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Erythrocytes from Cope's gray tree frog, Hyla chrysoscelis as a cell culture based model system to study the regulation of aquaglyceroporin, HC‐3 expression

Cope's Gray Tree Frog, Hyla chrysoscelis is a freeze tolerant anuran that accumulates glycerol during cold acclimation. Aquaporins, members of the MIP family of transmembrane water pores, may play an important role in the mechanism of freeze tolerance by mediating glycerol and water transport across cell membranes. We hypothesize that HC‐3, an ortholog of the aquaglyceroporin AQP3, enhances membrane permeability to glycerol facilitating the cellular response to osmotic gradients formed when extracellular water freezes. Erythrocytes of H. chrysoscelis were used to study the regulation of HC‐3 protein expression. Compared with warm‐acclimated frogs, cells from cold‐acclimated frogs had higher HC‐3 protein expression and enhanced membrane localization. Glycerol‐mediated hyperosmolarity, in vitro, induced enhanced glycosylation of HC‐3 and increased membrane localization. We tested the ability to knock down HC‐3 expression using morpholinos delivered via Endo‐Porter. Western blot analysis showed essentially complete knockdown of HC‐3 expression when using 3μM HC‐3 specific morpholino and 10μM Endo‐Porter. Taken together, these studies demonstrate the ability to induce both up‐ and down‐regulation of the HC‐3, thereby presenting a cell culture system suitable to study the regulation and functional role of this aquaglyceroporin. This research was supported by NSF Research Grant IOB‐0517301.