Curves for TMEM16A(abc) + CaM (dotted black collection) and TMEM16A (abc) + CaM1234(dotted red collection) are reproduced fromFig. chloride channel == Abstract == Ca2+-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, clean muscle mass contraction, olfactory transmission transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca2+is usually critical for defining their (patho)/physiological functions and for rationally targeting them therapeutically. Here, using a bioengineering approachchannel inactivation induced by membrane-tethering of an associated protein (ChIMP)we discovered that Ca2+-free calmodulin (apoCaM) is usually preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two unique 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 alternate splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segmentbselectively lost CDI, and segmentais 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. Calcium (Ca2+)-activated chloride (Cl) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1,2), neuronal excitability (35), easy muscle mass contraction (6), olfactory transduction (7,8), and cell proliferation (9,10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and malignancy (1,9,11). Three laboratories independently recognized the transmembrane protein TMEM16A as the molecular component of a CaCC (1214). TMEM16A belongs to a protein family with 10 users encoded by unique genes (1518). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2,1214,19). Consistent with this,TMEM16Aknockout mice displayed defective CaCC activity in a variety of epithelia (2022), and the olfactory CaCC current was completely abolished inTMEM16Bknockout BIIE 0246 mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2,19). Human TMEM16A has four alternatively spliced segments (ad), differential inclusion of which change voltage and Ca2+sensitivity of resultant channel splice variants (24). CaCCs are highly sensitive to intracellular [Ca2+], displaying graded increases in Clcurrent (ICl) amplitude as [Ca2+]iis raised from resting levels (100 nM) to the 1- to 2-M range. In some cases, high [Ca2+]i(>10 M) prospects to decreasedIClamplitude (inactivation) (2527). The Ca2+sensor(s) for Ca2+-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is usually unknown. You will find two possible nonexclusive mechanisms: (i) direct Ca2+binding to the channel or (ii) Ca2+binding through a separate Ca2+-sensing protein. The TMEM16A sequence does not reveal any canonical Ca2+-binding EF hand motifs (14,16,17). A sequence TSPAN32 in the first intracellular loop of TMEM16A resembling the Ca2+bowl in large conductance Ca2+-activated K+(BK) channels was disqualified by mutagenesis as the Ca2+sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca2+sensitivity of TMEM16A (29,30). Some reports have suggested involvement of calmodulin (CaM) in unique aspects of Ca2+-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segmentb(31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is usually contradicted by the strong CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segmentb(2,24). Recently, Ca2+CaM was found to bind TMEM16A(ac) in a Ca2+-dependent manner and result in an increased permeability of the channel to HCO3(32). Deleting the Ca2+CaM binding site did not impact CDA of TMEM16A(ac). Ca2+CaM regulation of TMEM16A HCO3permeability conforms to a traditional signaling mode where Ca2+binds to freely diffusing CaM to form a Ca2+CaM complex that then interacts with a target protein. There are several examples of an alternative mode of CaM signaling in which Ca2+-free CaM (apoCaM) is usually preassociated with BIIE 0246 target proteins under resting [Ca2+]iconditions BIIE 0246 and functions as a resident Ca2+sensor to regulate function of the host protein in response to increased [Ca2+]i(33). This mode of CaM signaling is used as the activating mechanism for small conductance K+channels (34) and Ca2+-dependent regulation of high voltage-gated Ca2+(CaV1 and CaV2) channels (35,36). A distinguishing feature of this mode of CaM signaling is usually that it is impervious to pharmacological inhibitors of Ca2+CaM,.