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Expression of the aquaglyceroporin HC3 and morpholino delivery in Hyla chrysoscelis erythrocyte cultures

Cold acclimation in Cope's gray treefrog, Hyla chrysoscelis entails changes in handling and distribution of water and glycerol with concomitant changes in aquaglyceroporin protein expression. We hypothesize that HC3, an ortholog of mammalian aquaporin 3, enhances membrane permeability to glycerol which acts as a natural cryoprotectant to regulate osmotic gradients formed by extracellular ice crystal formation during freezing. In this study, H. chrysoscelis erythrocytes cultured in complete cell media exhibited 85% viability after 72 hrs at 20°C. After 24 hrs, HC3 protein expression was reduced by 92%. HC3 membrane localization was confirmed by immunocytochemistry. HC3 expression increased by 1.7 fold in erythrocytes cultured for 24 hrs in media made hyperosmotic (400 mOsM) by the addition of glycerol as compared to control. Since no specific HC3 channel inhibitors exist, a morpholino‐mediated expression knockdown approach was optimized. Efficient uptake of 10 μM of fluorescein‐labeled control morpholino with 6 uM of the endosomal delivery vehicle Endo‐Porter (GeneTools®) was observed by fluorescent microscopy, with no evidence of compromised cell viability. These results show that HC3 protein expression in erythrocyte cultures from H. chrysoscelis can be induced by hyperosmolarity and potentially modulated by morpholino knockdown. This research was supported by NSF Research Grant IOB‐0517301.