Calmodulin-Dependent Regulation of Overexpressed but Not Endogenous TMEM16A Expressed in Airway Epithelial Cells

Regulation of the Ca²⁺-activated Cl⁻ channel TMEM16A by Ca²⁺/calmodulin (CAM) is discussed controversially. In the present study, we compared regulation of TMEM16A by Ca²⁺/calmodulin (holo-CAM), CAM-dependent kinase (CAMKII), and CAM-dependent phosphatase calcineurin in TMEM16A-overexpressing HEK293 cells and TMEM16A expressed endogenously in airway and colonic epithelial cells. The activator of the Ca²⁺/CAM-regulated K⁺ channel KCNN4, 1-EBIO, activated TMEM16A in overexpressing cells, but not in cells with endogenous expression of TMEM16A. Evidence is provided that CAM-interaction with TMEM16A modulates the Ca²⁺ sensitivity of the Cl⁻ channel. Enhanced Ca²⁺ sensitivity of overexpressed TMEM16A explains its activity at basal (non-elevated) intracellular Ca²⁺ levels. The present results correspond well to a recent report that demonstrates a Ca²⁺-unbound form of CAM (apo-CAM) that is pre-associated with TMEM16A and mediates a Ca²⁺-dependent sensitization of activation (and inactivation). However, when using activators or inhibitors for holo-CAM, CAMKII, or calcineurin, we were unable to detect a significant impact of CAM, and limit evidence for regulation by CAM-dependent regulatory proteins on receptor-mediated activation of endogenous TMEM16A in airway or colonic epithelial cells. We propose that regulatory properties of TMEM16A and and other members of the TMEM16 family as detected in overexpression studies, should be validated for endogenous TMEM16A and physiological stimuli such as activation of phospholipase C (PLC)-coupled receptors.