The Pannexin-1 (Panx1) channel is known to become activated under a variety of physiological conditions resulting in the release of medium-sized molecules such as ATP and amino acids from the cell. The detailed molecular mechanism of activation of the channel resulting in the opening of the Pannexin pore is poorly understood. The best-studied gating mechanism is caspase-3/7-mediated cleavage and truncation of the c-terminus. In the absence of caspase-cleavage, the c-terminal peptide maintains the channel in the closed state, possibly by directly plugging the pore from the intracellular side. We sought to understand in detail the part of the c-terminus necessary for this interaction by alanine-scanning and truncation mutagenesis of the c-terminal gating peptide. These experiments demonstrate that no single amino acid side-chain is necessary for this interaction. In fact, replacing blocks of 10-12 amino acids in different parts of the c-terminal peptide with alanines fails to disrupt the ability of the c-terminus to keep the channel closed. Surprisingly, even replacing the entire c-terminal gating peptide with a scrambled peptide of the same length maintains the interaction in some cases. Further analysis revealed that the interaction surface, while delocalized, is located within the amino-terminal two-thirds of the c-terminal peptide. Such a delocalized and potentially low-affinity interaction surface is allowed due to the high effective concentration of the c-terminal peptide near the inner vestibule of the pore and likely explains why this region is poorly conserved between species. This type of weak interaction with a tethered gating peptide may be required to maintain high-sensitivity to caspase-dependent activation.