Anoctamin 1 (ANO1)/transmembrane protein 16A (TMEM16A) is a calcium-activated anion channel that may play a role in HCO ₃ ⁻ secretion in epithelial cells. Here, we report that the anion selectivity of ANO1 is dynamically regulated by the Ca ²⁺ /calmodulin complex. Whole-cell current measurements in HEK 293T cells indicated that ANO1 becomes highly permeable to HCO ₃ ⁻ at high [Ca ²⁺ ] _i . Interestingly, this result was not observed in excised patches, indicating the involvement of cytosolic factors in this process. Further studies revealed that the direct association between ANO1 and calmodulin at high [Ca ²⁺ ] _i is responsible for changes in anion permeability. Calmodulin physically interacted with ANO1 in a [Ca ²⁺ ] _i -dependent manner, and addition of recombinant calmodulin to the cytosolic side of excised patches reversibly increased P _HCO3 /P _Cl . In addition, the high [Ca ²⁺ ] _i -induced increase in HCO ₃ ⁻ permeability was reproduced in mouse submandibular gland acinar cells, in which ANO1 plays a critical role in fluid secretion. These results indicate that the HCO ₃ ⁻ permeability of ANO1 can be dynamically modulated and that ANO1 may play an important role in cellular HCO ₃ ⁻ transport, especially in transepithelial HCO ₃ ⁻ secretion.

Aberrant membrane transport caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with a wide spectrum of respiratory and digestive diseases as well as cystic fibrosis. Using a gene scanning method, we found 11 polymorphisms and mutations of the CFTR gene in the Korean population. Individual variants at these sites were analyzed by conventional DNA screening in 117 control and 75 patients having bronchiectasis or chronic pancreatitis. In a haplotype determination based on a Bayesian algorithm, 15 haplotypes were assembled in the 192 individuals tested. Several haplotypes, especially with Q1352H, IVS8 T ₅ , and E217G, were found to have disease associations in a case–control study. Notably, a common polymorphism of M470V appears to affect the intensity of the disease association. Among the two haplotypes having IVS8 T ₅ , the T ₅ -V470 haplotype showed higher disease association than the T ₅ -M470 haplotype. In addition, a Q1352H mutation found in a V470 background showed the strongest disease association. The physiological significances of the identified mutations were rigorously analyzed. Non-synonymous E217G and Q1352H mutations in the M470 background caused a 60–80% reduction in CFTR-dependent Cl ⁻ currents and HCO ₃⁻ -transport activities. Surprisingly, the additional M470V polymorphic variant with the Q1352H mutation completely abolished CFTR-dependent anion transport activities. These findings provide the first evidence on the importance of CFTR mutations in the Asian population. Importantly, the results also reveal that interactions between multiple genetic variants in cis affect the final function of the gene products.

Background: Reactive oxygen species (ROS) and calcium ions (Ca ²⁺ ) are among the major effectors of Ang II (angiotensin II) in vascular smooth muscle cells. ROS are related to Ca ²⁺ signaling or contraction induced by Ang II, but little is known about their detailed functions. Here, NOX (NADPH oxidase), a major ROS source responsive to Ang II, was investigated regarding its contribution to Ca ²⁺ signaling. Methods: Vascular smooth muscle cells were primary cultured from rat aorta. Ca ²⁺ and ROS were monitored mainly using fura-2 and HyPer family probes‚ respectively. Signals activating NOX were examined with relevant pharmacological inhibitors and genetic manipulation techniques. Results: Ang II–induced ROS generation was found to be biphasic: the first phase of ROS production, which was mainly mediated by NOX1, was small and transient, preceding a rise in Ca ²⁺ , and the second phase of ROS generation, mediated by NOX1 and NOX4, was slow but sizeable, continuing over tens of minutes. NOX1-derived superoxide in the first phase is required for Ca ²⁺ influx through nonselective cation channels. AT1R (Ang II type 1 receptor)-G _βγ -PI3K _γ (phosphoinositide 3-kinase γ) signaling pathway was responsible for the rapid activation of NOX1 in the first phase, while in the second phase, NOX1 was further activated by a separate AT1R-Gα _q/11 -PLC (phospholipase C)-PKC _β (protein kinase C β) signaling axis. Consistent with these observations, aortas from NOX1-knockout mice exhibited reduced contractility in response to Ang II, and thus the acute pressor response to Ang II was also attenuated in NOX1-knockout mice. Conclusions: NOX1 mediates Ca ²⁺ signal generation and thereby contributes to vascular contraction and blood pressure elevation by Ang II.