The erythroid K-Cl cotransport inhibitor [(dihydroindenyl)oxy]acetic acid blocks erythroid Ca²⁺-activated K⁺ channel KCNN4

Abstract

Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca²⁺-activated K⁺ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K⁺ transporters and K⁺ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K⁺ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K⁺ uptake. In contrast, DIOA exhibited no inhibitory effect on K⁺ influx into A23187-treated red cells of Kcnn4^−/− mice. DIOA inhibition of human KCa3.1 was validated (IC₅₀ 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.

INTRODUCTION

The therapeutic regulation of erythrocyte volume has been acknowledged as a promising approach toward the development of adjunct treatments for sickle cell disease (1–3). The delay time for polymerization of deoxy-hemoglobin S (HbS) is an inverse function of the 30th–50th exponential power of deoxy-HbS concentration within the red blood cell (4). Red cell volume is controlled predominantly by K⁺ transporters and channels. The most important of these transmembrane erythroid K⁺ carriers are the K-Cl cotransporter KCC3 (with a lesser contribution from KCC1 and, in the human red cell, additional potential contribution from KCC4) (2, 5), and the Gardos channel—the intermediate conductance, Ca²⁺-activated K⁺ channel also known as KCNN4, KCa3.1, or IK1 (2, 6, 7). The Gardos channel is the principal (if not unique) K⁺ conductance of the red cell (8). The Gardos channel inhibitor clotrimazole (9) and its higher affinity and higher specificity congeners TRAM-34 (10) and senicapoc (11) have all been shown to regulate red cell volume, leading to proposed clinical use as adjunct treatments for sickle cell disease. Inhibitors of the K-Cl cotransporters should, in theory, be helpful in this strategy to control red cell volume. However, unlike for the neuronally expressed KCC2, specific inhibitors of high-affinity targeting KCC3, KCC1, and KCC4 are less extensively developed (12).

Several KCC inhibitors of low specificity and affinity have been used for pharmacological identification of K-Cl cotransport activity and to inhibit erythroid K-Cl cotransport activity during studies of other red cell K⁺ transport processes, including that mediated by the KCNN4 Gardos channel (12). The inexpensive loop diuretic bumetanide at the high concentration of 1 mM is commonly used to inhibit ∼80% of K-Cl cotransport while also completely inhibiting red cell NKCC1-mediated Na-K-Cl cotransport. However, DIOA has been shown to inhibit K-Cl cotransport with IC₅₀ of 10 µM (13), reportedly without inhibition of NKCC1 at the 100 µM concentration usually used for complete inhibition of K-Cl cotransport in red cells (14).

While assessing K-Cl cotransport in red cells from affected members of a family with hereditary xerocytosis (HX) secondary to KCNN4 heterozygous mutation V282M (15, 16), we noticed that the use of 100 µM DIOA appeared to be associated with inhibition of A23187-stimulated KCNN4 activity in normal and HX red cells. Since the activity of K-Cl cotransport is often pharmacologically blocked during radioisotopic assessment of KCNN4 activity, this observation led us to characterize DIOA as a potentially confounding inhibitor of KCNN4.

METHODS

Materials

Human blood obtained from research blood donors and from samples discarded after use for clinically indicated laboratory testing was used for these studies under protocols approved by the Institutional Review Boards of Boston Children’s Hospital and Beth Israel Deaconess Medical Center. ⁸³Rb was obtained from Meta Isotopes, LLC (Lubbock, TX). R-(+)-[(2-n-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid, also known as [(dihydroindenyl)oxy]acetic acid (DIOA) was from Sigma (St. Louis, MO). Senicapoc (SCP) was a gift from Pfizer, Inc. Salts and other drugs were obtained from Sigma.

Animals

Mouse blood was obtained by terminal exsanguination from mice bred, maintained, and used under Institutional Animal Care and Use Committee (IACUC) protocols approved by Beth Israel Deaconess Medical Center. C57Bl6/J wild-type (WT) mice were from our own colony. Kcc1^−/−;Kcc3^−/− mice (2, 5), Kcnn4^−/− mice (2, 17), and SAD mice (2) were maintained in our own colony, as previously described.

