Loss of the alpha subunit distal furin cleavage site blunts ENaC activation following Na+ restriction.

Epithelial Na+ channels (ENaCs) are activated by proteolysis of the α and γ subunits at specific sites flanking embedded inhibitory tracts. To examine the role of α subunit proteolysis in channel activation in vivo, we generated mice lacking the distal furin cleavage site in the α subunit (αF2M mice). On a normal Na+ control diet, no differences in ENaC protein abundance in kidney or distal colon were noted between wild-type (WT) and αF2M mice. Patch-clamp analyses revealed similar levels of ENaC activity in kidney tubules, while no physiologically relevant differences in blood chemistry or aldosterone levels were detected. Male αF2M mice did exhibit diminished ENaC activity in the distal colon, as measured by amiloride-sensitive short-circuit current (ISC). Following dietary Na+ restriction, WT and αF2M mice had similar natriuretic and colonic ISC responses to amiloride. However, single-channel activity was significantly lower in kidney tubules from Na+-restricted αF2M mice compared with WT littermates. ENaC α and γ subunit expression in kidney and distal colon were also enhanced in Na+-restricted αF2M vs. WT mice, in association with higher aldosterone levels. These data provide evidence that disrupting α subunit proteolysis impairs ENaC activity in vivo, requiring compensation in response to Na+ restriction. KEY POINTS: The epithelial Na+ channel (ENaC) is activated by proteolytic cleavage in vitro, but key questions regarding the role of ENaC proteolysis in terms of whole-animal physiology remain to be addressed. We studied the in vivo importance of this mechanism by generating a mouse model with a genetic disruption to a key cleavage site in the ENaC's α subunit (αF2M mice). We found that αF2M mice did not exhibit a physiologically relevant phenotype under normal dietary conditions, but have impaired ENaC activation (channel open probability) in the kidney during salt restriction. ENaC function at the organ level was preserved in salt-restricted αF2M mice, but this was associated with higher aldosterone levels and increased expression of ENaC subunits, suggesting compensation was required to maintain homeostasis. These results provide the first evidence that ENaC α subunit proteolysis is a key regulator of channel activity in vivo.

Extracellular intersubunit interactions modulate epithelial Na+ channel gating.

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the ß-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking ßE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging ßE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging ßE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1ß, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1ß. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

Effects of furosemide, acetazolamide and amiloride on renal cortical and medullary tissue oxygenation in non-anaesthetised healthy sheep.

It has been proposed that diuretics can improve renal tissue oxygenation through inhibition of tubular sodium reabsorption and reduced metabolic demand. However, the impact of clinically used diuretic drugs on the renal cortical and medullary microcirculation is unclear. Therefore, we examined the effects of three commonly used diuretics, at clinically relevant doses, on renal cortical and medullary perfusion and oxygenation in non-anaesthetised healthy sheep. Merino ewes received acetazolamide (250 mg; n = 9), furosemide (20 mg; n = 10) or amiloride (10 mg; n = 7) intravenously. Systemic and renal haemodynamics, renal cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ , and renal function were then monitored for up to 8 h post-treatment. The peak diuretic response occurred 2 h (99.4 ± 14.8 mL/h) after acetazolamide, at which stage cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ were not significantly different from their baseline levels. The peak diuretic response to furosemide occurred at 1 h (196.5 ± 12.3 mL/h) post-treatment but there were no significant changes in cortical and medullary tissue oxygenation during this period. However, cortical tissue P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ fell from 40.1 ± 3.8 mmHg at baseline to 17.2 ± 4.4 mmHg at 3 h and to 20.5 ± 5.3 mmHg at 6 h after furosemide administration. Amiloride did not produce a diuretic response and was not associated with significant changes in cortical or medullary tissue oxygenation. In conclusion, clinically relevant doses of diuretic agents did not improve regional renal tissue oxygenation in healthy animals during the 8 h experimentation period. On the contrary, rebound renal cortical hypoxia may develop after dissipation of furosemide-induced diuresis.

