Stimulation of Epithelial Sodium Channels in Endothelial Cells by Bone Morphogenetic Protein-4 Contributes to Salt-Sensitive Hypertension in Rats.

Previous studies have shown that high salt induces artery stiffness by causing endothelial dysfunction via increased sodium influx. We used our unique split-open artery technique combined with protein biochemistry and in vitro measurement of vascular tone to test a hypothesis that bone morphogenetic protein 4 (BMP4) mediates high salt-induced loss of vascular relaxation by stimulating the epithelial sodium channel (ENaC) in endothelial cells. The data show that high salt intake increased BMP4 both in endothelial cells and in the serum and that exogenous BMP4 stimulated ENaC in endothelial cells. The data also show that the stimulation is mediated by p38 mitogen-activated protein kinases (p38 MAPK) and serum and glucocorticoid-regulated kinase 1 (Sgk1)/neural precursor cell expressed developmentally downregulated gene 4-2 (Nedd4-2) (Sgk1/Nedd4-2). Furthermore, BMP4 decreased mesenteric artery relaxation in a benzamil-sensitive manner. These results suggest that high salt intake stimulates endothelial cells to express and release BMP4 and that the released BMP4 reduces artery relaxation by stimulating ENaC in endothelial cells. Therefore, stimulation of ENaC in endothelial cells by BMP4 may serve as another pathway to participate in the complex mechanism of salt-sensitive (SS) hypertension.

Lovastatin attenuates hypertension induced by renal tubule-specific knockout of ATP-binding cassette transporter A1, by inhibiting epithelial sodium channels.

BACKGROUND AND PURPOSE: We have shown that cholesterol is synthesized in the principal cells of renal cortical collecting ducts (CCD) and stimulates the epithelial sodium channels (ENaC). Here we have determined whether lovastatin, a cholesterol synthesis inhibitor, can antagonize the hypertension induced by activated ENaC, following deletion of the cholesterol transporter (ATP-binding cassette transporter A1; ABCA1). EXPERIMENTAL APPROACH: We selectively deleted ABCA1 in the principal cells of mouse CCD and used the cell-attached patch-clamp technique to record ENaC activity. Western blot and immunofluorescence staining were used to evaluate protein expression levels. Systolic BP was measured with the tail-cuff method. KEY RESULTS: Specific deletion of ABCA1 elevated BP and ENaC single-channel activity in the principal cells of CCD in mice. These effects were antagonized by lovastatin. ABCA1 deletion elevated intracellular cholesterol levels, which was accompanied by elevated ROS, increased expression of serum/glucocorticoid regulated kinase 1 (Sgk1), phosphorylated neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) and furin, along with shorten the primary cilium, and reduced ATP levels in urine. CONCLUSIONS AND IMPLICATIONS: These data suggest that specific deletion of ABCA1 in principal cells increases BP by stimulating ENaC channels via a cholesterol-dependent pathway which induces several secondary responses associated with oxidative stress, activated Sgk1/Nedd4-2, increased furin expression, and reduced cilium-mediated release of ATP. As ABCA1 can be blocked by cyclosporine A, these results suggest further investigation of the possible use of statins to treat CsA-induced hypertension.

Intracellular cholesterol stimulates ENaC by interacting with phosphatidylinositol­4,5­bisphosphate and mediates cyclosporine A-induced hypertension.

We have previously shown that blockade of ATP-binding cassette transporter A1 (ABCA1) with cyclosporine A (CsA) stimulates the epithelial sodium channel (ENaC) in cultured distal nephron cells. Here we show that CsA elevated systolic blood pressure in both wild-type and apolipoprotein E (ApoE) knockout (KO) mice to a similar level. The elevated systolic blood pressure was completely reversed by inhibition of cholesterol (Cho) synthesis with lovastatin. Inside-out patch-clamp data show that intracellular Cho stimulated ENaC in cultured distal nephron cells by interacting with phosphatidylinositol­4,5­bisphosphate (PIP2), an ENaC activator. Confocal microscopy data show that both α­ENaC and PIP2 were localized in microvilli via a Cho-dependent mechanism. Deletion of membrane Cho reduced the levels of γ­ENaC in the apical membrane. Reduced ABCA1 expression and elevated intracellular Cho were observed in old mice, compared to young mice. In parallel, cell-attached patch-clamp data from the split-open cortical collecting ducts (CCD) show that ENaC activity was significantly increased in old mice. These data suggest that elevation of intracellular Cho due to blockade of ABCA1 stimulates ENaC, which may contribute to CsA-induced hypertension. This study also implies that reduced ABCA1 expression may mediate age-related hypertension by increasing ENaC activity via elevation of intracellular Cho.