Solutions

Human chloride flux medium contained (in mM) 145 NaCl, 2 KCl, 1.5 CaCl₂, 0.15 MgCl₂, 10 glucose, 0.1 ouabain, 1.0 bumetanide, and 10 Tris-MOPS, pH 7.4 at 37°C. Human chloride-free sulfamate flux medium contained (in mM) 145 Na sulfamate, 2 KNO₃, 1.5 Ca(NO₃)₂, 0.15 Mg(NO₃)₂, 10 glucose, 0.1 ouabain, 1.0 bumetanide, and 10 Tris-MOPS, pH 7.4 at 37°C. All solutions for use with human cells exhibited osmolarities of 290–310 mosmol/kg H₂O by freezing point depression (Osmette A, Precision Instruments, Fisher Scientific). Mouse chloride flux medium contained 155 mM NaCl, and mouse chloride-free sulfamate flux medium contained 155 mM Na sulfamate. Other salt and drug concentrations were identical to their human counterparts except that ouabain was 1 mM in mouse solutions. All solutions for use with mouse cells exhibited osmolarities of 330–340 mosmol/kg H₂O by freezing point depression. 5 µM Ca²⁺ ionophore A23187 was added as specified. When in the presence of A23187, extracellular concentrations of CaCl₂ (in chloride media) and CaNO₃ (in chloride-free sulfamate media) were reduced to 100 µM. DIOA was used at 100 µM, Senicapoc was used at 200 nM, and YODA1 was used at 15 µM in both mouse and human solutions unless (in the case of DIOA) otherwise specified.

Unidirectional K⁺ Influx Studies

Whole blood was passed through cotton to remove the plasma and buffy coat. Red blood cells were washed 4× with isotonic choline chloride solution, and hematocrit was measured. Freshly isolated, washed red cells at 3% hematocrit were preincubated at 37°C in chloride-containing or chloride-free (sulfamate) transport media as described in Solutions. Influx was initiated by the addition of 30 µL packed, washed red cells to tubes containing transport media, drugs, and 5 µCi ⁸³RbCl (12) (total volume 1 mL) for 10 min (in the absence of A23187) or for 5 min (in the presence of A23187). Note that Rb⁺ salts have long been used to substitute for the higher energy, shorter-lived radioisotopes of K⁺ (12). For A23187 experiments, ionophore was added to a concentration of 5 µM at time zero, and aliquots of cell suspension removed after 5 min were immediately centrifuged through 0.8 mL cold transport medium containing 5 mM EGTA over a 0.3 mL cushion of n-butylphthalate. In the absence of A23187, aliquots were taken after 10-min incubation. Supernatants were aspirated, and the tube tip containing the cell pellet was cut off. Erythrocyte-associated radioactivity was counted in a gamma counter (Isomedic 41600 HE, ICN Biomedicals, Costa Mesa, CA) and expressed as mmol/L cell × hour. Each condition for each experimental sample was tested in quadruplicate, and the mean of the quadruplicate samples was taken as one experimental value for that condition. Concentration-response curves presented in Fig. 8 represent fits of the data to the Hill equation:

$$\text{Y}=\left[{\text{y}}_{\text{min}}+\left({\text{y}}_{\text{max}}-{\text{y}}_{\text{min}}\right)\right]/\text{1}+\text{X}/{\text{IC}}_{\text{5}0}.$$

Figure 8.

Concentration-response curves for DIOA inhibition of A23187-stimulated Gardos channel flux. A: DIOA concentration-response curve for inhibition of A23187-stimulated K⁺ influx into WT human red cells. B: DIOA concentration-response curve for inhibition of A23187-stimulated K⁺ influx into WT human red cells. DIOA, [(dihydroindenyl)oxy]acetic acid; WT, wild type.

Note that all influx values presented are measured in the presence of ouabain and bumetanide. Red cell exposure to A23187 in the presence of extracellular Ca²⁺ has been shown to hyperpolarize membrane potential by as much as ∼60 mV (18).

Electrophysiology

Whole cell patch clamp experiments on human embryonic kidney (HEK) cells stably expressing hKCa3.1 were performed with a HEKA EPC-10 amplifier, as previously described (19). Briefly, cells were clamped to a holding potential of −80 mV and K_Ca currents were elicited by 200-ms voltage ramps from −120 to +40 mV applied every 10 s. The extracellular solution contained 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES (pH 7.4, 300 mosmol/kg H₂O). The pipette (intracellular) solution contained 154 mM KCl, 10 mM HEPES, 10 mM EGTA, 1.75 mM MgCl₂, and 8.5 mM CaCl₂ (pH 7.2, 290 mosmol/kg H₂O), yielding a free [Ca²⁺] of 1 µM as calculated with MaxChelator. The concentration-response curve data were fit with the Hill equation (Fig. 10B) as described above for the flux data of Fig. 8 (see Unidirectional K⁺ Influx Studies).

Figure 10.

DIOA inhibits Ca²⁺-activated K⁺ currents in HEK-293 cells overexpressing recombinant human KCa3.1. A: current-voltage relationship of human KCa3.1 currents elicited by voltage ramps from −120 to +40 mV and recorded in whole cell patch mode in the absence (Control) and subsequent bath addition of DIOA first at 30 µM, and then at 100 µM. Cells were dialyzed with 1 mM free Ca²⁺ in the pipette solution B: normalized concentration-response curve showing DIOA inhibition of human KCa3.1-mediated K⁺ current. Values are means ± SE (n = 3). DIOA, [(dihydroindenyl)oxy]acetic acid.

