence of furosemide (100 μM) from the experiment shown in A. The continuous lines are the fit of Gaussian distributions to the data. The vertical dashed lines indicate the positions of the open and closed channels under control conditions; the closed-channel amplitude is shown on the right. (C) Effects of furosemide (100 μM) on i (top) and Po (bottom). Data are means + SEM (n = 6); *P < 0.05 versus the control.

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figure

Figure 5. Piretanide block of individual CFTR Cl channels. (A) Representative recordings show the effects of piretanide (100 μM) on a single CFTR Cl channel in an excised inside-out membrane patch from a C127 cell expressing wild-type human CFTR. ATP (0.3 mM) and PKA (75 nM) were continuously present in the intracellular solution. Beneath each prolonged 10 s recordings, 1 s portions indicated by the bars are shown on an expanded time scale. (B) Single-channel current amplitude histograms in the absence and presence of piretanide (100 μM) from the experiment shown in A. (C) Effects of piretanide (100 μM) on i (top) and Po (bottom). Data are means + SEM (n = 6); *P < 0.05 versus the control. Other details as in Figure 4.

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In the presence of piretanide (100 μM), bursts of channel openings were interrupted by many drug-induced blocking events (Figure 5A). Piretanide (100 μM) decreased markedly Po (54%; P < 0.05), but was without effect on i (P = 0.95) (Figure 5B and C). Thus, consistent with the time dependence of channel block following a hyperpolarizing voltage step, furosemide and piretanide have distinct effects on the single-channel activity of CFTR. Moreover, the effects of piretanide, but not furosemide, resemble those of glibenclamide (Schultz et al., 1996; Sheppard and Robinson, 1997).

Furosemide and piretanide alter the dwell-time distributions of CFTR

To determine how furosemide and piretanide decreased Po, we investigated gating kinetics using membrane patches that contained only a single active channel. Consistent with previous results (e.g. Winter et al., 1994), the open- and closed-time histograms of wild-type human CFTR were best fitted with one- and two-component exponential functions, respectively, described by the time constants τO2, τC1 and τC3 (Figure 6A and C; Supporting Information Table S2).

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Figure 6. Dwell-time histograms of CFTR inhibited by furosemide and piretanide. Representative dwell-time histograms of single wild-type CFTR Cl channels recorded in the absence (A, C) and presence of either furosemide (100 μM; B) or piretanide (100 μM; D). Data are from experiments in which membrane patches, from C127 cells expressing wild-type human CFTR, contained only one active channel studied using the experimental conditions described in Figure 4. The continuous lines are the fit of one-, two- or three-component exponential functions to the data and the dotted lines show the individual components of these functions. The vertical dashed lines indicate mean values of the open- (τO2) and closed- (τC1, τC3) time constants under control conditions. Logarithmic x-axes, with 10 bins per decade, were used for dwell-time histograms.

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Like meglitinide (Cai et al., 1999), in the presence of furosemide (100 μM), open- and closed-time histograms were best fitted with one- and two-component exponential functions, respectively (Figure 6B; Supporting Information Table S2); furosemide-induced blocking events could not be distinguished clearly from brief closures interrupting channel openings. Furosemide (100 μM) decreased the open-time constant (τO2) by 12.5-fold (Figure 6B; Supporting Information Table S2). It increased the fast closed-time constant (τC1) by 1.4-fold, while this time constant's share of the closed-time distribution expanded to 95% (Figure 6B; Supporting Information Table S2). However, furosemide (100 μM) was without effect on the slow closed-time constant (τC3), although this time constant's share of the closed-time distribution decreased to 5% (Figure 6B; Supporting Information Table S2).

In the presence of piretanide (100 μM), open and closed times were best fitted with two- and three-component exponential functions respectively (Figure 6D; Supporting Information Table S2). The new population of open times was described by a fast open-time constant (τO1), whereas the new population of closed times was described by an intermediate closed-time constant (τC2) (Figure 6D; Supporting Information Table S2). In addition, piretanide (100 μM) decreased τO2 by 1.3-fold and τC1 by 1.8-fold, but had little effect on τC3 (Figure 6D; Supporting Information Table S2). The share of the closed-time distribution occupied by τC1 decreased to 23%, whereas that occupied by τC3 was unaffected (Figure 6D; Supporting Information Table S2). As a result, τC2 occupied 45% of the closed-time distribution (Figure 6D; Supporting Information Table S2). Thus, with some differences in dwell-time duration, the effects of piretanide on CFTR gating kinetics are reminiscent of those of glibenclamide (Sheppard and Robinson, 1997).

