Diltiazem inhibits Ca(V)1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α₁ subunit of Ca(V)1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α₁ subunits.Ca(V)1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through Ca(V)1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).Quaternary derivative d-cis-diltiazem inhibited Ca(V)1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of Ca(V)1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.The data show that use-dependent block of in Ca(V)1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.
AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human tumor necrosis factor lectin-like domain (TIP domain) sequence, is currently being developed as a treatment for lung edema and has been shown to reduce extravascular lung water and improve lung function in mouse, rat, and pig models. The current paradigm for liquid homeostasis in the adult mammalian lung is that passive apical uptake of sodium via the amiloride-sensitive epithelial Na⁺ channel (ENaC) and nonselective cyclic-nucleotide-gated cation channels creates the major driving force for reabsorption of water through the alveolar epithelium in addition to other ion channels such as potassium and chloride channels. AP301 can increase amiloride-sensitive current in A549 cells as well as in freshly isolated type II alveolar epithelial cells from different species. ENaC is expressed endogenously in all of these cell types. Consequently, this study was undertaken to determine whether ENaC is the specific target of AP301. The effect of AP301 in A549 cells as well as in human embryonic kidney cells and Chinese hamster ovary cells heterologously expressing human ENaC subunits (α, β, γ, and δ) was measured in patch clamp experiments. The congener TIP peptide AP318 [Cyclo(4-aminobutanoic acid-GQRETPEGAEAKPWYD)] activated ENaC by increasing single-channel open probability. AP301 increased current in proteolytically activated (cleaved) but not near-silent (uncleaved) ENaC in a reversible manner. αβγ- or δβγ-ENaC coexpression was required for maximal activity. No increase in current was observed after deglycosylation of extracellular domains of ENaC. Thus, our data suggest that the specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s).
Capsaicin is a naturally occurring alkaloid derived from chili pepper which is responsible for its hot, pungent taste. It exerts multiple pharmacological actions, including pain-relieving, anti-cancer, anti-inflammatory, anti-obesity, and antioxidant effects. Previous studies have shown that capsaicin significantly affects the contractility and automaticity of the heart and alters cardiovascular functions. In this study, the effects of capsaicin were investigated on voltage-gated ion currents in rabbit ventricular myocytes. Capsaicin inhibited rapidly activated (IKr) and slowly activated (IKs) K+ currents and transient outward (Ito) K+ current with IC50 values of 3.4 µM,14.7 µM, and 9.6 µM, respectively. In addition, capsaicin, at higher concentrations, suppressed voltage-gated Na+ and Ca2+ currents and inward rectifier IK1 current with IC50 values of 42.7 µM, 34.9 µM, and 38.8 µM, respectively. Capsaicin inhibitions of INa, IL-Ca, IKr, IKs, Ito, and IK1 were not reversed in the presence of capsazepine (3 µM), a TRPV1 antagonist. The inhibitory effects of capsaicin on these currents developed gradually, reaching steady-state levels within 3 to 6 min, and the recoveries were usually incomplete during washout. In concentration-inhibition curves, apparent Hill coefficients higher than unity suggested multiple interaction sites of capsaicin on these channels. Collectively, these findings indicate that capsaicin affects cardiac electrophysiology by acting on a diverse range of ion channels and suggest that caution should be exercised when capsaicin is administered to carriers of cardiac channelopathies or to individuals with arrhythmia-prone conditions, such as ischemic heart diseases.
Pulmonary edema is a life threatening condition, and a frequent complication of acute lung injury, characterized by loss of epithelial sodium channel (ENaC) function and cell surface expression in diseased epithelia. AP301 peptide, a mimic of the lectin-like domain of TNF, is being developed as a therapy for pulmonary edema, having recently completed phase 2a clinical trials. We have previously shown that the proper glycosylation of H441 and A549 cells is crucial for its interaction with AP301 and initiates uptake of the peptide and interaction with the carboxyterminal domain of α-ENaC in H441 cells. In this study we identify a glycosylation-dependent mechanism that preserves ENaC expression and function. Single- and multi-N-glycosylation site mutations were generated in α(N232,293,312,397,511Q)- and δ(N166,211,384Q)-subunits. Therefore, αL576X and αN232,293,312,397,511Q,L576X mutants were generated by deletion of the carboxy terminal of wild type and quintuple (N232,293,312,397,511Q) mutant α-hENaC. These constructs were co-expressed in HEK-293 cells with βγ-hENaC. In α(N232,293,312,397,511Q),L576Xβγ-hENaC, AP301 activation, measured in the whole cell patch clamp mode, was abolished, and in αL576Xβγ-hENaC, it was attenuated. Taken together, our findings delineate an AP301 N-glycan dependent interaction leading to normalization of both sodium and fluid absorption in edematous alveoli to non-edematous levels. .
Significance Statement Rapid renal responses to ingested potassium are essential to prevent hyperkalemia and also play a central role in blood pressure regulation. Although local extracellular K + concentration in kidney tissue is increasingly recognized as an important regulator of K + secretion, the underlying mechanisms that are relevant in vivo remain controversial. To assess the role of the signaling kinase mTOR complex-2 (mTORC2), the authors compared the effects of K + administered by gavage in wild-type mice and knockout mice with kidney tubule-specific inactivation of mTORC2. They found that mTORC2 is rapidly activated to trigger K + secretion and maintain electrolyte homeostasis. Downstream targets of mTORC2 implicated in epithelial sodium channel regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. These findings offer insight into electrolyte physiologic and regulatory mechanisms. Background Increasing evidence implicates the signaling kinase mTOR complex-2 (mTORC2) in rapid renal responses to changes in plasma potassium concentration [K + ]. However, the underlying cellular and molecular mechanisms that are relevant in vivo for these responses remain controversial. Methods We used Cre-Lox–mediated knockout of rapamycin-insensitive companion of TOR (Rictor) to inactivate mTORC2 in kidney tubule cells of mice. In a series of time-course experiments in wild-type and knockout mice, we assessed urinary and blood parameters and renal expression and activity of signaling molecules and transport proteins after a K + load by gavage. Results A K + load rapidly stimulated epithelial sodium channel (ENaC) processing, plasma membrane localization, and activity in wild-type, but not in knockout, mice. Downstream targets of mTORC2 implicated in ENaC regulation (SGK1 and Nedd4-2) were concomitantly phosphorylated in wild-type, but not knockout, mice. We observed differences in urine electrolytes within 60 minutes, and plasma [K + ] was greater in knockout mice within 3 hours of gavage. Renal outer medullary potassium (ROMK) channels were not acutely stimulated in wild-type or knockout mice, nor were phosphorylation of other mTORC2 substrates (PKC and Akt). Conclusions The mTORC2-SGK1-Nedd4-2-ENaC signaling axis is a key mediator of rapid tubule cell responses to increased plasma [K + ] in vivo . The effects of K + on this signaling module are specific, in that other downstream mTORC2 targets, such as PKC and Akt, are not acutely affected, and ROMK and Large-conductance K + (BK) channels are not activated. These findings provide new insight into the signaling network and ion transport systems that underlie renal responses to K + in vivo .
