Pannexin-1 (Panx1) is a large-pore ion and metabolite channel with broad cell/tissue expression, including neurons and glia of the central nervous system. Panx1 is best known for its efflux of adenosine 5’-triphosphate (ATP), contributing to a multitude of pathophysiological mechanisms. However, there is no shortage of molecular diversity amongst the molecules that permeate these channels under numerous signalling conditions. Recently, the Panx1 cryo-EM structures were reported, uncovering unique gating mechanisms and insights into its biophysics that will help better understand its physiology. Given that Panx1 is an attractive drug target based on its involvement in pathophysiological mechanisms like cancer, ischemic stroke, and inflammation, it is important to understand the entire scope of molecules that permeate these channels during these mechanisms. Evidence from our lab has suggested that Panx1 is permeable to the endocannabinoid, anandamide (AEA) and cation, calcium (Ca2+) under different physiological settings. This thesis explores the biophysical gating mechanisms of Panx1 by undergoing a targeted mutational profile of its structural domains we hypothesize are important in AEA or Ca2+ permeability. My over-arching hypothesis is that the signalling lipid, AEA and cation, Ca2+ are permeants of Panx1. This thesis shows that in vitro Panx1 expression increases the uptake of a fluorescent AEA compound. Additionally, I identify key domains and residues that when mutated, create switch-of-function characteristics where a decrease in ion permeability results in increased AEA permeability and vice-versa. I propose that Panx1 rapidly switches between ion and AEA conducting states. Furthermore, I show that Panx1 is weakly permeable to cations and that mutagenesis of the Panx1 selectivity filter does not increase Ca2+ permeability. In conclusion, the work presented in this thesis has identified unique structural features of Panx1 that mediate the direct flux of AEA across the plasma membrane. Additionally, the structural domains that convey ion/size selectivity do not permit Ca2+, giving reason to believe that secondary signalling characteristics may give rise to this putative Panx1 function. Overall, this thesis uncovers a novel functionality of Panx1 that will have implications for its drug development and contributions to cellular physiology.