Expression of the Aquaglyceroporin HC-9 in a Freeze-tolerant Amphibian That Accumulates Glycerol Seasonally

As ambient temperatures fall in the autumn, freeze-tolerant Cope's gray treefrogs, (formerly ), accumulate glycerol as a cryoprotective agent. We hypothesized that these treefrogs express an ortholog of the mammalian aquaglyceroporin AQP9 and that AQP9 expression is upregulated in the cold to facilitate glycerol transport. We sequenced 1790 bp from cloned cDNA that codes for a 315 amino acid protein, HC-9, containing the predicted six transmembrane spanning domains, two Asn-Pro-Ala (NPA) motifs, and five amino acid residues characteristic of aquaglyceroporins. Functional characterization after heterologous expression of HC-9 cRNA in oocytes indicated that HC-9 facilitates glycerol and water permeability and is partially inhibited by 0.5 mmol/L phloretin or 0.3 mmol/L HgCl HC-9 mRNA (qPCR) and protein (immunoblot) were expressed in most treefrog tissues analyzed (muscle, liver, bladder, stomach, kidney, dorsal skin, and ventral skin) except the protein fraction of red blood cells. Contrary to our prediction, both mRNA and protein expression were either unchanged or downregulated in most tissues in response to cold, freezing, and thawing. A notable exception to that pattern occurred in liver, where protein expression was significantly elevated in frozen (~4-fold over warm) and thawed (~6-fold over warm) conditions. Immunofluorescence labeling of HC-9 protein revealed a signal that appeared to be localized to the plasma membrane of hepatocytes. Our results indicate that gray treefrogs express an AQP9-like protein that facilitates glycerol permeability. Both the transcriptional and translational levels of HC-9 change in response to thermal challenges, with a unique increase in liver during freezing and thawing.