title: Putative pore-loops of TMEM16/anoctamin channels affect channel density in cell membranes.
abstract: The recently identified TMEM16/anoctamin protein family includes Ca(2+)-activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5-TM6 flanking a re-entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca(2+) binding. The role of the putative pore-loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole-cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca(2+). Furthermore, whole-cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B-mediated currents. Replacement of the putative pore-loop of TMEM16A with that of TMEM16B (TMEM16A-B channels) reduced the currents by ∼six-fold, while the opposite modification (TMEM16B-A channels) produced a ∼six-fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca(2+) or voltage, nor were they due to changes in single-channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore-loop of TMEM16A and TMEM16B led to the identification of a region containing a non-canonical trafficking motif. Chimeras composed of the putative pore-loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore-loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore-loop of TMEM16A and TMEM16B channels in the control of the whole-cell Ca(2+)-activated Cl(-) conductance.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK.
authors.forename: Aiste
authors.initials: A
authors.lastname: Adomaviciene
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Smith
authors.forename: Hannah
authors.initials: H
authors.lastname: Garnett
authors.forename: Paolo
authors.initials: P
authors.lastname: Tammaro
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000737:chemistry* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000502:physiology; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D018118:Chloride Channels; D009363:Neoplasm Proteins; D002118:Calcium