Structural Basis of Pannexin Activation

Abstract

Pannexin 1 (Panx1) is a member of a family of large-pore ion channels distantly related to invertebrate gap junction channels, the innexins. Activation of Panx1 occurs under a variety of physiological processes, but the molecular mechanism of such activation has not in general been clearly established. Recently it was shown that Panx1 could be activated by direct cleavage near the c-terminus by caspase-3. This occurs during apoptosis and leads to the release of large intracellular molecules such as ATP, which act as “find-me” signals enabling nearby phagocytes to quickly clear dying cells. The C-terminal domain peptide seems to function as a tethered pore blocker and is thought to have a specific interaction with structural elements within the pore to inhibit the channel. However, the basis for this structural interaction is poorly understood. Here we dissect those interactions using progressive alanine substitutions and long alanine repeats within the region distal to the caspase cleavage site. Our results suggest that any specific interaction between the pore and the C-terminus is minimal since replacement of the amino acid residues in the region adjacent to the caspase cleavage site with alanines does not produce a constitutively open channel. The most important feature of the C-terminal inhibitory peptide appears instead to simply be its length. We therefore propose that the c-terminal peptides loop back into the pore to reach a constricted region, thereby blocking the pore by sterically interfering with ion permeation. Peptides not long enough to reach this constriction within the pore are consequently unable to block the channel while longer peptides are able to block the pore in a promiscuous fashion.