Molecular Modeling

The cryo-EM structures of the full-length KCa3.1 channel (20) in the closed state (PDB: 6CNM) were refined using the Rosetta cryo-EM refinement protocol. Conformers of DIOA were generated using Open Eye OMEGA software v. 2.5.1.4, randomly placed within either the pore or the fenestration binding pocket of KCa3.1, and then taken through the three stages of RosettaLigand modeling, progressing from low-resolution conformational sampling and scoring to full atom optimization using Rosetta’s all-atom energy function, as previously described in detail (19, 21). In total 10,000 models were generated for each site, from which the top 1,000 models of the lowest total energy score were selected. Among these 1,000 models, the top 10 models of lowest binding energy were identified and manually inspected for ligand/channel interactions and convergence. The lowest energy poses (typically also the most frequently sampled) are shown in Fig. 11.

Figure 11.

Molecular docking of DIOA to human KCa3.1. Transmembrane view of a Rosetta model of the cryo-EM structure of human KCa3.1 in the closed state (PDB:6CNM). The tetrameric channel is in light-brown ribbon presentation with the four associated calmodulins rendered in purple. The subunit in the back is shown semitransparent. The selectivity filter is indicated with a black arrow. The narrowest point of the inner pore is located at residue V282 (in purple at center of structure). DIOA is shown in stick presentation with carbon colored light blue, oxygen red, chlorine green, and hydrogen white. DIOA docks to the fenestration region with an energy of −15.8 REU (one of the four fenestrations is highlighted by a red box and shown magnified at left). DIOA can also dock within the inner pore (highlighted by black box, magnified at right) with an energy of −12.5 REU. At each docking site, the DIOA pose shown is that most frequently sampled and of lowest energy among the 10 poses of lowest energy. DIOA, [(dihydroindenyl)oxy]acetic acid; REU, Rosetta energy unit.

Statistical Analysis

Two-way comparisons were conducted by Mann–Whitney test. Comparisons among multiple samples were conducted by Kruskal–Wallis ANOVA (GraphPad PRISM 8.4.3).

RESULTS

DIOA Inhibits KCNN4 in a Cl⁻-Independent Manner in Human Red Cells

In the absence of A23187, and in the absence or presence of the ion transport inhibitors senicapoc or DIOA, basal K⁺ influx into normal (WT) human red cells was minimal in either chloride-containing or chloride-deficient (sulfamate-containing) medium (Fig. 1A). However, the calcium ionophore A23187 robustly stimulated K⁺ influx in a chloride-independent manner typical of Gardos channel activity, and that activity was almost completely inhibited by 200 nM senicapoc. Moreover, DIOA at the 100 µM concentration routinely used to maximally inhibit erythroid K-Cl cotransport also inhibited Gardos channel activity as completely as did senicapoc, and in a manner independent of extracellular chloride (Fig. 1B). The same patterns of inhibition were evident in tests of red cells from patients with hereditary xerocytosis (HX) secondary to the KCNN4 mutation V282M. (Fig. 1C and D). As we previously reported (15), KCNN4 V282M mutant HX red cells in this family exhibit reduced Gardos channel activity as measured by A23187-stimulated Gardos channel K⁺ influx assay. Both 100 µM DIOA and 200 nM senicapoc were equally effective as inhibitors of Gardos channel activity in both WT and HX human red cells. Levels of senicapoc-sensitive K⁺ influx and DIOA-sensitive K⁺ influx elicited by A23187 exposure were equivalent in WT human red cells (Fig. 2A and B) and (at the lower levels of activity noted above) in HX red cells with the heterozygous V282M mutation in KCNN4 (Fig. 2C and D).

Figure 1.

DIOA inhibits the Gardos channel in human red cells. A: basal K⁺ influx into WT human red cells in chloride or sulfamate media in the absence of A23187. Cells were exposed to no drug (dashed line), to senicapoc (SCP, 200 nM) or to DIOA (100 µM). B: A23187-stimulated K⁺ influx into WT human red cells in chloride or sulfamate media, in the presence of no drug, senicapoc or DIOA. C: basal K⁺ influx into human hereditary xerocytosis (HX) red cells carrying the KCNN4 mutation V282M, in chloride or sulfamate media in the absence of A23187. Cells were exposed to no drug, senicapoc, or DIOA. D: A23187-stimulated K⁺ influx into human HX V282M red cells in chloride or sulfamate media, in the presence of no drug, SCP or DIOA. *P < 0.05; **P < 0.01; ***P < 0.002 vs. no drug in chloride or in sulfamate, by Mann–Whitney test. DIOA, [(dihydroindenyl)oxy]acetic acid; NDA, no drug added; WT, wild type.