Development of UTX-143, a selective sodium-hydrogen exchange subtype 5 inhibitor, using amiloride as a lead compound.

NHE5, an isoform of the Na+/H+ exchanger (NHE) protein, is an ion-transporting membrane protein that regulates intracellular pH and is highly expressed in colorectal adenocarcinoma. Therefore, we hypothesized that NHE5 inhibitors can be used as anticancer drugs. However, because NHE1 is ubiquitously expressed in all cells, it is extremely important to demonstrate its selective inhibitory activity against NHE5. We used amiloride, an NHE non-selective inhibitor, as a lead compound and created UTX-143, which has NHE5-selective inhibitory activity, using a structure-activity relationship approach. UTX-143 showed selective cytotoxic effects on cancer cells and reduced the migratory and invasive abilities of cancer cells. These results suggest a new concept wherein drugs exhibit cancer-specific cytotoxic effects through selective inhibition of NHE5 and the possibility of UTX-143 as a lead NHE5-selective inhibitor.

Potency and specificity of amiloride and its analogues on branchial sodium fluxes in freshwater trout and goldfish.

There is a consensus that electroneutral Na+/H+ exchangers (NHEs) are important in branchial Na+ uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na+ uptake than amiloride. We evaluated this idea by testing the three drugs at 10-7 to 10-4 M, i.e. 0.1 to 100 µM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using 22Na+ to measure unidirectional Na+ influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na+ influx was HMA > amiloride > EIPA (IC50 values in the 10-70 µM range), very different from in mammals. At 100 µM, all three drugs inhibited Na+ influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 µM, all three drugs also stimulated unidirectional Na+ efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na+ efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.

Physiological insight into the conserved properties of Caenorhabditis elegans acid-sensing degenerin/epithelial sodium channels.

Acid-sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid sensitivity on all 30 subunits of the Caenorhabditis elegans DEG/ENaC family using two-electrode voltage clamp in Xenopus oocytes. We found two groups of acid-sensitive DEG/ENaCs characterised by being either inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified three new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs. KEY POINTS: Acidosis plays many roles in healthy physiology, including synaptic transmission and gut function, but is also a key feature of inflammatory pain, ischaemia and many other conditions. Cells monitor acidosis of their surroundings via pH-sensing channels, including the acid-sensing ion channels (ASICs). These are members of the degenerin/epithelial sodium channel (DEG/ENaC) family, along with, as the name suggests, vertebrate ENaCs and degenerins of the roundworm Caenorhabditis elegans. By screening all 30 C. elegans DEG/ENaCs for pH dependence, we describe, for the first time, three acid-activated members, as well as three additional acid-inhibited channels. We surveyed both groups for sensitivity to amiloride and zinc; like their mammalian counterparts, their currents can be blocked, enhanced or unaffected by these modulators. Likewise, they exhibit diverse ion selectivity. Our findings underline the diversity of acid-sensitive DEG/ENaCs across species and provide a comparative resource for better understanding the molecular basis of their function.

Accessibility of ENaC extracellular domain central core residues.

The epithelial Na+ channel (ENaC)/degenerin family has a similar extracellular architecture, where specific regulatory factors interact and alter channel gating behavior. The extracellular palm domain serves as a key link to the channel pore. In this study, we used cysteine-scanning mutagenesis to assess the functional effects of Cys-modifying reagents on palm domain ß10 strand residues in mouse ENaC. Of the 13 ENaC α subunit mutants with Cys substitutions examined, only mutants at sites in the proximal region of ß10 exhibited changes in channel activity in response to methanethiosulfonate reagents. Additionally, Cys substitutions at three proximal sites of ß and γ subunit ß10 strands also rendered mutant channels methanethiosulfonate-responsive. Moreover, multiple Cys mutants were activated by low concentrations of thiophilic Cd2+. Using the Na+ self-inhibition response to assess ENaC gating behavior, we identified four α, two ß, and two γ subunit ß10 strand mutations that changed the Na+ self-inhibition response. Our results suggest that the proximal regions of ß10 strands in all three subunits are accessible to small aqueous compounds and Cd2+ and have a role in modulating ENaC gating. These results are consistent with a structural model of mouse ENaC that predicts the presence of aqueous tunnels adjacent to the proximal part of ß10 and with previously resolved structures of a related family member where palm domain structural transitions were observed with channels in an open or closed state.