Depletion of Cholesterol Reduces ENaC Activity by Decreasing Phosphatidylinositol-4,5-Bisphosphate in Microvilli.

BACKGROUND/AIMS: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. METHODS: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. RESULTS: Here we show that extraction of membrane Cho with methyl-ß-cyclodextrin (MßCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP2 in the microvilli and that Cho was also co-localized with PIP2 in the microvilli. Either extraction of Cho with MßCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP2, and ENaC in the microvilli. CONCLUSIONS: Since PIP2 can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP2 in the microvilli.

TRUSS Exacerbates NAFLD Development by Promoting IκBα Degradation in Mice.

There is no effective treatment method for nonalcoholic fatty liver disease (NAFLD), the most common liver disease. The exact mechanism underlying the pathogenesis of NAFLD remains to be elucidated. Here, we report that tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein (TRUSS) acts as a positive regulator of NAFLD and in a variety of metabolic disorders. TRUSS expression was increased in the human liver specimens with NAFLD or nonalcoholic steatohepatitis, and in the livers of high-fat diet (HFD)-induced and genetically obese mice. Conditional knockout of TRUSS in hepatocytes significantly ameliorated hepatic steatosis, insulin resistance, glucose intolerance, and inflammatory responses in mice after HFD challenge or in spontaneous obese mice with normal chow feeding. All of these HFD-induced pathological phenotypes were exacerbated in mice overexpressing TRUSS in hepatocytes. We show that TRUSS physically interacts with the inhibitor of nuclear factor κB α (IκBα) and promotes the ubiquitination and degradation of IκBα, which leads to aberrant activation of nuclear factor κB (NF-κB). Overexpressing IκBαS32A/S36A , a phosphorylation-resistant mutant of IκBα, in the hepatocyte-specific TRUSS overexpressing mice almost abolished HFD-induced NAFLD and metabolic disorders. Conclusion: Hepatocyte TRUSS promotes pathological stimuli-induced NAFLD and metabolic disorders, through activation of NF-κB by promoting ubiquitination and degradation of IκBα. Our findings may provide a strategy for the prevention and treatment of NAFLD by targeting TRUSS.

Palmitate Stimulates the Epithelial Sodium Channel by Elevating Intracellular Calcium, Reactive Oxygen Species, and Phosphoinositide 3-Kinase Activity.

Previous studies indicate that the epithelial sodium channel (ENaC) in the kidney is upregulated in diabetes mellitus. Here, we show that ENaC single-channel activity in distal nephron cells was significantly increased by palmitate, a free fatty acid which is elevated in diabetes mellitus. We also show that palmitate increased intracellular Ca2+ and that after chelating intracellular Ca2+ with BAPTA-AM, palmitate failed to affect ENaC activity. Treatment of the cells with 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of IP3 receptors) abolished the elevation of both intracellular Ca2+ and ENaC activity. Treatment of the cells with apocynin (an NADPH oxidase inhibitor), dithiothreitol/NaHS (reducing agents), or LY294002 (a phosphoinositide 3-kinase (PI3K) inhibitor) prevented palmitate-induced ENaC activity, whereas thimerosal (an oxidizing agent) mimicked the effects of palmitate on ENaC activity. However, these treatments did not alter the levels of intracellular Ca2+, indicating that elevation of reactive oxygen species (ROS) and activation of PI3K are downstream of the signaling cascade. Since we have shown that ROS stimulate ENaC by activating PI3K, these data together suggest that palmitate first elevates intracellular Ca2+, then activates an NADPH oxidase to elevate intracellular ROS and PI3K activity, and finally increases ENaC activity via the activated PI3K.

Dietary salt blunts vasodilation by stimulating epithelial sodium channels in endothelial cells from salt-sensitive Dahl rats.

