Open AccessExpression of the aquaglyceroporin HC ‐9 in a freeze‐tolerant amphibian that accumulates glycerol seasonally
Author(s) -
Stogsdill Brian,
Frisbie James,
Krane Carissa M.,
Goldstein David L.
Publication year - 2017
Publication title -
physiological reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.918
H-Index - 39
ISSN - 2051-817X
DOI - 10.14814/phy2.13331
Subject(s) - complementary dna , biology , aquaporin , xenopus , messenger rna , glycerol , amino acid , microbiology and biotechnology , amphibian , signal peptide , phloretin , peptide sequence , biochemistry , gene , ecology
As ambient temperatures fall in the autumn, freeze‐tolerant Cope's gray treefrogs, Dryophytes chrysoscelis (formerly Hyla chrysoscelis ), accumulate glycerol as a cryoprotective agent. We hypothesized that these treefrogs express an ortholog of the mammalian aquaglyceroporin AQP 9 and that AQP 9 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 Xenopus laevis 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 2 . 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 AQP 9‐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.