Abstract
Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.
Highlights
Pannexin 1 (Panx1) is a widely expressed homo-heptameric membrane channel that plays a critical role in numerous physiological and pathophysiological processes
We used the Panx1 ortholog from Xenopus tropicalis that has been examined in recent structural studies (Deng et al, 2020; Michalski et al, 2020)
Purified fPanx1-enhanced green fluorescent protein (eGFP) was incorporated into proteoliposomes that appeared in fractions 1–3 of a co-floatation assay on a Nycodenz density gradient (Figure 1B,C; Hernandez et al, 2012)
Summary
Pannexin 1 (Panx1) is a widely expressed homo-heptameric membrane channel that plays a critical role in numerous physiological and pathophysiological processes. Among others, this includes cell clearance after apoptosis (Chekeni et al, 2010; Medina et al, 2020; Poon et al, 2014), blood pressure regulation (Billaud et al, 2011; Good et al, 2018a), stroke (Bargiotas et al, 2011; Good et al, 2018b; Thompson, 2015), and neuropathic pain (Bravo et al, 2014; Weaver et al, 2017; Zhang et al, 2015). Channel activation is associated with release of metabolites
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