Electrophysiology of Concatameric Pannexin 1 Channels Reveals the Stoichiometry of C-Terminal Autoinhibition

Abstract

Pannexin 1 (PANX1) is a non-selective ion channel that mediates the uptake of cyanine dyes and release of nucleotides and other metabolites. PANX1 activation and ATP release/dye uptake are regulated by diverse stimuli, including physicochemical factors (e.g., stretch, K+ ions) and signaling by various G protein-coupled and ionotropic receptors. We recently described a unique regulatory mechanism for the release of ATP from apoptotic cells: PANX1 is activated by caspase cleavage of a C-terminal autoinhibitory domain. Current evidence suggests that PANX1 channels are hexameric, and cleavage-resistant subunits can interfere with caspase-dependent activation in a dominant-negative fashion. To explore the subunit stoichiometry required for C-terminal autoinhibition, we engineered concatameric PANX1 constructs with different (from 0 to 6) combinations of truncated and full-length sequences. Formation of concatemers was assessed by the use of single-molecule photobleaching and protein cross-linking. Whole-cell recordings from concatenated PANX1 constructs suggest that at least four intact C-termini are required to inhibit channel activity. In addition, as the number of intact C-termini increased, there was a progressive decrease in single channel conductance, suggesting that individual C-termini may act within the multimeric channel to inhibit channel conductance. These results provide further mechanistic insights into the regulation of PANX1 channels by the C-terminal autoinhibitory domains.