Aquaporin 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 rapid 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 homologue 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 unique to aquaporins, 5 conserved residues typical of glyceroporins, a high homology (69%) to human AQP9, and a nearly identical homology (97%) to the hyla japonica. The mRNA strand also possesses a long untranslated tail unique to amphibians that appears to be necessary for translation. Furthermore, characterization using the Xenopus expression system has shown that HC‐9 is permeable to glycerol but has a poor water permeability compared to human AQP1.In warm‐acclimated animals, HC‐9 mRNA (RT‐PCR) and protein (western blotting) were expressed in many tissues, with some of the highest levels in liver, stomach, ventral skin, and muscle. Across most tissues, realtime mRNA expression (qRT‐PCR) decreased in cold‐acclimated (5ºC) and frozen (−2.5 ºC) treefrogs, with recovery in treefrogs returned to 5ºC for 24 hours. Meanwhile, protein expression increased in the liver 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 Treefrogs freeze tolerance mechanism. Contrary to our hypothesis, most tissues have decreased expression, which may reduce glycerol permeability to protect cells during the freeze process. Liver is a notable exception by increasing protein expression, which may facilitate the re‐uptake of glycerol during ice formation. Liver appears to play a critical role in the distribution of the cryoprotectant glycerol, and this study suggests HC‐9 facilitates in this role.Support or Funding InformationSupported by NSF IOS‐1121457.