Pannexin 1 activation and inhibition is permeant-selective

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. 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.