Figure 2.

DIOA-sensitive K⁺ influx in the absence or presence of A23187 is indistinguishable in magnitude from senicapoc-sensitive K⁺ influx. A: basal and A23187-stimulated SCP-sensitive K⁺ influx into human WT red cells. B: basal and A23187-stimulated DIOA-sensitive K⁺ influx into human WT red cells. *P < 0.001 vs. baseline, by Mann–Whitney test. C: basal and A23187-stimulated SCP-sensitive K⁺ influx into human HX V282M red cells. D: basal and A23187-stimulated DIOA-sensitive K⁺ influx into human HX V282M red cells. These data are replotted from the assays portrayed in Fig. 1. #P ≤ 0.01; ##P ≤ 0.0005 vs. WT RBC in same condition (in A and B), by Kruskal–Wallis test with Dunn’s correction. DIOA, [(dihydroindenyl)oxy]acetic acid; HX, hereditary xerocytosis; SCP, senicapoc; WT, wild type.

DIOA Inhibits KCNN4 in a Cl⁻-Independent Manner in Mouse Red Cells

In the absence of A23187, basal K⁺ influx into normal (WT) mouse red cells was minimal in media containing chloride or sulfamate, whether in the absence or presence of senicapoc or DIOA (Fig. 3A). However, A23187 robustly stimulated K⁺ influx in a chloride-independent manner typical of mouse red cell Gardos channel activity. That stimulated activity was inhibited to equivalent extents by 200 nM senicapoc and by 100 µM DIOA. The lower A23187-stimulated Gardos channel activity in mouse red cells as compared with human red cells (Fig. 3B) and the elevated senicapoc-resistant background activity evident in mouse RBC versus human RBC (Fig. 3B and C) are consistent with our previous observations (22). A23187-stimulated K⁺ influx sensitive to DIOA and that sensitive to senicapoc were indistinguishable in WT mouse red cells (Figs. 3C and D).

Figure 3.

DIOA inhibits the Gardos channel in mouse red cells. A: basal K⁺ influx into WT mouse red cells in chloride or sulfamate media in the absence of A23187. Cells were exposed to no drug (dash), to SCP (200 nM), or to DIOA (100 µM). #P < 0.02 vs. no drug in sulfamate (Mann–Whitney test). B: A23187-stimulated K⁺ influx into WT mouse red cells in chloride or sulfamate media, in the presence of no drug, SCP, or DIOA. C: basal and A23187-stimulated SCP-sensitive K⁺ influx into mouse WT red cells. D: basal and A23817-stimulated DIOA-sensitive K⁺ influx into mouse WT red cells. Data in C and D are derived from data in A and B. *P < 0.008; **P < 0.003 vs. no drug added (NDA) in B; and vs. corresponding baseline in C and D (Mann–Whitney test). DIOA, [(dihydroindenyl)oxy]acetic acid; HX, hereditary xerocytosis; SCP, senicapoc; WT, wild type.

DIOA Inhibition of KCNN4 Gardos Channel Activity in Mouse RBC Persists in Mouse RBC Devoid of K-Cl Cotransport and Requires KCNN4 Expression

Erythroid K-Cl cotransport in normal human RBC is nearly quiescent in the absence of activating stimuli that include hypotonic swelling, staurosporine, or urea (22, 23). However, mouse erythroid K-Cl cotransport exhibits a relatively higher basal activity in the absence of the same stimuli (22). To rule out a contribution from basal or atypically activated K-Cl cotransport to the DIOA-sensitive fraction of A23187-stimulated K⁺ influx, we examined K⁺ influx into Kcc1^−/−;Kcc3^−/− mouse red cells in the presence of extracellular chloride. These red cells lack nearly all K-Cl cotransport sensitive to hypotonic swelling, staurosporine, or urea and exhibit reduced levels of unstimulated K⁺ influx (2, 5). Figure 4A shows that Kcc1^−/−;Kcc3^−/− mouse red cells exhibit a 44.3% reduction in basal K⁺ influx insensitive to senicapoc or to DIOA. A23187 stimulated K⁺ influx 4.6-fold over baseline (Fig. 4A), roughly comparable to the 4.2-fold stimulation exhibited by WT mouse red cells. Senicapoc inhibited A23187-stimulated K⁺ influx by 64.3%, whereas inhibition by DIOA was 47.5% (Fig. 4A). Thus, senicapoc-sensitive and DIOA-sensitive fractions of A23187-stimulated K⁺ influx into red cells of Kcc1^−/−;Kcc3^−/− mice did not differ significantly (Fig. 4B and C).

Figure 4.

