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Romanovitch, Mallory

Biology

2012

MS

Paramecium tetraurelia, a ciliated unicellular eukaryote, is attracted to the chemical stimulus glutamate. A putative L-glutamate receptor, pGluR1, was previously identified in P. tetraurelia by its high homology to the glutamate-binding subunit ofan NMDA-like receptor. Down regulation of this sequence by RNA interference showed a decrease in chemoresponse to L-glutamate specifically. The L-glutamate receptor is proposed to be located in the cilia as ³[H]-L-glutamate binds to cilia. Using a vector containing a FLAG tag, a fusion protein was made with pGluR1. Using Western blot analysis and tandem mass spectrometry, pGluR1 can be found in the detergent soluble fraction of isolated pellicle and ciliary membranes.
Specifically in P. tetraurelia L-glutamate chemoresponse, there is an increase in intracellular cAMP upon glutamate stimulation implicating adenylyl cyclase downstream of the receptor. Adenylyl-cyclase-GFP and pGluRl-FLAG do not co-immunoprecipitate in the cilia from cells expressing both tagged proteins.
In the T-maze behavioral assay, wild-type cells were neutral towards glycine and Dserine. The combination of glycine or D-serine with glutamate in T-mazes did not affect the cell's chemoresponse toward glutamate. The work adds to the knowledge of the characteristics of the putative L-glutamate receptor in P. tetraurelia.