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Study of Amiloride Binding to Human Serum Albumin: Insights from Thermodynamic, Spectroscopic, and Molecular Docking Investigations.

This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of multi-spectroscopic techniques and computational methods were employed to identify the AML binding site on HSA and the forces responsible for the formation of the HSA-AML complex. Our findings revealed that AML specifically binds to Sudlow's site II, located in subdomain IIIA of HSA, and that the complex formed is stabilized using van der Waals hydrogen-bonding and hydrophobic interactions. FRET analysis showed that the distance between AML and Trp214 was optimal for efficient quenching. UV-Vis spectroscopy and circular dichroism indicated minor changes in the structure of HSA after AML binding, and molecular dynamics simulations (MDS) conducted over 100 ns provided additional evidence of stable HSA-AML-complex formation. This study enhances understanding of the interaction between AML and HSA and the mechanism responsible.

Bile acids regulate the epithelial Na+ channel in native tissues through direct binding at multiple sites.

Bile acids, originally known to emulsify dietary lipids, are now established signalling molecules that regulate physiological processes. Signalling targets several proteins that include the ion channels involved in regulating intestinal motility and bile viscosity. Studies show that bile acids regulate the epithelial sodium channel (ENaC) in cultured cell models and heterologous expression systems. ENaC plays both local and systemic roles in regulating extracellular fluids. Here we investigated whether bile acids regulate ENaC expressed in native tissues. We found that taurocholic acid and taurohyodeoxycholic acid regulated ENaC in both the distal nephron and distal colon. We also tested the hypothesis that regulation occurs through direct binding. Using photoaffinity labelling, we found evidence for specific binding to both the ß and γ subunits of the channel. In functional experiments, we found that the α subunit was sufficient for regulation. We also found that regulation by at least one bile acid was voltage-sensitive, suggesting that one binding site may be closely associated with the pore-forming helices of the channel. Our data provide evidence that bile acids regulate ENaC by binding to multiple sites to influence the open probability of the channel. KEY POINTS: Recent studies have shown that bile acids regulate the epithelial sodium channel (ENaC) in vitro. Here we investigated whether bile acids regulate ENaC in native tissues and whether bile acids directly bind the channel. We found that bile acids regulate ENaC expressed in the mouse cortical collecting duct and mouse colon by modulating open probability. Photoaffinity labelling experiments showed specific binding to the ß and γ subunits of the channel, while channels comprising only α subunits were sensitive to taurocholic acid in functional experiments using Xenopus oocytes. Taurocholic acid regulation of ENaC was voltage-dependent, providing evidence for binding to pore-forming helices. Our data indicate that bile acids are ENaC regulatory effectors that may have a role in the physiology and pathophysiology of several systems.

Amiloride is a suitable fluorescent substrate for the study of the drug transporter human multidrug and toxin extrusion 1 (MATE1).

Human multidrug and toxin extrusion 1 (MATE1; SLC47A1) is highly expressed in the kidneys and the liver. It plays a significant role in drug and endogenous compound disposition, and therefore, a rapid evaluation of its inhibition is important for drug development and for the understanding of renal and hepatic physiology. Amiloride is a potassium-sparing diuretic used for treating hypertension; it also demonstrates strong fluorescence in organic solvent or detergent solutions. In this study, we investigated the transport characteristics of amiloride by human MATE1. Cellular accumulation of amiloride was evaluated in control vector- or MATE1-transfected HEK293 cells. Cells were lysed with 1% sodium dodecyl sulfate, and fluorescence was measured using a microplate reader at wavelengths of 364ex and 409em. With ammonium prepulse-induced intracellular acidification, MATE1 transported amiloride at an extracellular pH of 7.4. The uptake demonstrated an overshoot phenomenon and saturated, with the Km and Vmax being 23.5 µM and 1.01 nmol/mg/min, respectively. MATE1-mediated amiloride transport also presented with a bell-shaped pH profile that reached a maximum pH value of 7.4. The inhibitor sensitivity of MATE1-facilitated amiloride transport was similar to those of known substrates, such as tetraethylammonium and metformin. Among the tested inhibitors, pyrimethamine demonstrated the most potent inhibition with an IC50 value of 0.266 µM. Furthermore, MATE1 was found to be inhibited by fampridine, which was previously considered to be a non-inhibitor of MATE1. This study demonstrates that amiloride is a suitable fluorescent substrate for the in vitro study of the transport activity of MATE1.