The data in Figure 6 and Supporting Information Table S2 demonstrate that furosemide and piretanide decreased the open time of CFTR, suggesting that like glibenclamide (Sheppard and Robinson, 1997), they might be open-channel blockers of CFTR. Open-channel block of CFTR is described by the simple kinetic model:

  • display math(1)

where C, O and B represent the closed, open and blocked states of the channel, respectively; kopen and kclose are the transition rates for channel opening and closing; kon is the second-order binding constant for drug binding to CFTR; and koff is the first-order rate constant for drug dissociation from CFTR. The equilibrium dissociation constant for drug binding to CFTR, Kd = koff/kon; koff = 1/τc and kon = (1/τo) × [drug]−1. For piretanide, using values of τO1 and τC2 from Supporting Information Table S2, we calculated values of kon = 1.67 × 106 M−1·s−1, koff = 156 s−1 and Kd = 89 μM at −50 mV. Comparison of these data with those of glibenclamide (kon = 3.5 × 106 M−1·s−1, koff = 92 s−1 and Kd = 26 μM; Sheppard and Robinson, 1997) reveals that glibenclamide is a more potent blocker than piretanide because it binds twofold faster and remains bound 40% longer.

Mechanism of loop diuretic inhibition of CFTR

CFTR is inhibited by open-channel and allosteric mechanisms (for review, see Li and Sheppard, 2009). To test whether loop diuretics are open-channel blockers of CFTR, we investigated the voltage dependence of macroscopic current blockade (Scott-Ward et al., 2004). Under control conditions, CFTR Cl currents exhibited weak inward rectification (Figure 7A and B; Cai et al., 2003). Consistent with our current relaxation data (Figure 3A and B), Figure 7A–D demonstrates that CFTR inhibition by both furosemide (100 μM) and piretanide (100 μM) is voltage dependent: at negative voltages, current magnitude was decreased strongly, whereas at positive voltages, inhibition was relieved robustly (piretanide) or current was augmented weakly above the control level (furosemide); enhancement of CFTR Cl current at positive voltages has been observed previously with some CFTR blockers [e.g. 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); Wang et al., 2005]. Using Equation (2), we calculated the voltage-dependent dissociation constant (Kd) for CFTR inhibition by furosemide and piretanide:

  • display math(2)

where Kd(V) is the voltage-dependent dissociation constant at voltage V, and I and Io are current values in the presence and absence of drug respectively. Figure 7E and F demonstrates that Kd values are voltage dependent and that furosemide is a weaker inhibitor than piretanide [furosemide, Kd(0 mV) = 369 ± 97 μM (n = 5); piretanide, Kd(0 mV) = 185 ± 25 μM (n = 5)]. However, both loop diuretics are weaker inhibitors than glibenclamide [Kd(0 mV) = 37 ± 6 μM (n = 5); Sheppard and Robinson, 1997].

figure

Figure 7. Voltage and external Cl concentration dependence of CFTR inhibition. (A, B) I−V relationships of CFTR Cl currents recorded in the absence and presence of furosemide (100 μM) and piretanide (100 μM), respectively, when membrane patches were bathed in symmetrical 147 mM Cl solutions. ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution. (C, D) Effects of voltage on CFTR Cl current inhibition by furosemide (100 μM) and piretanide (100 μM). The continuous lines are the fit of sigmoidal curves with three parameters to the data. (E, F) Relationship between the voltage-dependent dissociation constant (Kd) and voltage for CFTR inhibition by furosemide and piretanide, respectively, when the external Cl concentration was either 147 or 10 mM. All data were acquired using BHK cells expressing wild-type human CFTR. In C–F, data are means ± SEM (n = 5–6) at each voltage. In E and F, the continuous lines are the fits of first order regressions to Kd values over the range −100 to −40 mV extrapolated to 0 mV (furosemide: [Cl]external = 147 mM, r2 = 0.98; [Cl]external = 10 mM, r2 = 0.99; piretanide: [Cl]external = 147 mM, r2 = 0.98; [Cl]external = 10 mM, r2 = 0.99).

