Pannexin 1 activation and inhibition is permeant‐selective

Abstract

Key points The large‐pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C‐terminal cleavage led to a Panx1 open‐state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1‐mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. Abstract Pannexin 1 (Panx1) is a large‐pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant‐selectivity of Panx1 activation and inhibition, we employed Panx1‐expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C‐terminal truncation or cell shrinkage elevated Panx1‐mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K+]o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl−, lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1‐mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant‐selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.

A novel voltage clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

“…Consistent with this, alterations in the putative pore residue V37 of Cx30 increase atomic ion flux without altering permeability to ethidium (Nielsen et al, 2017). Interestingly, pharmacological blockers of connexin or pannexin channels can selectively inhibit atomic ion permeation but not molecular uptake (Nielsen et al, 2019a;Nielsen et al, 2019b). Consistent with this, we note that 1 mM extracellular Ca 2+ reduces ~95% of ionic current but only half of the YO-PRO permeation through CALHM1 channels (Fig.…”

“…For example, significant Cx43-mediated ethidium permeability was reported at negative resting membrane potential where no atomic ion currents were detected even at the single-channel level (Contreras et al, 2003). Recently, the same phenomenon was observed in pannexin-1 channels (Nielsen et al, 2019a).…”

“…Nevertheless, recent evidence suggests that permeability to molecules cannot be extrapolated to atomic ion permeation and vice versa. The uptake of molecular dyes through connexin and pannexin channels has been described at resting membrane potentials where atomic ion currents are not detected (Contreras et al, 2003;Nielsen et al, 2019a). Furthermore, pharmacological blockade of connexins or pannexins have been shown to differentially affect atomic ion and molecular permeation (Nielsen et al, 2019a;Nielsen et al, 2019b).…”

Uncoupled permeation through large‐pore channels: ions and molecules don't always ride together

“…In this issue of The Journal of Physiology , Nielsen et al . () evaluate permeation of atomic ions and ethidium through pannexin 1 channels using different stimuli to gate these channels. Combining two different heterologous expression systems, Xenopus oocytes and HEK cells, they report basal pannexin 1‐mediated ethidium permeability in the absence of detectable ionic currents at resting membrane potentials.…”

ATP triggers macropinocytosis that internalizes and is regulated by PANX1

“…PANX1 forms ubiquitously-expressed heptameric channels that are permeable to small (Cl -) and large (e.g. ATP) anions [38][39][40][41][42][43][44]. Additionally, PANX1 may also regulate the flux of small cations (Ca 2+ ) [45] and release of large cations (spermidine) [46].…”