Glyceroporin HC‐9 in the Freeze Tolerance Mechanism of Cope's Gray Treefrogs

Abstract

Cope's gray treefrogs Hyla chrysoscelis accumulate glycerol in response to cold as part of their freeze tolerance mechanism. This glycerol may derive from glucose stores in the liver that are broken down and released through glycerol transporters. We therefore hypothesized that these treefrogs would express aquaporin 9 (AQP9), a transmembrane protein from the aquaglyceroporin family that facilitates glycerol transport, predominately in the liver. We also hypothesized that this protein would be up‐regulated during cold acclimation to promote glycerol permeability. To test this hypothesis, we sequenced the AQP9 homolog HC‐9, analyzed RNA and protein expression, and characterized water and glycerol permeability through a Xenopus oocyte expression system.HC‐9 has 315 amino acids which include 2 conserved NPA motifs characteristic of aquaporins, 5 conserved residues typical of glyceroporins, and high sequence homology to human AQP9 (69%) and to AQP9 from Hyla japonica (97%). The mRNA strand also possesses a long untranslated tail that appears to be unique to amphibians and necessary for translation. Characterization using the Xenopus oocyte expression system indicates that HC‐9 is permeable to glycerol but has a poor water permeability compared with human AQP1.In warm‐acclimated treefrogs, HC‐9 mRNA (RT‐PCR) and protein (Western blotting) were expressed in many tissues, with highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5°C) and frozen (−2.5°C) treefrogs, with return to levels near those in warm acclimated animals after return to 5°C for 24 hours. In contrast, protein expression increased in the liver and ventral skin of cold and frozen treefrogs, but remained unchanged or decreased in all other tissues.HC‐9 expression is clearly regulated as a part of the Cope's gray treefrog freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression of HC‐9 in the cold, which may reduce glycerol permeability to protect cells during the freezing process. Liver is a notable exception by increasing protein expression from warm to cold to frozen conditions. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol during the freeze‐thaw cycle, and this study suggests HC‐9 facilitates in this role.Support or Funding InformationSupported by NSF IOS‐1121457