DIOA inhibits the Gardos channel in mouse red cells genetically devoid of K-Cl cotransport activity. A: basal and A23187-stimulated K⁺ influx into Kcc1^−/−;Kcc3^−/− mouse red cells in chloride media. **P < 0.008 vs. baseline NDA; #P < 0.04 and ##P < 0.008 vs A23187 NDA by Mann–Whitney test. B: SCP-sensitive basal and A23187-stimulated K⁺ influx into Kcc1^−/−;Kcc3^−/− mouse red cells. C: DIOA-sensitive basal and A23187-stimulated K⁺ influx into Kcc1^−/−;Kcc3^−/− mouse red cells. Data in B and C are derived from data in A. *P < 0.04; **P < 0.008 vs baseline by Mann–Whitney test. DIOA, [(dihydroindenyl)oxy]acetic acid; NDA, no drug added; SCP, senicapoc.

In contrast, A23187 failed to stimulate K⁺ influx in red cells from mice with genetically inactivated expression of the Kcnn4 gene, as we previously reported (2, 17). Moreover, neither senicapoc nor DIOA acted as inhibitors of K⁺ influx in the absence (Fig. 5A) or presence of A23187 (Fig. 5B). Unexpectedly, DIOA in the absence of A23187 slightly increased K⁺ influx into Kcnn4^−/− red cells from chloride and from sulfamate solutions (Fig. 5A), an effect magnified two- to threefold in the presence of A23187 (Fig. 5B and D). Curiously, senicapoc also increased K⁺ influx into Kcnn4^−/− red cells in chloride solution in the presence of A23187 (Fig. 5C).

Figure 5.

Gardos channel Inhibitory activity of DIOA is absent in red cells from mice in which Kcnn4 has been genetically inactivated. A: basal K⁺ influx into Kcnn4^−/− mouse red cells in chloride or sulfamate media in the absence of A23187. Cells were exposed to no drug (dash), to SCP (200 nM), or to DIOA (100 µM). **P ≤ 0.007 vs. no drug or SCP (both in sulfamate) by Mann–Whitney test. B: A23187-stimulated K⁺ influx into Kcnn4^−/− mouse red cells in chloride or sulfamate media, exposed to no drug, senicapoc, or DIOA. *P < 0.025 vs. no drug in chloride, by Mann–Whitney test. C: SCP-sensitive K⁺ influx into Kcnn4^−/− mouse red cells in the absence (baseline) and presence of A23187. *P < 0.005 by Mann–Whitney test. D: DIOA-stimulated K⁺ influx into Kcnn4^−/− mouse red cells in the absence and presence of A23187. Data in C and D are derived from the chloride media data of A and B. DIOA, [(dihydroindenyl)oxy]acetic acid; NDA, no drug added; SCP, senicapoc.

DIOA Inhibits YODA-Stimulated K⁺ Influx in a Chloride-Independent Manner in Normal Human Red Cells

YODA1 is a small molecule activator of PIEZO1, the mechanosensitive Ca²⁺-permeable cation channel of red cells (24, 25). YODA1-stimulated K⁺ influx into normal human red cells was more variable (Fig. 6A) than A23187-stimulated K⁺ influx into human cells. However, YODA1-stimulated K⁺ influx into human cells was statistically indistinguishable in chloride or in sulfamate solutions (Fig. 6A and C). Although YODA1-stimulated K⁺ influx was considerably reduced in HX red cells, DIOA inhibition of that K⁺ influx measured in chloride solution achieved statistical significance (Fig. 6B and D). The data were consistent with inhibition of Gardos channel activity by DIOA and by senicapoc, each downstream of PIEZO1 activation by YODA1. Apparently incomplete inhibition of K⁺ influx by either senicapoc or DIOA likely represents YODA-stimulated K⁺ influx directly mediated by PIEZO1.

Figure 6.

DIOA inhibits K⁺ influx stimulated by the PIEZO1 activator YODA1 in human red cells. A: YODA1-stimulated K⁺ influx into WT human red cells in chloride or sulfamate media, in the presence of no drug, SCP, or DIOA. *P < 0.02; **P ≤ 0.004 vs. WT in same solution, by Kruskal–Wallis test. %%P < 0.01 by Mann–Whitney test. B: YODA1-stimulated K⁺ influx into human HX V282M red cells in chloride or sulfamate media, in the presence of no drug, SCP, or DIOA. %P = 0.032 by Mann–Whitney test. C: SCP-sensitive, YODA1-stimulated K⁺ influx into human WT and HX V282M red cells, in chloride and sulfamate media. D: DIOA-sensitive, YODA1-stimulated K⁺ influx into human WT and HX V282M red cells, in chloride and sulfamate media. Data from C and D are derived from data in A and B. ##P ≤ 0.01; #P < 0.03 vs. WT in same solution, by Mann–Whitney test. DIOA, [(dihydroindenyl)oxy]acetic acid; NDA, no drug added; HX, hereditary xerocytosis; SCP, senicapoc WT, wild type.

