Characterization of the Zebrafish Panx1a Phosphorylation Profile and Interactome in the Regulation of the Channel Function and Trafficking

Pannexin-1 (Panx1) is an integral membrane protein that forms heptameric channels, involved in the communication between the intracellular and the extracellular environment. Its primary function is to passage small molecules, such as ATP, in and out of the cell, which can trigger vital cellular responses like cell death. Panx1s have been implicated in various diseases, including epilepsy, ischemia, and inflammation. They also play roles in the sensory processing systems, such as vision, hearing, olfaction, and taste. Although it is clear that Panx1s are vital to the function of biological processes, the exact mechanism of how these channels operate and what activates/inactivates them is still under investigation. Using the zebrafish orthologues of Panx1, we explore the structural properties responsible for the trafficking of the channel while maintaining a focus on the phospho-proteomics and the kinase-dependent signaling involving Panx1. We identified a critical residue highly conserved in the third transmembrane domain, Y205 of the zebrafish Panx1a protein, which plays a role in the stabilization of the protein, likely through aromatic-aromatic interactions. Our results show that this residue is not subject to phosphorylation but is vital for the localization of the channel at the cell surface. Using protein purification techniques combined with mass spectrometry analysis, we identified several phosphorylated residues in the cytoplasmic loop of Panx1a and explored the potential interacting kinases of the protein. We studied the interactions between Panx1a with two kinases: ERK2 and CaMKIIa. Our results indicated that a strong interaction exists with CaMKIIa, which plays a role in the trafficking dynamics of Panx1a to the cell membrane. The overexpression of CaMKIIa led to an accumulation of Panx1a in the intracellular compartments. We investigated the effects of various CaMKIIa functional and structural mutants on expression and interaction with Panx1a. We found that the constitutively active T286D CaMKIIa mutant leads to a partial rescue in Panx1a expression, suggesting that Panx1a requires phosphorylation by CaMKIIa for proper cell membrane localization. Together, the research in this thesis provides novel findings that contribute to the phospho-proteomic exploration of Panx1 channel activity and regulation.