BACKGROUND AND PURPOSE: Our recent studies show that the reduced activity of epithelial sodium channels (ENaC) in endothelial cells accounts for the adaptation of vasculature to salt in Sprague-Dawley rats. The present study examines a hypothesis that enhanced ENaC activity mediates the loss of vasorelaxation in Dahl salt-sensitive (SS) rats. EXPERIMENTAL APPROACH: We used the cell-attached patch-clamp technique to record ENaC activity in split-open mesenteric arteries. Western blot and immunofluorescence staining were used to evaluate the levels of aldosterone, ENaC, eNOS and NO. Blood pressure was measured with the tail-cuff method and the artery relaxation was measured with the wire myograph assay. KEY RESULTS: High-salt (HS) diet significantly increased plasma aldosterone and ENaC activity in the endothelial cells of Dahl SS rats. The endothelium-dependent artery relaxation was blunted by HS challenge in these rats. Amiloride, a potent blocker of ENaC, increased both phosphorylated eNOS and NO and therefore prevented the HS-induced loss of vasorelaxation. As, in SS rats, endogenous aldosterone was already elevated by HS challenge, exogenous aldosterone did not further elevate ENaC activity in the rats fed with HS. Eplerenone, a mineralocorticoid receptor antagonist, attenuated the effects of HS on both ENaC activity and artery relaxation. CONCLUSIONS AND IMPLICATIONS: These data suggest that HS diet blunts artery relaxation and causes hypertension via a pathway associated with aldosterone-dependent activation of ENaC in endothelial cells. This pathway provides one of the mechanisms by which HS causes hypertension in Dahl SS rats. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Oxidized low-density lipoprotein stimulates epithelial sodium channels in endothelial cells of mouse thoracic aorta.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is expressed in endothelial cells and acts as a negative modulator of vasodilatation. Oxidized LDL (ox-LDL) is a key pathological factor in endothelial dysfunction. In the present study we examined the role of ENaC in ox-LDL-induced endothelial dysfunction and its associated signal transduction pathway. EXPERIMENTAL APPROACH: Patch clamp techniques combined with pharmacological approaches were used to examine ENaC activity in the endothelial cells of a split-open mouse thoracic aorta. Western blot analysis was used to determine ENaC expression in the aorta. The aorta relaxation was measured using a wire myograph assay. KEY RESULTS: Ox-LDL, but not LDL, significantly increased ENaC activity in the endothelial cells attached to split-open thoracic aortas, and the increase was inhibited by a lectin-like ox-LDL receptor-1 (LOX-1) antagonist (κ-carrageenan), an NADPH oxidase inhibitor (apocynin), and a scavenger of ROS (TEMPOL). Sodium nitroprusside, an NO donor, diminished the ox-LDL-mediated activation of ENaC, and this effect was abolished by inhibiting soluble guanylate cyclase (sGC) and PKG. Ox-LDL reduced the endothelium-dependent vasodilatation of the aorta pectoralis induced by ACh, and this reduction was partially restored by blocking ENaC. CONCLUSION AND IMPLICATIONS: Ox-LDL stimulates ENaC in endothelial cells through LOX-1 receptor-mediated activation of NADPH oxidase and accumulation of intracellular ROS. Since the stimulation of ENaC can be reversed by elevating NO, we suggest that both inhibition of ENaC and an elevation of NO may protect the endothelium from ox-LDL-induced dysfunction. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

ZNF307 (Zinc Finger Protein 307) Acts as a Negative Regulator of Pressure Overload-Induced Cardiac Hypertrophy.

