The Life Cycle of Pannexins: Trafficking, Cell Surface Dynamics, Turnover and Degradation

Author

Ruchi Gehi, The University of Western OntarioFollow

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Dr. Dale Laird

Abstract

Pannexins (Panxs) are a recently identified family of channel-forming glycoproteins, comprised of three members: Panx1, Panx2 and Panx3. Panxs were classified as gap junction (GJ) proteins based on their shared sequence homology to the invertebrate GJ protein, innexins. While no sequence homology exist between Panxs and the GJ proteins, connexins (Cxs), they share similar topology with four transmembrane domains, two extracellular loops, an intracellular loop and cytoplasmic exposed amino and carboxy terminal tails. In this study, we investigated if Panx1 and Panx3 exhibit unique or shared properties of cell surface delivery, mobility, cytoskeletal dependency, internalization and degradation, to the well characterized Cx43 GJ protein. Over-expression of a dominant-negative Sar1 mutant revealed that, like Cx43, Panx1 and Panx3 followed the classical secretory pathway en route to the cell surface. At the plasma membrane, Panx1 and Panx3 distributed in a relatively uniform pattern independent of the presence of an adjoining cell, a feature distinct from the punctate localization of Cx43 at cell-cell apposition. Rapid time-lapse imaging and photo-bleaching revealed high mobility of Panx1 and Panx3 at the cell surface, unlike Cx43-GJs. Using pharmacological inhibitors of microtubule and microfilament polymerization, Panx1 and Panx3 were identified to rely primarily on actin filaments for their overall cell surface stability. Interestingly, co-sedimentation and co-immunoprecipitation assays revealed that actin bound directly to the C-terminal tail of Panx1. Truncation of the Panx1 C-terminal tail at residue 307 (Panx1T307) inhibited its trafficking to the cell surface causing it to be retained within the endoplasmic reticulum. Co-expression of full length Panx1 could not rescue the delivery of Panx1T307 to the cell surface but exhibited limited co-interaction. Blocking of protein synthesis and secretion revealed a prolonged half-life of Panx1 in comparison to Cx43. Rapid time-lapse imaging of cells expressing Panx1-GFP uncovered a unique pattern of Panx1 internalization through dynamic tubular-like extensions. Chloroquine-induced inhibition of lysosomal function revealed that Panx1 is degraded by lysosomes. Collectively, our studies show that Panx1 and Panx3 exhibit many properties that are distinct from Cx43, further supporting that pannexins forms a unique class of channel proteins that should be considered separate from connexin-based GJ channels.

Recommended Citation

Gehi, Ruchi, "The Life Cycle of Pannexins: Trafficking, Cell Surface Dynamics, Turnover and Degradation" (2011). Electronic Thesis and Dissertation Repository. 87.
https://ir.lib.uwo.ca/etd/87