YODA1 also stimulated K⁺ influx into mouse red cells (Fig. 7A), in a manner independent of expression of K-Cl cotransporters (Fig. 7B) or of the Gardos channel/KCNN4 (Fig. 7C). Senicapoc only modestly inhibited YODA1-stimulated K⁺ influx in WT mouse red cells (Fig. 7A), likely reflecting a substantial fraction of PIEZO1-mediated K⁺ influx. This was supported by undiminished YODA1-stimulated K⁺ influx in the absence of KCNN4 function (Fig. 7C). YODA1-stimulated K⁺ influx in red cells from WT mice and from Kcc1^−/−;Kcc3^−/− mice trended toward inhibition by DIOA, but this apparent decrease did not achieve statistical significance (Fig. 7A and B). However, DIOA significantly inhibited YODA-stimulated K⁺ influx into red cells of Kcnn4^−/− mice (Fig. 7C), suggesting possible upregulation of K-Cl cotransport and/or another K⁺ transport pathway (22).

Figure 7.

DIOA inhibits YODA1-stimulated K⁺ influx in mouse red cells. A: basal and YODA1-stimulated K⁺ influx into WT mouse red cells in chloride medium, in the presence of no drug, senicapoc, or DIOA. B: basal and YODA1-stimulated K⁺ influx into Kcc1^−/−;Kcc3^−/− mouse red cells in chloride medium in the presence of no drug, senicapoc, or DIOA. C: basal and YODA1-stimulated K⁺ influx into Kcnn4^−/− mouse red cells in chloride medium in the presence of no drug, senicapoc, or DIOA. ***P < 0.0001; **P < 0.008; *P < 0.04 vs. baseline in the presence of the same drug condition; #P < 0.05 vs. +YODA1-NDA, all by Mann–Whitney test. D: SCP-sensitive or DIOA-sensitive fractions of YODA1-stimulated K⁺ influx into WT mouse red cells or red cells from Kcc1^−/−;Kcc3^−/− or Kcnn4^−/− mice. Data in D is derived from data in A–C. *P < 0.025 vs. same genotype in senicapoc, by Mann–Whitney test. DIOA, [(dihydroindenyl)oxy]acetic acid; NDA, no drug added; HX, hereditary xerocytosis; SCP, senicapoc WT, wild type.

As shown in Fig. 8, DIOA inhibition of KCNN4 is similar in human and mouse red cells. The IC₅₀ values for DIOA inhibition of KCNN4 were 27 ± 4.2 µM in human red cells (n = 3) and 44 ± 13 µM (SE) in mouse red cells (n = 4, n.s.). The Hill coefficients for inhibition by DIOA were 1.5 + 0.25 in humans and 2.0 ± 1.1 in mouse cells. Figure 9 demonstrates that DIOA at 100 µM inhibits A23187-stimulated K⁺ influx (KCNN4 activity) in human sickle red cells to the same degree as in normal human red cells (Fig. 1). KCNN4 activity in red cells from the SAD mouse model of sickle cell disease is similarly inhibited by 100 µM DIOA (Fig. 9).

Figure 9.

DIOA inhibits A23187-stimulated Gardos channel flux in human and mouse sickle red cells. A: DIOA and SCP inhibit A23187-stimulated K⁺ influx into human sickle cell disease (SS) red cells. B: inhibitor-sensitive fraction of A23187-stimulated K⁺ influx into human SS red cells. C: DIOA and SCP inhibit A23187-stimulated K⁺ influx into red cells of the SAD mouse model of sickle cell disease. D: inhibitor-sensitive fraction of A23187-stimulated K⁺ influx into red cells of the SAD mouse model of sickle cell disease. DIOA, [(dihydroindenyl)oxy]acetic acid; SCP, senicapoc.

DIOA Inhibits Currents from Heterologously Expressed KCNN4 Channels

To confirm that DIOA directly interacts with KCa3.1, we performed whole cell patch clamp experiments on HEK-293 cells stably expressing human KCa3.1/KCNN4 (Fig. 10). DIOA blocked currents elicited by voltage ramps in the presence of 1 µM free intracellular Ca²⁺ with IC₅₀ of 42 µM (n = 3). The Hill coefficient of the concentration-response curve was ∼2, consistent with the interaction of two or more DIOA molecules per tetrameric KCa3.1/KCNN4 channel.

