Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

Abstract

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

Preassociated apocalmodulin mediates Ca 2+ -dependent sensitization of activation and inactivation of TMEM16A/16B Ca 2+ -gated Cl − channels

“…Interestingly, reconstituted purified TMEM16A (abc) lacks CDI, suggesting the sensor for this phenomenon is not encoded within the channel's primary sequence (30). Recent studies indicate that Ca 2+ -CaM regulates the HCO 3 − permeability (32) and run-up/run-down of TMEM16A (40). These modes of CaM signaling are different from what we report here, which features apoCaM preassociated to TMEM16A/16B channel complexes as a resident Ca 2+ sensor.…”

“…Previous biochemical pull-down studies suggest that TMEM16A does not bind apoCaM (32,40). Additionally, whether TMEM16A binds Ca 2+ -CaM is controversial-some biochemical pull-down studies support an interaction (31,32), whereas others do not (40).…”

“…Additionally, whether TMEM16A binds Ca 2+ -CaM is controversial-some biochemical pull-down studies support an interaction (31,32), whereas others do not (40). Notably, pull-down methods can be notoriously inconsistent with regards to confirming apoCaM/Ca 2+ -CaM association with ion channels even when these are functionally known to occur (35,41).…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…126 Two putative CaM binding sites were identified on ANO1, which led to the proposal that Ca 2C sensitivity is mediated by calmodulin. 46,127 Recent papers did however present strong evidence against the possible role of calmodulin. 128,129 The most direct evidence that the Ca 2C sensitivity is intrinsic and not dependent on calmodulin comes from the observation that purified ANO1 reconstituted in liposomes recapitulates the functional properties of ANO1.…”

Activation of Ca2+‐activated Cl− channel ANO1 by localized Ca2+ signals

“…2011; Vocke et al . 2013) or anion permeability (Jung et al . 2013) but these findings are being disputed (Terashima et al .…”