Temperature‐Induced Changes to Aquaporin Localization in the Freeze‐Tolerant Cope's Gray Treefrog

Cope's gray treefrogs alter their physiology in response to cold as part of their freeze tolerance mechanism. One such change involves the accumulation, both extracellularly and intracellularly, of cryoprotective glycerol, which likely crosses cellular membranes using glycerol‐permeable channels called aquaglyceroporins (GLPs). Three GLPs (HC‐3, HC‐7, and HC‐9) have been identified in the Cope's gray treefrog, representing homologs to the mammalian AQP3, AQP7, and AQP9, respectively. Regulation of transmembrane glycerol diffusion depends on GLP expression in the plasma membrane resulting from changes to transcriptional and translational output of the GLPs and/or by changes in protein trafficking to the plasma membrane. We have previously reported that GLP mRNA and protein expression are modulated during cold acclimation and freezing; however, localization of GLP proteins in the plasma membrane has not been studied thoroughly. We hypothesized that changes in acclimation temperature would alter the localization of the GLPs HC‐3 and HC‐9 in the plasma membrane and confirmed this with immunofluorescence, plasma membrane fractionation, and biotinylation. For HC‐9 in liver, quantitation of protein expression (by immunoblot) showed a 4‐ to 6‐fold increase in the higher molecular weight band of frozen (−2.5°C for 24 hours) and thawed (5°C for 24 hours after freezing) over warm (20°C) treefrogs. In immunohistochemistry images, HC‐9 signal appeared to be localized to the plasma membrane of hepatocytes of warm‐ and cold‐acclimated animals but dispersed intracellularly in frozen treefrog hepatocytes. In preliminary studies, fractionation of cellular protein (Biovision, Milpitas, CA) indicated that between freezing and thawing, the nominal (~34 kDa) molecular weight band of HC‐9 diminishes in the plasma membrane fraction while the (putatively) glycosylated high molecular weight band of HC‐9 increases. For HC‐3 in erythrocytes, protein expression is enhanced by cold acclimation and by incubation in medium containing glycerol. We incubated erythrocytes taken from warm‐acclimated animals at 4°C for 48 hours and biotinylated the surface proteins. In immunoblots of the biotinylated fraction probed with anti‐HC‐3 primary antibody, the high molecular weight (glycosylated) band was enriched and the nominal (~32 kDa) molecular weight band was diminished. These results suggest that water and glycerol permeability during cold acclimation and freezing can be modulated by protein trafficking, which may be influenced by the post‐translational modification of glycosylation. Support or Funding Information Supported by NSF IOS‐1121457.