Docking of DIOA into the KCa3.1/KCNN4 Structure Identifies Two Potential Binding Sites

The KCNN4 structure (20) contains several binding pockets for small molecules (Fig. 11). Inhibitors of higher affinity and specificity such as senicapoc and TRAM-34 bind in the inner pore lumen, whereas inhibitors of lower affinity and specificity such as the dihydropyridine nifedipine interact with residues in the fenestration region (21). Docking of DIOA in 10,000 random starting positions with the RosettaLigand method identified low energy binding poses in both the pore lumen (−12.4 REU) and the fenestration site (−15.8 REU) (As the Rosetta energy function is a combination of both physics-based and statistics-based potentials, Rosetta energies are reported on an arbitrary scale referred to as Rosetta energy units or REUs). These modeling results allow us to suggest that two DIOA molecules bind to KCNN4 in two of the tetrameric protein’s four fenestration sites. Also possible would be one DIOA molecule bound in the single pore site, with a second DIOA engaging one of the fenestration sites. We note in this context that DIOA interacts with the KCC3 K-Cl cotransporter polypeptide with a 1:1 stoichiometry, as reflected in a recent cryo-EM structure of KCC3 in its inward-open state (26), in which two DIOA molecules are bound within the interprotomeric central cleft of the homodimeric transporter imaged as a single particle (26).

DISCUSSION

We have reported here that DIOA used at 100 µM nearly completely blocks erythroid KCNN4/Gardos channel activity maximally stimulated by A23187 in human and mouse red cells. This Gardos channel inhibitor activity of DIOA is chloride-independent and persists in mouse red cells genetically devoid of both the K-Cl cotransporters KCC3 and KCC1. The Gardos channel inhibitor activity of DIOA is appropriately abrogated in mouse red cells genetically devoid of the KCNN4/Gardos channel. The degree of inhibition is comparable in human sickle red cells and nearly so in SAD mouse red cells that model the red cell dehydration of sickle cell disease. The results are consistent with a previous report of DIOA blockade of a Cl⁻-independent, Ca²⁺-activated K⁺ conductance in human lens epithelial cells, with apparent IC₅₀ of ∼75 µM (27).

DIOA inhibition of KCa3.1 is likely mediated through the binding of two DIOA molecules at two of the four fenestration sites present in the KCa3.1 tetramer. The binding of two inhibitor drugs per tetramer corresponds to the Hill coefficients of ∼2 exhibited by DIOA inhibition of recombinant human KCa3.1 K⁺ channel activity stimulated by 1 µM free Ca²⁺ in whole cell patch clamp records of KCa3.1-overexpressing HEK 293 cells, as well as by DIOA inhibition of A23187-stimulated K⁺ influx into red cells of both mouse and human. Predicted DIOA binding to the KCa3.1 fenestration site resembles dihydropyridine binding to the fenestration sites of voltage-gated Ca²⁺ channels (28). Interestingly, DIOA binding to electroneutral cotransporter KCC3 is also located outside its ion translocation pathway (26). The inner pore lumen of KCa3.1, the nM affinity binding site for the specific and potent inhibitors TRAM-34 and senicapoc (20, 29), is predicted by the present study to serve as an energetically less favorable docking site for DIOA. The potential importance of this likely secondary DIOA binding site for DIOA inhibition of KCa3.1 activity remains to be determined.

K-Cl cotransport mediated predominantly by KCC3 and KCC1 is a major component of red cell volume regulation, along with the Ca²⁺-activated K⁺ channel activity of KCNN4/KCa3.1, also known as the Gardos channel. Inhibitor modulation of these volume regulatory activities in sickle erythrocytes is a therapeutic objective for adjunct treatment of sickle cell disease. Indeed, the potent Gardos channel inhibitor senicapoc has already been shown to swell red cells and decrease mean corpuscular hemoglobin concentration in mouse models of sickle cell disease (11), and in human red cells in clinical trials (30, 31).

The development of inhibitors of KCC3 and KCC1 with potency and specificity sufficient for clinical testing (12) has lagged behind the development of KCNN4 inhibitors. However, blockade of erythroid K-Cl cotransport is important during experimental tests of erythroid Gardos channel activity. Experimental K-Cl cotransport blockade is often accomplished by the use of 1 mM bumetanide, but bumetanide also inhibits the NKCC1 Na-K-2Cl cotransporter of the red cell. Garay et al. (13, 14) in their study therefore proposed the use of DIOA as an inhibitor of erythroid K-Cl cotransport. With a red cell IC₅₀ of 10 µM, 100 µM DIOA blocked erythroid K-Cl cotransport nearly completely, without apparent inhibition of erythroid Na-K-2Cl cotransport. However, DIOA was later found to inhibit Na-K-2Cl cotransport activity mediated in HEK-293 cells by overexpressed recombinant human NKCC1 with IC₅₀ of 23 µM, at least as potently as DIOAs 57 µM IC₅₀ for N-ethylmaleimide-stimulated recombinant rabbit KCC1 similarly overexpressed in HEK-293 cells (32).

