Purification and Functional Reconstitution of the TMEM16A Ca2+ Activated Cl− Channel

Abstract

Ca2+-activated Cl− channels (CaCCs) play numerous physiological roles ranging from electrolyte secretion in epithelia and glands, to muscle contraction, olfactory transduction and nociception. Despite their physiological importance the molecular identity of these proteins remained elusive until recent work showed that TMEM16A and B, two members of the TMEM16 family of membrane proteins, are critical components of CaCCs. Heterologous expression of these two genes in oocytes and cells results in currents closely resembling native CaCCs. It is however unknown whether these proteins alone are necessary and sufficient to form functional Ca2+ activated Cl− channels, or whether association to other subunits is required. For example, neither TMEM16A nor B bear any conventional Ca2+ binding motifs and studies suggest that association of a TMEM16A isoform with Calmodulin is required for Ca2+ sensitivity.To investigate whether TMEM16A alone forms functional CaCCs or if association to other partner proteins is required we expressed, purified and reconstituted TMEM16A in proteoliposomes. We found that purified TMEM16A mediates Ca2+ dependent Cl− fluxes with an apparent Km of ∼300 nM, a value comparable to that measured in patch clamp experiments. Channel opening is also promoted by the application of high positive voltages, consistent with electrophysiological measurements. Channel activity is diminished by CaCC inhibitors such as Niflumic Acid, NPPB, NPA and DIDS with K1/2's comparable to those measured for native and heterologous CaCCs. Mutating two conserved glutamates in the TM5-6 intracellular loops abolishes Ca2+ sensitivity of the fluxes, in a manner similar to what was reported for heterologously expressed TMEM16A.In conclusion, our results demonstrate that the purified TMEM16A protein recapitulates all the fundamental biophysical and pharmacological properties of native and heterologously expressed CaCC currents indicating that association with other partner proteins is not strictly required for function.