Panx1 mutants are generally within a factor of 2-fold relative to the FL Panx1 channel with a few exceptions. In general, constitutively active mutants usually have lower surface expression than inactive mutants. To examine whether differences in surface expression between mutants might effect our conclusions, we re-analyzed our data correcting for measured relative surface expression levels. We find that our conclusions remain unchanged with this analysis. none of the double-alanine or pAla mutants are constitutively active, pAlaExt3 is still active, 2 of 4 scrambled mutants are inactive, truncation mutants shorter than D407 are active while longer truncations are active, and truncations in the context of the pAlaExt mutant have an activity length curve shifted to the right by about amino acids. The two scrambled mutants that are partially active appear less active when correcting for surface expression since these two mutants appear to have higher levels of surface expression relative to other mutants. Such a large, non-specific and delocalized interaction surface contained within the residues downstream of the caspasecleavage site does not necessarily imply a low affinity interaction since many low affinity interactions within this large region could in theory add up to a high affinity interaction. However, a low affinity interaction may be tolerated in the case of Panx1 since the channel contains six identical subunits. This means that six 48amino acid c-terminal gating peptides are positioned within interaction distance to the vestibule of the pore. We calculate that the approximate concentration of c-termini within the inner vestibule region to be on the order. Therefore a low affinity interaction would still allow near 100% occupancy, especially if we assume that only a single c-terminus is required to block the pore at any one time. Thus, there is little evolutionary pressure to enhance the affinity or specificity of this interaction. This is likely the reason that this 48-residue c-terminus is very poorly conserved across different vertebrate species despite the fact that the cterminus plays such a critical role in the gating and function of the Panx1 channel. In fact there may be a selection pressure to maintain the low affinity nature of this interaction allowing the channel to be more sensitive to activation by caspase3/7, as these enzymes would not need to remove all six c-termini to achieve channel opening. While we can speculate that the interaction between pore and cterminal gating peptide is likely very low affinity, measuring the actual affinity will be difficult. Experiments where the gating peptide is added exogenously will be complicated by the fact that very high concentrations likely are required to achieve the effective concentration normally present at the inner vestibule of the pore.