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The electrical distance across the membrane sensed by blocking ions is calculated using the relationship (Woodhull, 1973):

  • display math(3)

where z′ is the apparent valency of the blocking ion [defined as the actual valency of the blocking ion (z) multiplied by the electrical distance across the membrane experienced by the blocking ion (δ)], and F, R and T are the Faraday constant, gas constant and absolute temperature respectively. Using the data in Figure 7E and F and assuming single binding sites for furosemide, z′ = 0.37 ± 0.07 (n = 5) and for piretanide, z′ = 0.25 ± 0.04 (n = 5), measured from the inside of the membrane. For comparison, for glibenclamide, z′ = 0.48 ± 0.08 (n = 5) (Sheppard and Robinson, 1997). While these data demonstrate that furosemide penetrates deeper into the transmembrane electric field than piretanide, comparison with glibenclamide is complicated because furosemide and piretanide possess two negative charges located on different parts of their structures, whereas glibenclamide has a single negative charge.

Next, we investigated whether Cl flow through the CFTR pore prevents macroscopic current blockade (Scott-Ward et al., 2004). When the external Cl concentration was reduced to 10 mM by substituting Cl with the impermeant anion aspartate, the potency of furosemide inhibition was enhanced, whereas the electrical distance sensed was reduced (external [Cl] = 147 mM, Kd(0 mV) = 369 ± 97 μM; z′ = 0.37 ± 0.07; n = 5; external [Cl] = 10 mM, Kd(0 mV) = 136 ± 23 μM; z′ = 0.29 ± 0.02; n = 6; P < 0.05) (Figure 7E). However, piretanide inhibition of CFTR was unaffected (external [Cl] = 147 mM, Kd(0 mV) = 185 ± 25 μM; z′ = 0.25 ± 0.04; external [Cl] = 10 mM, Kd(0 mV) = 236 ± 27 μM; z’ = 0.18 ± 0.02; n = 5 for all values; P > 0.11) (Figure 7F). Thus, like glibenclamide (Sheppard and Robinson, 1997), furosemide, but not piretanide, might directly or indirectly compete with Cl for a common binding site within the CFTR pore.

CFTR blockade by some allosteric inhibitors is relieved by increasing the intracellular ATP concentration (Li and Sheppard, 2009). Supporting Information Figure S3 demonstrates that CFTR inhibition by furosemide (100 μM) and piretanide (100 μM) was unaffected by raising the intracellular ATP concentration from 0.3 to 5 mM. This result suggests that like glibenclamide (Sheppard and Robinson, 1997), neither loop diuretic inhibits CFTR by interfering with ATP-dependent channel gating. However, some allosteric inhibitors interfere with CFTR gating without competing with ATP [e.g. GaTx1 (Fuller et al., 2007); CFTRinh-172 (Kopeikin et al., 2010)], whereas other agents (e.g. thyroid hormones, Cai et al., 2013) act at multiple steps within the CFTR gating pathway. Therefore, we investigated whether loop diuretic-blocked channels would first reopen before closing on washout of loop diuretic, PKA and ATP from the intracellular solution (see the Supporting Information Results and Scott-Ward et al., 2004). Supporting Information Figure S4 demonstrates that on washout of furosemide, i returned to its control level before channels became quiescent. This suggests that furosemide accesses its binding site on CFTR from the open channel configuration.

Loop diuretics inhibit weakly CFTR Cl channels in the apical membrane of FRT epithelia

To evaluate the effects of loop diuretics on CFTR during transepithelial Cl transport, we studied CFTR-mediated apical membrane Cl current (IClapical) in FRT epithelia heterologously expressing wild-type human CFTR. Figure 8 shows that each loop diuretic (tested at 100 μM) caused a time-dependent attenuation of IClapical, blocking ∼30% of the control current 20 min after drug addition. As controls, we demonstrated that IClapical was unaffected by the Na+-K+ ATPase inhibitor ouabain (1 μM), but abolished by the thiazolidinone CFTR inhibitor CFTRinh-172 (10 μM) (Ma et al., 2002) (Figure 8). Thus, like glibenclamide (e.g. Sheppard et al., 1994; Li et al., 2004), bumetanide, furosemide, piretanide and xipamide are open-channel blockers that attenuate weakly CFTR-mediated transepithelial Cl transport.

figure

Figure 8. Loop diuretics inhibit apical membrane CFTR Cl currents. (A–D) Time courses of IClapical in FRT epithelia expressing wild-type human CFTR. During the periods indicated by the bars: (i) forskolin (Fk, 10 μM) and genistein (Gen, 50 μM); (ii) loop diuretics (100 μM); (iii) ouabain (1 μM); and (iv) CFTRinh-172 (C172, 10 μM) were present in the apical and basolateral solutions. Prior to the recording, the basolateral membrane was permeabilized with nystatin (0.36 mg·mL−1). Transepithelial voltage was clamped at 0 mV and there was a large Cl concentration gradient across epithelia (basolateral [Cl], 149 mM; apical [Cl], 14.8 mM). (E) Effects of loop diuretics on IClapical. Values represent IClapical measured 20 min after loop diuretic addition normalized to control values recorded immediately before addition and corrected for the spontaneous decay of IClapical in the absence of loop diuretics at this time point (34 ± 6%; n = 6). Data are means + SEM (n = 6, except piretanide where n = 8); *P < 0.05 versus the control.

