The Epithelial Na⁺ Channel (ENaC) plays a central role in control of epithelial surface hydration and vascular volume. Similar to other ion channels, ENaC activity is regulated, in part, by cortical cytoskeleton. Besides, the cytoskeleton is an established target for small G proteins signaling. Here we studied whether ENaC activity is modulated by changes in the state of the cytoskeleton and whether cytoskeletal elements are involved in small G protein mediated increase of ENaC activity. First, the functional importance of the cytoskeleton was established with whole-cell patch clamp experiments recording ENaC reconstituted in CHO cells. Pretreatment with Cytochalasin D (CytD; 10 µg/ml; 1–2 h) or colchicine (500 µM; 1–3 h) to disassembly F-actin and destroy microtubules, respectively, significantly decreased amiloride sensitive current. However, acute application of CytD induced rapid increase in macroscopic current. Single channel measurements under cell-attached conditions revealed similar observations. CytD rapidly increased ENaC activity in freshly isolated rat collecting duct, polarized epithelial mouse mpkCCD_c14 cells and HEK293 cells transiently transfected with ENaC subunits. In contrast, colchicine did not have an acute effect on ENaC activity. Small G proteins RhoA, Rac1 and Rab11a markedly increase ENaC activity. 1–2 h treatment with colchicine or CytD abolished effects of these GTPases. Interestingly, when cells were coexpressed with ENaC and RhoA, short-term treatment with CytD decreased ENaC activity. We conclude that cytoskeleton is involved in regulation of ENaC and is necessary for small G protein mediated increase of ENaC activity.

The epithelial Na⁺ channel (ENaC) is an essential channel responsible for Na⁺ reabsorption in the aldosterone-sensitive distal nephron. Consequently, ENaC is a major effector impacting systemic blood volume and pressure. We have shown recently that Rac1 increases ENaC activity, whereas Cdc42 fails to change channel activity. Here we tested whether Rac1 signaling plays a physiological role in modulating ENaC in native tissue and polarized epithelial cells. We found that Rac1 inhibitor NSC23766 markedly decreased ENaC activity in freshly isolated collecting ducts. Knockdown of Rac1 in native principal cells decreased ENaC-mediated sodium reabsorption and the number of channels at the apical plasma membrane. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in the control of the actin cytoskeleton. N-WASP functions downstream of Cdc42, whereas WAVEs are effectors of Rac1 activity. N-WASP and all 3 isoforms of WAVE significantly increased ENaC activity when coexpressed in Chinese hamster ovary cells. However, wiskostatin, an inhibitor of N-WASP, had no effect on ENaC activity. Immunoblotting demonstrated the presence of WAVE1 and WAVE2 and absence of N-WASP and WAVE3 in mpkCCD_c14 and M-1 principal cells. Immunohistochemistry analysis also revealed localization of WAVE1 and WAVE2 but not N-WASP in the cortical collecting duct of Sprague-Dawley rat kidneys. Moreover, patch clamp analysis revealed that Rac1 and WAVE1/2 are parts of the same signaling pathway with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.

The epithelial sodium channel (ENaC) is believed to represent the rate-limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector affecting systemic blood volume and pressure. Sodium and water transport are dysregulated in diabetes mellitus. Peroxisome proliferator-activated receptor γ (PPARγ) agonists are currently used in the treatment of type 2 diabetes, although their use remains limited by fluid retention. The effects of PPARγ agonists on ENaC activity remain controversial. Although PPARγ agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via the serum- and glucocorticoid-regulated kinase 1, other studies reported that the PPARγ agonist-induced fluid retention is independent of ENaC activity. Here we confirmed that four chemically distinct PPARγ agonists [pioglitazone, rosiglitazone, troglitazone, and 15-deoxy-Δ^12,14-prostaglandin J₂ (PGJ₂)] do not enhance Na⁺ transport in cultured renal collecting duct principal mpkCCD_c14 cells, as assessed by short-circuit current measurements. However, the PPARγ antagonist 2-chloro-5-nitro-N-4-pyridinyl-benzamide (T0070907), and to a lesser extent 2-chloro-5-nitrobenzanilide (GW9662), were found to decrease Na⁺ reabsorption across mpkCCD_c14 cell layers. Furthermore, pretreatment of monolayers with T0070907 diminished the insulin-stimulated sodium transport. PPARγ agonist PGJ₂ did not enhance insulin-stimulated Na⁺ flux via ENaC. We also show that PPARγ enhances ENaC activity when all three subunits are reconstituted in Chinese hamster ovary (CHO) cells. GW9662 inhibits ENaC activity when ENaC subunits are coexpressed in CHO cells with PPARγ. In contrast, rosiglitazone has no effect on ENaC activity. We conclude that PPARγ activity is important for maintaining basal and insulin-dependent transepithelial Na⁺ transport and ENaC activity.

Epithelial cells are exposed to a variety of mechanical stimuli. Epithelial Na+ channels (ENaC) mediate sodium transport across apical membranes of epithelial cells that line the distal nephron, airway and alveoli, and distal colon. Early investigations into stretch sensitivity of ENaC were controversial. However, recent studies are supportive of ENaC's mechanosensitivity. This work studied whether flow-dependent activation of ENaC is modulated by changes in the state of the actin cytoskeleton and whether small GTPase RhoA is involved in flow-mediated increase of ENaC activity.

Empagliflozin, an inhibitor of sodium-glucose co-transporter 2 (iSGLT2), improves cardiovascular outcomes in patients with and without diabetes and possesses an antiarrhythmic activity. However, the mechanisms of these protective effects have not been fully elucidated. This study aimed to explore the impact of empagliflozin on ion channel activity and electrophysiological characteristics in the ventricular myocardium. The main cardiac ionic currents (I_Na, I_CaL, I_CaT, I_Kr, I_Ks) and action potentials (APs) were studied in zebrafish. Whole-cell currents were measured using the patch clamp method in the isolated ventricular cardiomyocytes. The conventional sharp glass microelectrode technique was applied for the recording of APs from the ventricular myocardium of the excised heart. Empagliflozin pretreatment compared to the control group enhanced potassium I_Kr step current density in the range of testing potentials from 0 to +30 mV, I_Kr tail current density in the range of testing potentials from +10 to +70 mV, and I_Ks current density in the range of testing potentials from −10 to +20 mV. Moreover, in the ventricular myocardium, empagliflozin pretreatment shortened AP duration APD as shown by reduced APD50 and APD90. Empagliflozin had no influence on sodium (I_Na) and L- and T-type calcium currents (I_CaL and I_CaT) in zebrafish ventricular cardiomyocytes. Thus, we conclude that empagliflozin increases the rapid and slow components of delayed rectifier K⁺ current (I_Kr and I_Ks). This mechanism could be favorable for cardiac protection.