Functional data show that even though hPANX1 channels can work as hexamers, the native form is heptameric. (A) Diagram depicting the experimental strategy used by Gupta et al. (2025) to generate the desired forms of hPANX1 channels and the corresponding single-channel activity (obtained from excised inside-out patches at +80 mV). Top: Native hPANX1 channel formed by hPANX1 expressed as monomers. The full-length C-terminal (CT) domain (red) is depicted as ending in a pore-lining ball to indicate a nonconductive state. Caspase truncation of the CT domain activates hPANX1 channels. In Gupta et al., this activating truncation was achieved by TEV protease cleavage at an engineered TEV cleavage site (orange-filled circle). Middle: Hexameric concatemer in which the same portion of the CT was deleted, and followed by a TEV cleavage site and short linker (green) to the N-terminus of the adjacent subunit. TEV cleavage of the linker was required to activate the channels. Lower: Heptameric concatemer in which the same portion of the CT was deleted, followed by a TEV cleavage site and short linker to the N-terminus of the adjacent subunit. TEV cleavage of the linker was required to activate the channels. (Channel traces shown were adapted from Gupta et al. [2025] Fig. S3 B [native], Fig. 3 B [hexamer], and Fig. 4 B [heptamer]). (B) Comparison of properties of native hPANX1 channels (N; hPANX1 expressed as monomers) activated by CT truncation, with those of concatemeric hexamers (6) and heptamers (7) with CT truncations, active after intersubunit linker cleavage. From left to right: Graphs show unitary conductance and mean open times obtained from single-channel recordings in excised patches (+80 mV) and normalized ATP release. Bars show means and SE. (Conductance data shown are as stated in the paper; mean open times and normalized ATP release data are taken from Gupta et al. (2025) Fig. 5, D and E; and Fig. 6 A, respectively, and replotted as means and SE).