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Discussion and conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

This study investigated how loop diuretics inhibit CFTR and explored structure–activity relationships. We demonstrated that bumetanide, furosemide, piretanide and xipamide are open-channel blockers of CFTR. With distinct kinetics, they impede Cl flow by occluding the intracellular vestibule of the CFTR pore.

Comparison of the effects of loop diuretics on NKCCs (SLC12A1 and SLC12A2) (Hebert et al., 2004) and CFTR (ABCC7) highlights similarities and differences in their mechanism(s) of action, as discussed in detail below.

Similarities between NKCCs and CFTR

The first similarity is stoichiometry of blockade. For both NKCCs and CFTR, the stoichiometry of loop diuretic blockade is one inhibitor per transport protein (Forbush and Palfrey, 1983; present study).

The second similarity is the state dependence of blockade. Inhibition of NKCCs by loop diuretics requires the simultaneous presence of Na+, K+ and Cl, suggesting inhibition of active transporters (Forbush and Palfrey, 1983; Hannaert et al., 2002). Similarly, loop diuretics block active CFTR Cl channels (Venglarik, 1997; Reddy and Quinton, 1999; present study). The data suggest that loop diuretics access their binding site within the CFTR pore from the open-channel configuration. However, it is possible that loop diuretics might bind this site while the channel is closed because there is evidence that the intracellular vestibule is accessible in the closed-channel configuration (El Hiani and Linsdell, 2010).

The third similarity is competition with Cl ions. Early studies suggested that loop diuretics compete directly or indirectly with Cl for a common binding site on NKCC1 (Ludens, 1982; Haas and McManus, 1983). Following the molecular identification of NKCC1 (Delpire et al., 1994), the binding sites for Cl and bumetanide were localized to the membrane-spanning domain (MSD) of NKCC1 using human–shark chimeric constructs (Isenring and Forbush, 1997). Within this region, residues in transmembrane segment 2 (M2), M4 and M7 determine the difference in Cl-binding affinity between human and shark NKCC1, whereas residues in M2, M4, M7, M11 and M12 specify the difference in bumetanide binding (Isenring et al., 1998a,b). For CFTR, block by furosemide, but not piretanide, is relieved by inwardly directed Cl flow. The simplest interpretation of these data is that furosemide and Cl might directly or indirectly compete for a common binding site within the CFTR pore. However, reducing the external Cl concentration likely alters electrostatic potentials within the CFTR pore (Liu et al., 2003), complicating data interpretation.

Differences between NKCCs and CFTR

The first difference is the site of blockade. Loop diuretics interact with a site accessible from the extracellular side of the membrane to inhibit NKCCs (Forbush and Palfrey, 1983; Isenring and Forbush, 1997). By contrast, the slow onset of CFTR inhibition when loop diuretics are added to the outside of cells, but the rapid development of blockade in excised membrane patches (Reddy and Quinton, 1999; present study), suggests that loop diuretics access their binding site on CFTR from the intracellular side of the membrane. Consistent with this idea, the voltage dependence of loop diuretic inhibition of CFTR argues that these agents are open-channel blockers that occlude the intracellular vestibule of the CFTR pore (Li and Sheppard, 2009; present study). Moreover, the time dependence of piretanide blockade following a hyperpolarizing voltage step suggests that like glibenclamide (Zhang et al., 2004a), it might interact with multiple sites to prevent Cl flow.

