Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold‐ and warm‐acclimated cope's gray treefrog, Hyla chrysoscelis

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Abstract
Cope's gray treefrog, Hyla chrysoscelis,is a freeze‐tolerant anuran which accumulates and distributes glycerol as a cryoprotectant before freezing. We hypothesize that HC‐3, an aquaglyceroporin member of the MIP family of water pores, may play an important role in the process of freeze tolerance by mediating transmembrane passage of glycerol and water during cold‐acclimation. The objectives of this study were two‐fold: to examine HC‐3 protein abundance and cellular localization in erythrocytes from cold‐ and warm‐acclimated frogs and to develop and characterize an erythrocyte cell culture system for examining HC‐3 gene regulation. Compared with warm‐acclimated frogs, erythrocytes from cold‐acclimated frogs had higher HC‐3 protein expression and enhanced plasma membrane localization. Furthermore, erythrocytes from cold‐ and warm‐acclimated frogs maintained in culture at 4 and 20°C exhibited time‐ and temperature‐dependent regulation of HC‐3 expression and an increase in the abundance of high molecular weight immunoreactive species within 24 hr of culture at 20°C. Deglycosylation of erythrocyte proteins resulted in the disappearance of the high molecular weight species, indicating that HC‐3 is post‐translationally modified by N‐linked glycosylation. Erythrocytes cultured in media containing glycerol also showed an increased abundance of the high molecular weight bands and enhanced plasma membrane localization of HC‐3, suggesting a role for glycerol in regulating HC‐3 subcellular trafficking. Thus, the development of this erythrocyte cell culture system from H. chrysoscelis opened an opportunity to study the properties of cells with changing expression of an aquaglyceroporin, HC‐3, and to explore the factors regulating that expression. J. Exp. Zool. 315:424–437, 2011.

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