Acute decrease of urine calcium by amiloride in healthy volunteers under high-sodium diet.

BACKGROUND: Amiloride is a competitive blocker of the epithelial sodium (Na) channel in the renal collecting duct. It is a less potent diuretic than thiazides or loop diuretics, but is often used in association with its potassium (K)-sparing profile. Whether amiloride has a hypocalciuric effect similar to thiazides remains unclear. Animal studies and experiments on cell lines suggested that amiloride increases calcium (Ca) reabsorption in the distal nephron, but human studies are scarce. METHODS: We performed a post hoc analysis of a study with 48 healthy males (mean ± standard deviation age, 23.2 ± 3.9 years) who were assigned to a high-Na/low-K diet for 7 days before receiving 20 mg of amiloride orally. Urinary excretions of electrolytes were measured at 3 and 6 h afterwards; we calculated the relative changes in urinary excretion rates after amiloride administration. RESULTS: The high-Na/low-K diet led to an expected suppression of plasma renin and aldosterone. Amiloride showed a mild natriuretic effect associated with a decreased kaliuresis. Urinary Ca excretion dropped substantially (by 80%) 3 h after amiloride administration and remained low at the sixth hour. At the same time, fractional excretion of lithium decreased by a third, reflecting an increased proximal tubular reabsorption. CONCLUSIONS: During a high-Na/low-K diet, amiloride had a strong acute hypocalciuric effect, most probably mediated by increased proximal Ca reabsorption, even though a distal effect cannot be excluded. Further studies should establish if chronic amiloride or combined amiloride/thiazide treatment may decrease calciuria more efficiently and be useful in preventing kidney stones.

Incidence and risk factors for hyperkalaemia in patients treated for COVID-19 with nafamostat mesylate.

WHAT IS KNOWN AND OBJECTIVE: Nafamostat mesylate (NM) is used clinically in combination with antiviral drugs to treat coronavirus disease (COVID-19). One of the adverse events of NM is hyperkalaemia due to inhibition of the amiloride-sensitive sodium channels (ENaC). The incidence and risk factors for hyperkalaemia due to NM have been studied in patients with pancreatitis but not in COVID-19. COVID-19 can be associated with hypokalaemia or hyperkalaemia, and SARS-CoV-2 is thought to inhibit ENaC. Therefore, frequency and risk factors for hyperkalaemia due to NM may differ between COVID-19 and pancreatitis. Hyperkalaemia may worsen the respiratory condition of patients. The objective of this study was to determine the incidence and risk factors for hyperkalaemia in COVID-19 patients treated with favipiravir, dexamethasone and NM. METHODS: This retrospective study reviewed the records of hospitalized COVID-19 patients treated with favipiravir and dexamethasone, with or without NM, between March 2020 and January 2021. Multivariable logistic regression analysis was performed to identify the risk factors for hyperkalaemia. RESULTS AND DISCUSSION: Of 45 patients who received favipiravir and dexamethasone with NM for the treatment of COVID-19, 21 (47%) experienced hyperkalaemia. The duration of NM administration was a significant predictor of hyperkalaemia (odds ratio: 1.55, 95% confidence interval: 1.04-2.31, p = 0.031). The receiver-operating characteristic curve analysis determined that the cut-off value for predicting the number of days until the onset of hyperkalaemia was 6 days and the area under the curve was 0.707. WHAT IS NEW AND CONCLUSION: This study revealed that the incidence of hyperkalaemia is high in patients treated for COVID-19 with NM, and that the duration of NM administration is a key risk factor. When NM is administered for the treatment of COVID-19, it should be discontinued within 6 days to minimize the risk of hyperkalaemia.

