Abstract
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.
Highlights
In our earlier study we analyzed the regulation of the Ca2+-activated Cl− channel (CaCC) TMEM16A
While overexpressed TMEM16A was potently activated by the inositol phosphate INO-4995, endogenous TMEM16A expressed in airway and colonic epithelial cells was not
As we did not want to rely on results obtained in one cell line only, we compared results obtained in overexpressing HEK293 cells with those obtained in cell expressing endogenous TMEM16A channels (CFBE, BCi-NS1, HT29 cells)
Summary
In our earlier study we analyzed the regulation of the Ca2+-activated Cl− channel (CaCC) TMEM16A. We observed that regulation of TMEM16A by INO-4995, a lipid inositol phosphate, differs depending on whether the channel is overexpressed in mammalian cells such as HEK293, or whether regulation of endogenous TMEM16A is examined [23]. While overexpressed TMEM16A was potently activated by the inositol phosphate INO-4995, endogenous TMEM16A expressed in airway and colonic epithelial cells was not. Other previously reported activators of TMEM16A such as melittin or cinnamaldehyde failed to activate endogenous TMEM16A These differences prompted us to compare the role of CAM for acute receptor-mediated activation of TMEM16A in overexpressing cells and epithelial cells with endogenous expression of TMEM16A. The data suggest only a minor role of CAM and the CAM-dependent proteins CAMKII and calcineurin for activation of endogenous TMEM16A, while CAM is likely to enhance Ca2+ sensitivity of overexpressed TMEM16A
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