Abstract
Ca2+-activated Cl- channels (CaCCs) play important roles in various cellular mechanisms, including fluid secretion in epithelia, sensory transduction, and regulation of neuronal and smooth muscle excitability. Molecular identity of this type of channels was controversial. Our group has recently identified TMEM16A as a possible CaCC. The aim of our present study is to characterize the properties of the Cl- currents associated with TMEM16A expression and to compare them with those of classical CaCCs described in several previous studies. For this purpose, we have used the patch clamp technique in the whole-cell configuration on FRT cells stable-transfected with the TMEM16A(abc) isoform. To analyse the ion channel selectivity, we substituted Cl- in the extracellular solution with other anions (I-, Br-, SCN-, and gluconate) and we measured the resulting shift in the reversal potential of membrane currents. Our data indicate that TMEM16A-dependent channels have ion selectivity properties similar to those of native CaCCs (Hartzell, Putzier and Arreola, Annu. Rev. Physiol. 2005. 67:719-58). We also studied the Ca2+-dependence of TMEM16A channels by changing the cytosolic free Ca2+-concentration in the 0.017 - 1.35 ?M range. By plotting the maximal current elicited at + 100 mV versus the cytosolic Ca2+ concentration, and fitting the data with a Hill function, we found a Kd = 91.8 nM and a Hill coefficient nH = 2.32. These values are close to those published previously for CaCCs (Kd = 61 nM, nH = 2.7; Arreola, Melvin and Begenisish, J. Gen. Physiol. 1996. 108:35-47). In conclusion, our results confirm that TMEM16A is a membrane protein involved in Ca2+-dependent Cl- transport. This remark evidences that TMEM16A may represent an important pharmacological target to treat cystic fibrosis in which activation of an alternative Cl- channel may compensate for the defective CFTR activity