In Silico Evaluation of Hexamethylene Amiloride Derivatives as Potential Luminal Inhibitors of SARS-CoV-2 E Protein.

The coronavirus E proteins are small membrane proteins found in the virus envelope of alpha and beta coronaviruses that have a high degree of overlap in their biochemical and functional properties despite minor sequence variations. The SARS-CoV-2 E is a 75-amino acid transmembrane protein capable of acting as an ion channel when assembled in a pentameric fashion. Various studies have found that hexamethylene amiloride (HMA) can inhibit the ion channel activity of the E protein in bilayers and also inhibit viral replication in cultured cells. Here, we use the available structural data in conjunction with homology modelling to build a comprehensive model of the E protein to assess potential binding sites and molecular interactions of HMA derivatives. Furthermore, we employed an iterative cycle of molecular modelling, extensive docking simulations, molecular dynamics and leveraging steered molecular dynamics to better understand the pore characteristics and quantify the affinity of the bound ligands. Results from this work highlight the potential of acylguanidines as blockers of the E protein and guide the development of subsequent small molecule inhibitors.

Epithelial Sodium Channel Inhibition by Amiloride Addressed with THz Spectroscopy and Molecular Modeling.

THz spectroscopy is important for the study of ion channels because it directly addresses the low frequency collective motions relevant for their function. Here we used THz spectroscopy to investigate the inhibition of the epithelial sodium channel (ENaC) by its specific blocker, amiloride. Experiments were performed on A6 cells' suspensions, which are cells overexpressing ENaC derived from Xenopus laevis kidney. THz spectra were investigated with or without amiloride. When ENaC was inhibited by amiloride, a substantial increase in THz absorption was noticed. Molecular modeling methods were used to explain the observed spectroscopic differences. THz spectra were simulated using the structural models of ENaC and ENaC-amiloride complexes built here. The agreement between the experiment and the simulations allowed us to validate the structural models and to describe the amiloride dynamics inside the channel pore. The amiloride binding site validated using THz spectroscopy agrees with previous mutagenesis studies. Altogether, our results show that THz spectroscopy can be successfully used to discriminate between native and inhibited ENaC channels and to characterize the dynamics of channels in the presence of their specific antagonist.

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types.

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomA/PomB stator complex of Vibrio spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types and in pathogenic and nonpathogenic strains. IMPORTANCE Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 µM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.

Acid-sensing ion channel 1 contributes to pulmonary arterial smooth muscle cell depolarization following hypoxic pulmonary hypertension.

Pulmonary hypertension is characterized by sustained vasoconstriction and remodelling of the small pulmonary arteries, which is associated with persistent depolarization of the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs). It is well-known that the underlying mechanism of this depolarization includes inhibition of K+ channels; however, whether other ion channels contribute to this depolarization is unknown. We previously reported that acid-sensing ion channel 1 (ASIC1), a non-selective cation channel (NSCC) that conducts both Na+ and Ca2+ , is present in PASMCs and contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension. Therefore, we tested the hypothesis that ASIC1-mediated Na+ influx contributes to PASMC Em regulation following CH-induced pulmonary hypertension. Using sharp electrode intracellular recordings in isolated, pressurized small pulmonary arteries from rats and mice, we show that exposure to CH leads to PASMC membrane depolarization compared with control animals, and this is independent of intraluminal pressure-induced depolarization. In addition to a decrease in PASMC whole-cell K+ currents following CH, we demonstrate that whole-cell NSCC currents are increased and essential to the persistent CH-induced Em depolarization in PASMCs. Both the specific inhibitor of ASIC1, psalmotoxin 1, and global knockout of ASIC1 (Asic1-/- ) prevents CH-induced Em depolarization and largely inhibits whole-cell NSCC currents, without affecting whole-cell K+ currents. Our results show a combination of factors, including inhibition of K+ efflux and augmented Na+ influx, mediate CH-induced PASMC depolarization. Furthermore, this study demonstrates a novel role for ASIC1 in the regulation of Em in PASMCs during CH-induced pulmonary hypertension. KEY POINTS: In pulmonary hypertensive patients and animal models of pulmonary hypertension, the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs) is persistently depolarized. In addition to the well-established reduction of K+ conductance, we show that non-selective cation channel currents are increased and essential to the persistent Em depolarization in PASMCs following chronic hypoxia (CH)-induced pulmonary hypertension. The current study provides novel evidence that acid-sensing ion channel 1 (ASIC1)-mediated Na+ influx induces membrane depolarization and regulates Em in PASMCs following CH exposure. Although fairly quiescent under control conditions, our findings demonstrate a pathological function of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension.