Since DIOA blocks neuron-specific KCC2-mediated K-Cl cotransport with similar potency (IC₅₀ 13 µM) (33) and KCC2 controls the equilibrium chloride potential of neuronal membranes, DIOA has also been useful for modulation of neuronal GABA(A) responses (34). Moreover, 100 µM DIOA has been used to inhibit KCC3 in MCF7 mammary carcinoma cells (35). In lipopolysaccharide-stimulated neutrophils and other mononuclear cells, DIOA has been used to inhibit KCC3-mediated K-Cl cotransport (36) that in some conditions may contribute to the cellular K loss required for WNK1-regulated NLRP3 inflammasome activation (37).

The ability of DIOA to block the KCNN4/Gardos channel is not the first demonstration of “off-target” activities of DIOA. DIOA has been shown to inhibit the cystic fibrosis transmembrane regulator (CFTR) with IC₅₀ of 17 µM, a potency indistinguishable from the 16 µM IC₅₀ of traditional chloride channel blocker, 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) (38), which also happens to block KCa3.1 at a concentration of 100 µM (39). DIOA at 50–100 µM concentrations has also been shown to block swelling-activated chloride currents in retinal pigment epithelial cells (40) and in osteoblast-like cells (41), as well as NPPB-sensitive chloride currents in human glioma cells (42) and anion exchange activity in lamprey red cells (43). Moreover, DIOA has also been shown to inhibit P-type ATPases at comparable or lower potency, including dog kidney Na⁺,K⁺-ATPase (IC₅₀ 53 µM), hog gastric H⁺,K⁺-ATPase (IC₅₀ 97 µM), and rabbit muscle Ca²⁺-ATPase (IC₅₀ 127 µM).

Low-K sheep red cells lacking Gardos channel activity (44) were originally reported to be insensitive to intracellular Ca²⁺ (45). However, subsequent observations detected biphasic regulation of K-Cl cotransport activity by A23187 in low-K sheep red cells, such that reduction of intracellular [Ca²⁺] further increased anisosmotically stimulated K-Cl cotransport, but not cotransport stimulated by N-ethylmaleimide. In contrast, elevated intracellular [Ca²⁺] in low-K sheep red cells nearly completely inhibited K-Cl cotransport stimulated either anisosmotically or by N-ethylmaleimide (46, 47). Both inhibitory and activating effects of A23187-mediated manipulation of intracellular [Ca²⁺] were fully reversible, and reversibly abolished in the setting of cellular ATP depletion (47). Later studies confirmed K-Cl cotransport inhibition by elevated intracellular [Ca²⁺] in Gardos channel-expressing red cells from humans with sickle cell disease (48).

Tests of erythroid K-Cl cotransport during periods of deoxygenation should be accompanied in a consistent fraction of the erythroid population by elevated intracellular [Ca²⁺], reflecting the deoxygenation-activated cation conductance known as Psickle (49). Ca²⁺ entry into red cells mediated by Psickle should in turn activate KCNN4/Gardos channel. Use of DIOA to monitor oxygen-sensitive K-Cl cotransport (50) during cyclic oxygenation/deoxygenation may thus include a fraction of DIOA-sensitive K⁺ transport mediated by KCNN4/Gardos channel, although possibly not in conditions of continuous deoxygenation (51). Knowledge of the ability of DIOA to block KCNN4 may prevent inadvertent inhibition of KCNN4 during experiments in red cells or other pertinent cell types designed with the intention of specific inhibition of K-Cl cotransporters, or may modify interpretation of experimental results.

GRANTS

This work was supported by research funds from Quest Diagnostics. Joshua. A. Nasburg was supported by the National Institute of General Medical Sciences-funded Pharmacology Training Program [T32GM099608].

DISCLOSURES

Seth L. Alper received research support from and consulted with Quest Diagnostics, Inc. Heike Wulff consulted with Saniona, Inc. L. Michael Snyder consulted with Quest Diagnostics, Inc. Jay G. Wohlgemuth and Jeffrey S. Dlott are employees and stockholders of Quest Diagnostics, Inc. Jason Kitten is the founder and employee of Meta Isotopes LLC. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

A.R. and S.L.A. conceived and designed research; A.R., J.A.N., H.S., and B.E.S. performed experiments; A.R., J.A.N., H.S., B.E.S., and H.W. analyzed data; A.R., J.A.N., H.S., J.K., H.W., and S.L.A. interpreted results of experiments; A.R., J.A.N., H.S., and H.W. prepared figures; S.L.A. drafted manuscript; A.R., J.G.W., J.S.D., L.M.S., C.B., H.W., and S.L.A. edited and revised manuscript; A.R., J.G.W., J.S.D., L.M.S., C.B., H.W., and S.L.A. approved final version of manuscript.