The Effect of Acute Temperature Change on the Localization of Aquaporin‐3 in the Erythrocytes of the Freeze‐tolerant Cope's Gray Treefrog

Abstract

Cope's gray treefrogs (Dryophytes chrysoscelis) accumulate cryoprotective glycerol (>100 mM) throughout their body in response to falling temperatures, which enables them to tolerate freezing. The most likely pathway for the transmembrane transport of glycerol is through glycerol‐permeable aquaporins such as HC‐3, a homolog of Aquaporin‐3, which increases in both gene and protein expression for erythrocytes during cold acclimation. Much of the increase in immunoblot signal intensity occurs not at the nominal molecular weight of HC‐3 (~31.5 kDa), but at a higher molecular weight (~65–100 kDa) shown to be glycosylated HC‐3. We hypothesized that glycosylation of HC‐3 is involved in trafficking of the protein to the plasma membrane. To test this hypothesis, we established conditions in culture that enhance expression of HC‐3, and we assessed expression of glycosylated and un‐glycosylated HC‐3 in the plasma membrane via biotinylation and immunoblot.Erythrocytes were cultured at 20°C and 5°C over the course of 48 hours. Cell surface expression was assessed by biotinylation, which resulted in total and biotinylated (plasma membrane, PM) fractions assayed by immunoblot for HC‐3 and quantified with densitometry. Erythrocytes incubated at 20°C for 48 hours yielded total protein with 4‐fold greater glycosylated HC‐3 compared with 5°C‐incubated erythrocytes, but no change in the nominal molecular weight (unglycosylated) band. The biotinylated fraction of protein from 20°C‐incubated erythrocytes had another 4‐fold increase in glycosylated HC‐3 over the total, suggesting enrichment of glycosylated protein in HC‐3 localized to the plasma membrane. In 5°C‐incubated erythrocytes, total expression of HC‐3 was diminished compared with 20°C. In contrast to RBCs incubated at 20°C, cells at 5°C were enriched for glycosylated HC‐3 in the non‐biotinylated protein fraction compared with PM‐localized protein. Thus, while glycosylation is enhanced in the plasma membrane fraction of HC‐3 in 20°C‐incubated erythrocytes, the opposite occurs in cells incubated at 5°C, suggesting that while the glycosylation of HC‐3 isn't required for localization to the plasma membrane, its expression is variably regulated in response to environmental and humoral factors.We further hypothesized that epinephrine could induce changes in membrane localization of HC‐3 in cultured erythrocytes similar to those observed during cold acclimation. When erythrocytes incubated at 5°C for 48 hours were treated with epinephrine (1 μM) for 60 minutes, the expression of glycosylated protein in the plasma membrane fraction of HC‐3 increased >10‐fold compared with 5°C‐incubated erythrocytes without epinephrine treatment; the nominal MW band was not present in the plasma membrane fraction from cells incubated with epinephrine. Our data suggest that the enhanced expression of HC‐3 observed in erythrocytes from long‐term cold‐acclimated animals is not induced by a decrease in temperature, but changes to HC‐3 expression and subsequent trafficking to the plasma membrane can be activated by epinephrine.Support or Funding InformationNSF grant (IOS‐1121457)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.