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

Large-pore plasma membrane channels, including those formed by pannexins, connexins, LRRC8 and CALHM proteins, are found in many vertebrate cell types and are important for development and organ function. Unlike conventional ion channels, large-pore channels are permeable to both atomic ions and small molecules (usually less than 1 kDa). They also display ionic and molecular selectivity that might underlie their distinct physiological functions. In particular, pannexin channels are thought to be major conduits for ATP release in many cell types. Because ATP is critical for purinergic signalling transmission, pannexin channels have been associated with various physiological processes and pathologies, such as cell migration, inflammation and epilepsy. Nonetheless, how the permeability of pannexins to either atomic ions or molecules is altered during physiological or pathophysiological states remains a largely unexplored area of research. To study opening of large-pore channels, including pannexins, conventional electrophysiological techniques and dye uptake assays are commonly used for evaluating ionic currents and molecular permeability to fluorescent dyes, respectively. Although both approaches are accepted as measurements of channel activity, it is unclear whether permeability to fluorescent dyes can be extrapolated to atomic ion permeation and vice versa. Previous studies on Connexin 43 (Cx43) hemichannels have shown that the rate of uptake for ethidium bromide is proportional to its concentration, exposure time and, importantly, to the number of active channels at the cell surface. However, while high permeability rates for ethidium uptake are observed at negative membrane potentials, ionic current is not detected (Contreras et al. 2003; Hansen et al. 2014). This is not the case for Cx30 hemichannels, in which ionic current and ethidium permeability correlate positively (Hansen et al. 2014). Nevertheless, in this case, ionic current but not ethidium uptake mediated by Cx30 hemichannels was sensitive to flufenamic acid, a classic connexin blocker (Nielsen et al. 2019b). These properties support a model by which different mechanisms underlie ionic and molecular flux through Cx43 and Cx30 hemichannels. Whether these properties are unique to connexins or could be extended to other large-pore channels is still unknown, but such information is critical for assessing channel activation. In this issue of The Journal of Physiology, Nielsen et al. (2020) 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. They also demonstrate that at least four commonly used pharmacological blockers of pannexin 1, carbenoxolone, probenecid, panx and spironolactone, inhibit ionic currents but not ethidium permeability observed at resting membrane potentials. Based on this finding, the authors highlight that the efficiency of pannexin inhibitors cannot be extrapolated to ethidium transport. In addition, Nielsen et al. systematically and rigorously tested multiple activation stimuli to determine differences in ionic and ethidium permeation. They assessed gating by high extracellular potassium, pH (alkalization and acidification), truncation of the C-terminal region, and hypotonic/hypertonic-dependent mechanical stress. Notably, the authors found that for the latter three activation stimuli, pannexin 1-mediated ethidium permeation is not correlated with ionic current. As for high extracellular K+ ions, the authors did not observe ionic current or ethidium uptake. This finding is not a surprise, given that activation of pannexin 1 by extracellular K+ has been controversial, with differences attributed to species-specific isoforms. However, Nielsen et al. report lack of activation for both mouse and rat pannexin 1 channels, suggesting that the mechanism by which K+ activates pannexin 1 is probably a secondary effect of K+ ions on the channel. Nielsen et al. further enhance our understanding of the pannexin 1 permeability profile using ion substitution experiments, which confirm previous reports suggesting that pannexin 1 channels are highly permeable to chloride anions at positive membrane potentials. The authors also provide evidence indicating that pannexin 1 is not water permeable. Finally, they demonstrate that pannexin 1 channels are permeable to lactate and glutamate, but not glucose, using C-radiolabelled molecules. Importantly, the pannexin 1 inhibitor carbenoxolone does not block the transport of lactate or glutamate, which reinforces the notion of separate mechanisms of action for the blockade of ionic currents and molecule transport. The results presented by Nielsen et al. suggest that molecules and atomic ions can permeate using different mechanisms in pannexin channels, similar to that reported for connexins. Distinctive inhibition of ethidium uptake and ionic permeation by pannexin channel blockers warn us about the methodologies used to evaluate activity of these channels. The concept that an intrinsic wide pore confers a free diffusion pathway for ions and molecules is changing towards a more complex and fascinating scenario. Interestingly, high-resolution structures of large-pore channels (connexin, innexin, LRCC8) show striking pore similarities (Deneka et al. 2018) which, at face value, would suggest that ions and molecules use the same permeation pathways. It is time for biophysicists and physiologists to tackle the following questions: (1) what are the molecular mechanisms underlying the uncoupling between atomic ion and molecule permeation, and (2) are there physiological consequences for cellular or organ function for this activationdependent, selective permeability?