Destination and consequences of Panx1 and mutant expression in polarized MDCK cells

The channel-forming membrane glycoprotein pannexin 1 (Panx1) is best characterized as an ATP release channel. To investigate the trafficking and sorting of Panx1, we used polarized MDCK cells and non-polarized BICR-M1R_k cells to track the fate of GFP-tagged Panx1. In non-polarized cells, Panx1 was found throughout the plasma membrane, including the lamellipodia of the tumor cells and the cell surface-targeting domain was mapped to residues 307–379. Panx1 was preferentially enriched at the apical membrane domain of polarized MDCK cells grown as monolayer sheets or as spheroids. Residual Panx1 localized within basolateral membranes of polarized MDCK cells was independent of a putative dileucine sorting motif LL365/6 found within the C-terminal of Panx1. Unexpectedly, stable expression of a Panx1 mutant, where a putative tyrosine-based basolateral sorting motif (YxxØ) was mutated (Y308F), or a truncated Δ379 Panx1 mutant, caused MDCK cells to lose cell-cell contacts and their ability to polarize as they underwent a switch to a more fibroblast-like phenotype. We conclude that Panx1 is preferentially delivered to the apical domain of polarized epithelial cells, and Panx1 mutants drive phenotypic changes to MDCK cells preventing their polarization.