Activating Individual Subunits of TMEM16A Calcium-Activated Chloride Channels

Ca2+-activated Cl− channels in the TMEM16 family are transmembrane proteins formed by two identical subunits. They participate in various physiological functions such as transepithelial Cl- transport and olfactory signal transduction. Site-directed mutagenesis studies of TMEM16A have identified several acidic amino acids, the mutations of which alter the apparent affinity of the channel for Ca2+, suggesting a Ca2+-binding site formed by these acidic residues. A recent crystallization study has further revealed that these acidic residues indeed coordinate bound Ca2+ in the TMEM16 molecules. Here, we created constructs with two linked TMEM16A subunits: a high Ca2+ affinity wild-type subunit and a low Ca2+ affinity subunit with mutation of one of the aforementioned acidic residues. The dose-response curve of the heterodimer appears to be a summation of two Hill equations, one similar to the dose-response curve of WT TMEM16A and the other similar to that of the mutant with a low Ca2+ affinity. The independent activation of the two subunits was also clearly observed when Ca2+ was washed out from the channels. These results indicate that Ca2+ binding to one subunit of TMEM16A can significantly activate the channel current. However, activations of heterodimers with a Ca2+ concentration that saturates the WT channel but rarely opens the mutant channel generate currents below or above 50 % of the current in different heterodimers, suggesting that inter-subunit interaction may play a minor role in the cooperativity of channel activation by multiple Ca2+ bindings. That activation of a single subunit can generate significant current diverges from the scenario in voltage-gated cation channels where all subunits must be activated before channel opens. Rather, separate activation of individual subunits may suggest that the principle of TMEM16A gating is more similar to that of CLC chloride channels.