Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels

Ca2+-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca2+ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach-channel inactivation induced by membrane-tethering of an associated protein (ChIMP)-we discovered that Ca2+-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca2+-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM1234. These effects are Ca2+-dependent sensitization of activation (CDSA) and Ca2+-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca2+-activated chloride channels.