title: The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop.
abstract: Ca(2+)-activated Cl(-) channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure-function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca(2+) dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca(2+) concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates (386)EEEEE(390) and (399)EYE(401). The EYE deletion did not significantly modify the apparent Ca(2+) dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca(2+) affinity but modified the voltage dependence, shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and (386)EEEEE(390) in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure-function study for this channel.
authors.affiliation: Neurobiology Sector, International School for Advanced Studies, and Italian Institute of Technology, SISSA Unit, 34136 Trieste, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Cenedese
authors.forename: Giulia
authors.initials: G
authors.lastname: Betto
authors.forename: Fulvio
authors.initials: F
authors.lastname: Celsi
authors.forename: O Lijo
authors.initials: OL
authors.lastname: Cherian
authors.forename: Simone
authors.initials: S
authors.lastname: Pifferi
authors.forename: Anna
authors.initials: A
authors.lastname: Menini
mesh_terms: D000595:Amino Acid Sequence; D000075368:Anoctamins; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: C532161:ANO2 protein, mouse; D000075368:Anoctamins; D018118:Chloride Channels