Pathological cardiac hypertrophy is a key risk factor for heart failure. We found that the protein expression levels of the ZNF307 (zinc finger protein 307) were significantly increased in heart samples from both human patients with dilated cardiomyopathy and mice subjected to aortic banding. Therefore, we aimed to elucidate the role of ZNF307 in the development of cardiac hypertrophy and to explore the signal transduction events that mediate the effect of ZNF307 on cardiac hypertrophy, using cardiac-specific ZNF307 transgenic (ZNF307-TG) mice and ZNF307 global knockout (ZNF307-KO) mice. The results showed that the deletion of ZNF307 potentiated aortic banding-induced pathological cardiac hypertrophy, fibrosis, and cardiac dysfunction; however, the aortic banding-induced cardiac hypertrophic phenotype was dramatically diminished by ZNF307 overexpression in mouse heart. Mechanistically, the antihypertrophic effects mediated by ZNF307 in response to pathological stimuli were associated with the direct inactivation of NF-κB (nuclear factor-κB) signaling and blockade of the nuclear translocation of NF-κB subunit p65. Furthermore, the overexpression of a degradation-resistant mutant of IκBα (IκBαS32A/S36A) reversed the exacerbation of cardiac hypertrophy, fibrosis, and dysfunction shown in aortic banding-treated ZNF307-KO mice. In conclusion, our findings demonstrate that ZNF307 ameliorates pressure overload-induced cardiac hypertrophy by inhibiting the activity of NF-κB-signaling pathway.

Transient Receptor Potential Melastatin 4 (TRPM4) Contributes to High Salt Diet-Mediated Early-Stage Endothelial Injury.

BACKGROUND/AIMS: The present study investigated whether the transient receptor potential melastatin 4 (TRPM4) channel plays a role in high salt diet (HSD)-induced endothelial injuries. METHODS: Western blotting and immunofluorescence were used to examine TRPM4 expression in the mesenteric endothelium of Dahl salt-sensitive (SS) rats fed a HSD. The MTT, TUNEL, and transwell assays were used to evaluate the cell viability, cell apoptosis, and cell migration, respectively, of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were used to determine the concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), and E-selectin. Carboxy-H2DCFDA, a membrane-permeable reactive oxygen species (ROS)-sensitive fluorescent probe, was used to detect intracellular ROS levels. RESULTS: TRPM4 was mainly expressed near the plasma membrane of mesenteric artery endothelial cells, and its expression level increased in SS hypertensive rats fed a HSD. Its protein expression was significantly upregulated upon treatment with exogenous hydrogen peroxide (H2O2) and aldosterone in cultured HUVECs. Cell viability decreased upon treatment with both agents in a concentration-dependent manner, which could be partially reversed by 9-phenanthrol, a specific TRPM4 inhibitor. Exogenous H2O2 induced apoptosis, enhanced cell migration, and increased the release of adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, all of which were significantly attenuated upon treatment with 9-phenanthrol. Aldosterone and H2O2 induced the accumulation of intracellular ROS, which was significantly inhibited by 9-phenanthrol, suggesting that oxidative stress is one of the mechanisms underlying aldosterone-induced endothelial injury. CONCLUSIONS: Given the fact that oxidative stress and high levels of circulating aldosterone are present in hypertensive patients, we suggest that the upregulation of TRPM4 in the vascular endothelium may be involved in endothelial injuries caused by these stimuli.

AMP-Activated Protein Kinase Attenuates High Salt-Induced Activation of Epithelial Sodium Channels (ENaC) in Human Umbilical Vein Endothelial Cells.

Recent studies suggest that the epithelial sodium channel (ENaC) is expressed in the endothelial cells. To test whether high salt affects the NO production via regulation of endothelial ENaC, human umbilical vein endothelial cells (HUVECs) were incubated in solutions containing either normal or high sodium (additional 20 mM NaCl). Our data showed that high sodium treatment significantly increased α-, ß-, and γ-ENaC expression levels in HUVECs. Using the cell-attached patch-clamp technique, we demonstrated that high sodium treatment significantly increased ENaC open probability (P O ). Moreover, nitric oxide synthase (eNOS) phosphorylation (Ser 1177) levels and NO production were significantly decreased by high sodium in HUVECs; the effects of high sodium on eNOS phosphorylation and NO production were inhibited by a specific ENaC blocker, amiloride. Our results showed that high sodium decreased AMP-activated kinase (AMPK) phosphorylation in endothelial cells. On the other hand, metformin, an AMPK activator, prevented high sodium-induced upregulation of ENaC expression and P O . Moreover, metformin prevented high salt-induced decrease in NO production and eNOS phosphorylation. These results suggest that high sodium stimulates ENaC activation by negatively modulating AMPK activity, thereby leading to reduction in eNOS activity and NO production in endothelial cells.

Angiotensin-Converting Enzyme 3 (ACE3) Protects Against Pressure Overload-Induced Cardiac Hypertrophy.