The second difference is blocker affinity and rank order of inhibition. Reminiscent of the effects of glibenclamide on pancreatic beta cell ATP-sensitive K+ channels and CFTR (Inagaki et al., 1995; Sheppard and Robinson, 1997), a notable difference between NKCCs and CFTR is affinity of block. For example, bumetanide inhibits recombinant human NKCC1 with 200-fold greater potency than recombinant human CFTR [NKCC1, Ki(bumetanide) = 0.25 μM (Isenring et al., 1998a); CFTR, Ki(bumetanide) = 56 μM (excised membrane patches; present study)]. This difference in blocker affinity is accentuated when CFTR is studied in polarized epithelia (Reddy and Quinton, 1999; present study), arguing that CFTR inhibition is an unlikely consequence of diuretic therapy. Loop diuretics inhibit NKCCs in the rank order: bumetanide > piretanide > furosemide = xipamide with block by bumetanide substantially stronger than that of furosemide (Piyasena et al., 1975; Schlatter et al., 1983; Hannaert et al., 2002). By contrast, loop diuretics inhibit CFTR with little difference in potency (rank order: xipamide ≥ bumetanide = piretanide ≥ furosemide) (present study). Thus, the structural features of loop diuretics, which determine CFTR blockade, are likely to differ from those that confer NKCC inhibition.

The third difference is the voltage dependence of blockade. NKCCs are electroneutral transporters (Haas and Forbush, 2000), suggesting that membrane voltage has little influence on loop diuretics (but, see Paredes et al., 2006). In contrast to NKCCs, anion flow through CFTR is driven by the transmembrane electrochemical gradient, and channel blockade by loop diuretics is voltage dependent (present study). Comparison with other CFTR blockers [e.g. NPPB (Zhang et al., 2000); glibenclamide (Sheppard and Robinson, 1997)] suggests that the anionic form of loop diuretics inhibits CFTR. Consistent with this idea, under the experimental conditions used, the carboxyl moieties of bumetanide, furosemide and piretanide, and the phenolic group of xipamide will all be deprotonated and carry negative charges (Ruiz-Angel et al., 2004).

Structure–activity analysis of NKCC and CFTR inhibition by loop diuretics

Structure–activity studies of NKCC inhibition by loop diuretics suggest that potent NKCC2 inhibition requires a phenyl ring possessing (i) a sulfonamide group at position 5; (ii) an acidic group (e.g. carboxylate) at position 1; (iii) a secondary or tertiary amine at positions 2 or 3 (furosemide, furfurylamine; bumetanide, butylamine; piretanide, pyrrolidine); and (iv) an apolar group at position 4 (furosemide, chloro; bumetanide and piretanide, phenoxy) (Schlatter et al., 1983; Cabantchik and Greger, 1992). Moreover, using NKCC1 structure–function data (Isenring et al., 1998a,b), Hannaert et al. (2002) speculated that position 1 interacts with the Cl binding site of NKCC1; positions 2 and 3, the cation binding sites, and positions 4 and 5, NKCC1 MSD sequences distinct from the Na+, K+ and Cl binding sites (see figure 4 of Hannaert et al., 2002).

The present data suggest that the chemical structure of loop diuretics influences the kinetics of CFTR inhibition. Furosemide and xipamide, which possess a chloro group at position 4, block CFTR with ‘very fast’ speed [residency time (1/koff) ∼1 ms; see the Supporting Information Results) and block by furosemide is time independent following a hyperpolarizing voltage step. By contrast, bumetanide and piretanide, which carry a phenoxy group at position 4, inhibit CFTR with ‘intermediate’ speed (piretanide residency time ∼6 ms) and inhibition by piretanide is time dependent following a hyperpolarizing voltage step. These data argue that the phenoxy group might slow loop diuretic access to its binding site and stabilize docking within the CFTR pore, possibly through interactions with aromatic residues. We speculate that piretanide, with its larger size and greater lipophilicity, wedges more tightly into the CFTR pore than furosemide (molar volume: piretanide, 256 cm3·mol−1; furosemide, 206 cm3·mol−1; polar surface area: piretanide, 181 Å2; furosemide, 131 Å2). This difference might also explain three further distinctions between furosemide and piretanide: (i) the deeper penetration of furosemide into the transmembrane electric field to reach its binding site within the intracellular vestibule of the CFTR pore; (ii) the relief by inwardly directed Cl flow of block by furosemide, but not piretanide; and (iii) the asymmetric time dependence of block onset and relief for piretanide, but not furosemide, following voltage steps.

