Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods, we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods, we found that TACAN, at high protein concentrations, produces heterogeneous

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... onduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single-particle cryo-electron microscopy and observed that it is a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme A cofactor, confirmed by mass spectrometry. The TACAN protomer is related in three-dimensional structure to a fatty acid elongase, ELOVL7. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel. Results Functional analysis in cells and reconstituted membranes We sought to reproduce the mechanically evoked currents reported when TACAN is expressed in cells (Beaulieu-Laroche et al., 2020) . Using CHO cells, similar to those used in the original study, we did not observe pressure-evoked currents in excised membrane patches ( Figure 1A,B) . Background channels that were not sensitive to the pressure steps were observed in CHO cells expressing either TACAN or the M2 muscarinic receptor as a control. Similarly, TACAN expressed in a Piezo1 knockout HEK cell line did not elicit pressure-activated channels ( Figure 1C,D) . Purified TACAN protein reconstituted into giant unilamellar vesicles (GUVs) of soy L-α-phosphatidylcholine (soy-PC) also did not yield pressure-activated channels in membrane patches isolated from the GUVs ( Figure 1E ). We note that previously we have successfully recorded mechanosensitive TRAAK channels in GUVs using the identical approach (Brohawn et al., 2014) . When TACAN was expressed, purified, and reconstituted into both GUVs and planar lipid bilayers at high protein-to-lipid ratios (≥1:100, m:m), transient currents were observed, as shown in Figure 2 .