BACKGROUND: Angiotensin-converting enzyme 3 (ACE3) is a recently defined homolog of ACE. However, the pathophysiological function of ACE3 is largely unknown. Here, we aim to explore the role of ACE3 in pathological cardiac hypertrophy. METHODS AND RESULTS: Neonatal rat cardiomyocytes (NRCMs) with gain and loss of function of ACE3 and mice with global knockout or cardiac-specific overexpression of ACE3 were used in this study. In cultured cardiomyocytes, ACE3 conferred protection against angiotensin II (Ang II)-induced hypertrophic growth. Cardiac hypertrophy in mice was induced by aortic banding (AB) and the extent of hypertrophy was analyzed through echocardiographic, pathological, and molecular analyses. Our data demonstrated that ACE3-deficient mice exhibited more pronounced cardiac hypertrophy and fibrosis and a strong decrease in cardiac contractile function, conversely, cardiac-specific ACE3-overexpressing mice displayed an attenuated hypertrophic phenotype, compared with control mice, respectively. Analyses of the underlying molecular mechanism revealed that ACE3-mediated protection against cardiac hypertrophy by suppressing the activation of mitogen-activated protein kinase kinase (MEK)-regulated extracellular signal-regulated protein kinase (ERK1/2) signaling, which was further evidenced by the observation that inhibition of the MEK-ERK1/2 signaling by U0126 rescued the exacerbated hypertrophic phenotype in ACE3-deficient mice. CONCLUSIONS: Our comprehensive analyses suggest that ACE3 inhibits pressure overload-induced cardiac hypertrophy by blocking the MEK-ERK1/2 signaling pathway.

Dietary salt regulates epithelial sodium channels in rat endothelial cells: adaptation of vasculature to salt.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is expressed in vascular endothelial cells and is a negative modulator of vasodilation. However, the role of endothelial ENaCs in salt-sensitive hypertension remains unclear. Here, we have investigated how endothelial ENaCs in Sprague-Dawley (SD) rats respond to high-salt (HS) challenge. EXPERIMENTAL APPROACH: BP and plasma aldosterone levels were measured. We used patch-clamp technique to record ENaC activity in split-open mesenteric arteries (MAs). Western blot and Griess assay were used to detect expression of α-ENaCs, eNOS and NO. Vasorelaxation in second-order MAs was measured with wire myograph assays. KEY RESULTS: Functional ENaCs were observed in endothelial cells and their activity was significantly decreased after 1 week of HS diet. After 3 weeks of HS diet, ENaC expression was also reduced. When either ENaC activity or expression was reduced, endothelium-dependent relaxation (EDR) of MAs, in response to ACh, was enhanced. This enhancement of EDR was mimicked by amiloride, a blocker of ENaCs. By contrast, HS diet significantly increased contractility of MAs, accompanied by decreased eNOS activity and NO levels. However, ACh-induced release of NO was much higher in MAs isolated from HS rats than those from NS rats. CONCLUSIONS AND IMPLICATIONS: HS intake increased the BP of SD rats, but simultaneously enhanced EDR by reducing ENaC activity and expression due to feedback inhibition. Therefore, ENaCs may play an important role in endothelial cells allowing the vasculature to adapt to HS conditions.

Characterization of Cardiac Anoctamin1 Ca²âº-Activated Chloride Channels and Functional Role in Ischemia-Induced Arrhythmias.

Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride (Cl(-)) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca(2+)-activated Cl(-) current (I(Cl.Ca)) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of I(Cl.Ca), which was inhibited by a specific ANO1 inhibitor, T16A(inh)-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper "spike and dome" compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower "spike and dome". We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of I(Cl.Ca) in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias.

Hypoxia augments the calcium-activated chloride current carried by anoctamin-1 in cardiac vascular endothelial cells of neonatal mice.

BACKGROUND AND PURPOSE: The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). EXPERIMENTAL APPROACH: Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. KEY RESULTS: Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca(2+) and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl ( C a) detected in CVECs was significantly inhibited by T16Ainh -A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl ( C a) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl ( C a) was inhibited by T16Ainh -A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl ( C a) in Ano1 gene knockdown CVECs. CONCLUSIONS AND IMPLICATIONS: Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl ( C a) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca(2+) . Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.