Knowledge of CFTR inhibition by loop diuretics and other open-channel blockers might guide molecular modelling efforts to define the architecture of the intracellular vestibule and identify drug-binding sites within the CFTR pore (Zhou et al., 2001; Linsdell, 2005; St. Aubin et al., 2007; Dalton et al., 2012; Norimatsu et al., 2012a,b). They might also inform the design of innovative small molecule CFTR inhibitors for the treatment of secretory diarrhoea, autosomal dominant polycystic kidney disease and some reproductive disorders (Chan et al., 2009; Li and Sheppard, 2009; Verkman and Galietta, 2009). The widely used open-channel blocker glibenclamide might be used as a framework to engineer higher affinity blockers of the intracellular vestibule. Glibenclamide penetrates deeply into the intracellular vestibule and binds tightly by interacting at multiple sites (Cai et al., 1999; Zhou et al., 2002; Zhang et al., 2004a,b; Dalton et al., 2012). Nevertheless, tighter binding, and hence, higher affinity might be achieved by matching its size to better fit the intracellular vestibule and modifying its charge and/or lipophilicity. Previously, Bachmann et al. (1999) reported that two metabolites of glibenclamide hydroxylated at either the 3-cis or 4-trans positions of the cyclohexyl ring (see Figure 1) had equi- or lower potency than glibenclamide. Because glibenclamide and piretanide inhibit CFTR with similar kinetics, it is unclear whether substitution of the chloro group in glibenclamide by a phenoxy group would enhance further potency, as suggested by the present results. Alternatively, incorporation of additional halide moieties might achieve this goal. In support of this idea, the glycine hydrazide, GlyH-101, the open-channel blocker that occludes the extracellular vestibule by wedging its hydrophobic tail into the pore constriction, possesses two bromo groups (Muanprasat et al., 2004; Norimatsu et al., 2012b). Moreover, phloxine B, a CFTR potentiator that also acts as an open-channel blocker of the intracellular vestibule, possesses multiple halogen groups (Cai and Sheppard, 2002). Identification of the pharmacophore for open-channel blockers that occlude the intracellular vestibule should be a priority for future studies.

In conclusion, CFTR inhibition by furosemide and piretanide resembles block by tolbutamide and glibenclamide. Furosemide and tolbutamide block single channels with ‘very fast’ speed, and blockade of macroscopic currents is time independent following a hyperpolarizing voltage step (Venglarik et al., 1996; Cui et al., 2012; present study). By contrast, piretanide and glibenclamide block single channels with ‘intermediate’ speed, and blockade of macroscopic currents is time dependent following a hyperpolarizing voltage step (Sheppard and Robinson, 1997; Cui et al., 2012; present study). Previous work argues that glibenclamide interacts with multiple sites within the intracellular vestibule of the CFTR pore (Cai et al., 1999; Zhou et al., 2002; Zhang et al., 2004a,b; Dalton et al., 2012). The same is likely the case for loop diuretics, which, with the exception of xipamide, possess both benzenesulfonamide moieties and carboxyl groups, negatively charged side chains predicted to interact with pore-lining residues. In the case of bumetanide and piretanide, their phenoxy group is likely to provide an additional site of interaction to stabilize binding within the intracellular vestibule of the CFTR pore. This locking mechanism is absent in furosemide and xipamide, which lack the phenoxy group. We conclude that differences in molecular dimensions and lipophilicity affect open-channel blockade of CFTR by loop diuretics.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information

We thank HR de Jonge and our departmental colleagues for valuable discussions and MD Amaral, LJV Galietta, CR O'Riordan, Dishman Europe Ltd. and Sanofi-Aventis Deutschland GmbH for generous gifts of cells and small molecules. This work was supported by the Cystic Fibrosis Trust; J. L. was supported by scholarships from the University of Bristol and the Overseas Research Students Awards Scheme of Universities UK and P. K. by the Strategic Scholarships Fellowships Frontier Research Networks, Office of the Higher Education Commission of Thailand.

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  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion and conclusions
  7. Acknowledgements
  8. Conflicts of interest
  9. References
  10. Supporting Information
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bph12458-sup-0001-si.tif257K

Figure S1 Shows overlays of the chemical structures of the four loop diuretics studied.

bph12458-sup-0002-si.tif2011K

Figure S2 Uses noise analysis to explore the kinetics of CFTR block by loop diuretics.

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Figure S3 Investigates whether loop diuretics are allosteric blockers of CFTR by exploring whether changing the intracellular ATP concentration affects the efficacy of channel block.

bph12458-sup-0004-si.tif15586K

Figure S4 Examines whether loop diuretic-blocked channels reopen first before closing on washout of loop diuretic, PKA and ATP from the intracellular solution.

bph12458-sup-0005-si.docx40K

Table S1 Effects of loop diuretics on corner frequencies derived from spectral analysis of CFTR Cl currents.

Table S2 Effects of furosemide and piretanide on the open- and closed-time constants of wild-type CFTR.

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