Vascular control of kidney epithelial transporters.

A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT1) receptors in the kidney, stimulating Na+ reabsorption. AT1 receptors in tubular epithelia control expression and stimulation of Na+ transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT1 receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na+ excretion in SMKO mice, we considered whether alterations in Na+ transporter expression might also play a role; therefore, we carried out proteomic analysis of key Na+ transporters and associated proteins. Here, we show that levels of Na+-K+-2Cl- cotransporter isoform 2 (NKCC2) and Na+/H+ exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, we found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser71), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na+ channel. However, the increases in α- and γ-epithelial Na+ channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na+ excretion observed after cell-specific deletion of AT1 receptors from smooth muscle cells is associated with altered Na+ transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na+ transporters and contributing to the regulation of pressure natriuresis.NEW & NOTEWORTHY The use of drugs to block the renin-angiotensin system to reduce blood pressure is common. However, the precise mechanism for how these medications control blood pressure is incompletely understood. Here, we show that mice lacking angiotensin receptors specifically in smooth muscle cells lead to alternation in tubular transporter amount and function. Thus, demonstrating the importance of vascular-tubular cross talk in the control of blood pressure.

Non-thiazide diuretics and hospitalization due to hyponatraemia: A population-based case-control study.

OBJECTIVE: Diuretics are often implicated in hyponatraemia. While thiazides constitute one of the most common causes of hyponatraemia, data on loop diuretics and potassium-sparing agents are limited and partly conflicting. The objective of this investigation was to study the association between use of different types of non-thiazide diuretics and hospitalization due to hyponatraemia. DESIGN, PATIENTS AND MEASUREMENTS: This was a register-based case-control study on the adult Swedish population. By linking national registers, patients hospitalized with a principal diagnosis of hyponatraemia (n = 11,213) from 1 October 2005 through 31 December 2014 were compared with matched controls (n = 44,801). Multivariable logistic regression, adjusted for multiple confounders, was used to analyse the association between use of diuretics and hyponatraemia. In addition, newly initiated use (≤90 days) and ongoing use were examined separately. RESULTS: Adjusted odds ratios (aORs) (95% CI) were 0.61 (0.57-0.66) for the use of furosemide, 1.69 (1.54-1.86) for the use of amiloride and 1.96 (1.78-2.18) for the use of spironolactone and hospitalization due to hyponatraemia. For newly initiated therapy, aORs ranged from 1.23 (1.04-1.47) for furosemide to 3.55 (2.75-4.61) for spironolactone. The aORs for ongoing use were 0.52 (0.47-0.57) for furosemide, 1.62 (1.47-1.79) for amiloride and 1.75 (1.56-1.98) for spironolactone. CONCLUSIONS: Ongoing use of furosemide was inversely correlated with hospitalization due to hyponatraemia, suggesting a protective effect. Consequently, if treatment with furosemide precedes the development of hyponatraemia by some time, other causes of hyponatraemia should be sought. Spironolactone and amiloride may both contribute to hyponatraemia; this effect is most prominent early in treatment.