N. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca 2ϩ levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca 2ϩ -activated ClϪ channels (Cl Ca), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl Ca channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca 2ϩ -activated Cl Ϫ current [ICl(Ca)] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I Cl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl Ca channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 M), an L-type Ca 2ϩ channel blocker, and NFA (30 or 100 M, with or without 10 M indomethacin to inhibit cyclooxygenases) or T16A Inh-A01 (10 M), TMEM16A/Cl Ca channel inhibitors, than that of control animals. In conclusion, augmented Cl Ca/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. pulmonary arterial tone; TMEM16A; anoctamin-1; Ca 2ϩ -activated Cl Ϫ channel; patch-clamp technique PULMONARY HYPERTENSION (PH) is defined as a sustained high blood pressure (Ͼ25 mmHg at rest and Ͼ30 mmHg during exercise) in the main pulmonary artery (PA) that ultimately leads to failure of the right hand side of the heart and death (4). Characteristic pathophysiological manifestations of PH are enhanced vasoconstriction, thickening of the arterial muscle wall, and a propensity for thrombosis, as a result of changes in all layers of the blood vessel, but little is known about the molecular mechanisms that drive these pathological responses. It is well established that pulmonary arterial smooth muscle cells (PASMCs) from animal models of PH and human PH patients are more depolarized and exhibit a higher intracellular calcium concentration ([Ca 2ϩ ] i ) than cells from healthy individuals and several ionic conductances are altered in PASMCs from animal models of PH and PH patients (4,13,29,43,68,70). Except for one recent study carried out using the chronic hypoxic model of PH in the rat (58), there is little information regarding the potential role of Ca 2ϩ -activa...

The mechanism of KCNQ1 inactivation and its modulation by external K þ are dissimilar to the mechanism described for C-type inactivation in Shaker-like K þ channels (Gibor et al., 2007). Further, inactivation of wild-type (WT) KCNQ1 channels becomes evident only in the characteristic hooked tail currents which reflect recovery from inactivation (Abitbol et al., 1999; Pusch et al., 1998; Tristani-Firouzi and Sanguinetti, 1998). We use a combination of functional-structural analysis combined with mathematical and 3D-structural modeling to gain insights into the structural rearrangements during KCNQ1-inactivation. We show that the Kv7.1 a-subunits act in a concerted way to initiate KCNQ1-inactivation.

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Ca2+‐dependent Cl− currents (IClCa) are down regulated by phosphorylation in arterial smooth muscle cells. We recently showed that niflumic acid (NFA), an inhibitor of IClCa, is less efficacious at blocking the current in conditions promoting phosphorylation. This study aimed to assess whether another Cl− channel blocker, anthracene‐9‐carboxylic acid (A9C), is also affected by channel phosphorylation. A9C blocks IClCa at positive potentials but paradoxically stimulates the inward IClCa tail after repolarization to negative potentials. IClCa was evoked by pipette solutions containing 500 nM free Ca2+ with or without 5 mM ATP to alter the state of phosphorylation. A9C (10‐500 μM) produced voltage‐dependent block of IClCa that was more potent in cells dialyzed with ATP vs. 0 ATP. A9C enhanced IClCa tail at ‐80 mV by causing a large negative shift in voltage‐dependence in both cell groups. Finally, phosphorylation greatly attenuated the rate of recovery of IClCa at +80 mV upon washout of A9C, but not IClCa tail at ‐80 mV. As for NFA, the complex actions of A9C with IClCa are profoundly influenced by the state of channel phosphorylation and we propose the existence of multiple binding sites for A9C.