The TMEM16 family encompasses Ca²⁺-activated Cl^- channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca²⁺ affinities. The dose-response curve of the heterodimer appears to be a weighted sum of two dose-response curves-one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca²⁺ activation curve for the heterodimer at a low Ca²⁺ concentration range ([Ca²⁺] < 5 µM) is similar to that of the wild-type channel-the Hill coefficients in both cases are significantly greater than one. This suggests that Ca²⁺ binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca²⁺-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca²⁺ affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a "double-barrel" pore architecture, similar to that found in CLC channels and transporters.

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