Cloning and Characterization of Glutamate Receptor Subunit 4 (GLUA4) and its Alternatively Spliced Isoforms in Turtle Brain

Abstract

Ionotropic glutamate receptors sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), GluAs, play an important role in neural development, synaptic plasticity, and neurodegeneration. Previous studies using an in vitro model of eyeblink classical conditioning in pond turtles suggested that acquisition of conditioning is associated with synaptic delivery of AMPA receptors containing GluA4 subunits. However, sequences of the GluA4 subunit, expression profile, and its alternatively spliced isoforms in turtle brain have not been previously determined. The sequence and domain structure of turtle GluA4 (tGluA4) and its splice variants was characterized. We found ten isoforms of tGluA4 including several previously unidentified truncated variants. Analysis of the nucleotide sequences of tGluA4 flip/flop, tGluA4c flip/flop, and tGluA4s showed they are highly similar to known isoforms of the GluA4 subunit identified in chick. Examination of the relative abundance of mRNA expression for the tGluA4 variants showed that the flip and flop versions of tGluA4 and tGluA4c, and a novel truncated variant, tGluA4trc1, which is also expressed as protein, are major forms in the adult turtle brain. Identification of these alternatively spliced isoforms of tGluA4 will provide a unique opportunity to assess their role in synaptic plasticity through the application of short interfering RNAs.