Reverse Transcription PCR Of A Molluscan Aquaporin

Yekaterina Shishkina, Lee Nachtigal, Barry Hoopengardner, and Martin Kapper, Department of Biomolecular Sciences, Central Connecticut State University

During adaptation to altered salinities, intertidal invertebrates typically modulate the concentration of intracellular solutes to match the concentration of the ambient seawater. The solutes most often used are free amino acids. For example, the ribbed mussel, Geukensia demissa will accumulate large concentrations of alanine while adapting to high salinity. Coincident with the adjustment of intracellular osmolality is a transient osmotic flux as water first leaves the cells down the osmotic gradient, and then returns during a volume regulatory stage. Since cell membranes are generally impermeant to water, we hypothesize that modulation of aquaporin (specific water channel proteins) content or function is involved in lessening or delaying large-scale changes in cellular osmolality due to water fluxes. Since preliminary experiments using Western blotting to follow the concentration of aquaporin-2 during the course of adaptation from 15o/ooS to 35o/ooS were inconclusive, we are currently developing protocols to amplify the AQP-2 message by reverse-transcription polymerase chain reaction. Following extraction of total mRNA from approximately 100 mg of gill tissue, a cDNA was generated by standard reverse transcription procedures. Degenerate PCR primers were designed using published sequence information from mammalian aquaporin-2. Supported by CSU-AAUP research and faculty-student grants to MAK.

Presented April 21, 2007 at the 61st Annual Eastern Colleges Science Conference, College of Mount Saint Vincent, Riverdale NY