Modulating Ca²âº signals: a common theme for TMEM16, Ist2, and TMC.

Since the discovery of TMEM16A (anoctamin 1, ANO1) as Ca(2+)-activated Cl(-) channel, the protein was found to serve different physiological functions, depending on the type of tissue. Subsequent reports on other members of the anoctamin family demonstrated a broad range of yet poorly understood properties. Compromised anoctamin function is causing a wide range of diseases, such as hearing loss (ANO2), bleeding disorder (ANO6), ataxia and dystonia (ANO3, 10), persistent borrelia and mycobacteria infection (ANO10), skeletal syndromes like gnathodiaphyseal dysplasia and limb girdle muscle dystrophy (ANO5), and cancer (ANO1, 6, 7). Animal models demonstrate CF-like airway disease, asthma, and intestinal hyposecretion (ANO1). Although present data indicate that ANO1 is a Ca(2+)-activated Cl(-) channel, it remains unclear whether all anoctamins form plasma membrane-localized or intracellular chloride channels. We find Ca(2+)-activated Cl(-) currents appearing by expression of most anoctamin paralogs, including the Nectria haematococca homologue nhTMEM16 and the yeast homologue Ist2. As recent studies show a role of anoctamins, Ist2, and the related transmembrane channel-like (TMC) proteins for intracellular Ca(2+) signaling, we will discuss the role of these proteins in generating compartmentalized Ca(2+) signals, which may give a hint as to the broad range of cellular functions of anoctamins.

Identification of determinants of lipid and ion transport in TMEM16/anoctamin proteins through a Bayesian statistical analysis.

The TMEM16/Anoctamin protein family (TMEM16x) is composed of members with different functions; some members form Ca2+-activated chloride channels, while others are lipid scramblases or combine the two functions. TMEM16x proteins are typically activated in response to agonist-induced rises of intracellular Ca2+; thus, they couple Ca2+-signalling with cell electrical activity or plasmalemmal lipid homeostasis. The structural domains underlying these functions are not fully defined. We used a Naïve Bayes classifier to gain insights into these domains. The method enabled identification of regions involved in either ion or lipid transport, and suggested domains for possible pharmacological exploitation. The method allowed the prediction of the transport property of any given TMEM16x. We envisage this strategy could be exploited to illuminate the structure-function relationship of any protein family composed of members playing different molecular roles.

Ca2+ Dependence of Volume-Regulated VRAC/LRRC8 and TMEM16A Cl- Channels.

All vertebrate cells activate Cl- currents (ICl ,swell) when swollen by hypotonic bath solution. The volume-regulated anion channel VRAC has now been identified as LRRC8/SWELL1. However, apart from VRAC, the Ca2+-activated Cl- channel (CaCC) TMEM16A and the phospholipid scramblase and ion channel TMEM16F were suggested to contribute to cell swelling-activated whole-cell currents. Cell swelling was shown to induce Ca2+ release from the endoplasmic reticulum and to cause subsequent Ca2+ influx. It is suggested that TMEM16A/F support intracellular Ca2+ signaling and thus Ca2+-dependent activation of VRAC. In the present study, we tried to clarify the contribution of TMEM16A to ICl ,swell. In HEK293 cells coexpressing LRRC8A and LRRC8C, we found that activation of ICl ,swell by hypotonic bath solution (Hypo; 200 mosm/l) was Ca2+ dependent. TMEM16A augmented the activation of LRRC8A/C by enhancing swelling-induced local intracellular Ca2+ concentrations. In HT29 cells, knockdown of endogenous TMEM16A attenuated ICl ,swell and changed time-independent swelling-activated currents to VRAC-typical time-dependent currents. Activation of ICl ,swell by Hypo was attenuated by blocking receptors for inositol trisphosphate and ryanodine (IP3R; RyR), as well as by inhibiting Ca2+ influx. The data suggest that TMEM16A contributes directly to ICl ,swell as it is activated through swelling-induced Ca2+ increase. As activation of VRAC is shown to be Ca2+-dependent, TMEM16A augments VRAC currents by facilitating Hypo-induced Ca2+ increase in submembraneous signaling compartments by means of ER tethering.