The membrane protein Pannexin1 forms two open-channel conformations depending on the mode of activation

Abstract

Pannexin1 (Panx1) participates in several signaling events that involve ATP release, including the innate immune response, ciliary beat in airway epithelia and oxygen supply in the vasculature. The view that Panx1 forms a large ATP-release channel has been challenged by the association of a low conductance, small anion-selective channel with the presence of Panx1. We showed that Panx1 membrane channels can function in two distinct modes with different conductances and permeabilities when heterologously expressed in Xenopus oocytes. When stimulated by potassium ions (K + ), Panx1 formed a high conductance channel of ~500 pS that was permeable to ATP. Various physiological stimuli can induce this ATP-permeable conformation of the channel in several cell types. In contrast, the channel had a low conductance (~50 pS) with no detectable ATP permeability when activated by voltage in the absence of K + . The two channel states were associated with different reactivities of the terminal cysteine of Panx1 to thiol reagents, suggesting different conformations. Single particle electron microscopic analysis revealed that K + stimulated the formation of channels with a larger pore diameter than those formed in the absence of K + . These data suggest that different stimuli lead to distinct channel structures with distinct biophysical properties.

A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction

“…The reversal potentials of R217H (Ϫ71.77 Ϯ 4.415, n ϭ 6) and PANX1 currents (Ϫ61.83 Ϯ 3.508, n ϭ 6) were comparable, suggesting that reduced currents in R217H-expressing cells could not be attributed to a change in ionic permeability. Consistent with previous reports (7,34,40), PANX1-mediated currents were augmented by treatment with high potassium, most notably at negative holding potentials (Fig. 4C).…”

“…Functional Analysis of the R217H Mutant-Because PANX1 channels are large-pore channels (7,9,32) suitable for the uptake of small fluorescent dyes (38, 39), we assessed whether channels formed from the R217H mutant were functionally compromised. Ad293 cells expressing wild-type PANX1 or the R217H mutant (Fig.…”

“…3A) were assessed for ethidium bromide (EtBr) dye uptake (9) after channels were induced open in high potassium medium (Fig. 3B) (7,34,40). Quantitative assessment of EtBr uptake revealed that cells expressing the R217H mutant exhibited significantly reduced dye uptake over a time course of 5-40 min (Fig.…”

“…Briefly, cells were sparsely subcultured on 35-mm glass-bottom dishes (MatTek Corp., Ashland, MA) for 1 day prior to the medium being replaced with 5% FBS (heat-inactive), DMEM containing 140 mM potassium gluconate, 100 mM KCl, and 10 M ethidium bromide (EtBr) (7,33,34). To block PANX1 channels, cells were treated with 1 mM probenecid (35).…”

“…Rodent Panx1 is an ϳ41-48-kDa protein with its broad range in size due to the fact that it is post-translationally modified in what is now referred to as Gly0, Gly1, and Gly2 species to reflect the degree of glycosylation (4 -6). PANX1 oligomerizes into a hexamer that contains a large pore functioning at the cell surface to allow the passage of small molecules below 1000 daltons in size (7,8). Although the scope of molecules that pass through PANX1 pores is likely broad (9,10), the functional consequence of ATP release via these channels is best understood (11).…”