MOLECULAR CHARACTERIZATION OF GLUTAMATE RECEPTOR EXPRESSION IN BRAIN

点击翻译按钮获取中文摘要

Glutamate receptors (GluRs) have been implicated in simple forms of learning and memory and are the site of action of several psychoactive drugs. Initially it had been proposed that glutamate activates three distinct pharmacologically sites known as the kainate, AMPA and NMDA receptors, however, recent findings demonstrate that kainate and AMPA can compete for a common receptor site. A family of four genes coding for AMPA/Kainate receptors, called GluR 1-4 (or G]uR A-D), has been recently described. Alternative splicing generates two isoforms of each GluR subunit, designated flip and flop, that when expressed in transfected cells form channels that exhibit different electrophysiological properties. Sequences from the GluR-1 subunit cDNA were used to clone a cDNA originating from a third type of GluR 4 transcript; the cDNA was called GluR-4c because it codes for a protein with a distinct C-terminus. We have shown that GluR-4c mRNAs arise from yet another type of differential RNA processing; alternate C-termini may not be restricted to GluR-4 but also present on proteins derived from other GluR genes. Results obtained using a Xenopus oocyte expression system, in conjunction with two-electrode voltage clamp, demonstrate that the transcripts coded by the GluR-4c cDNA form functional homo-oligomeric channels in injected oocytes that open in response to kainate, AMPA and glutamate application. The EC50 of the kainate response is 44 microM and the inward currents elicited by kainate application are greatly reduced by coapplication of quisqualate or AMPA, suggesting a common binding site on GluR-4c receptors for these agonists. Transcripts originating from the GluR-4 gene were analyzed on Northern blots, and by in situ hybridization, using oligonucleotide probes specific to the different splice variants. Three bands of 6.2,4.2, and 3.0 Kb were observed when probes corresponding to sequences present in the proposed N-terminal extracellular domain of the receptor were used to hybridize blots containing RNA from cerebellum or cultured granule cells. In contrast, the smaller transcript was not observed in blots containing RNA from cerebellar astrocytes. Hybridization with probes specific to the flip, flop and C-terminal domains was associated exclusively to the two higher molecular weight bands. In situ hybridization revealed that the GluR-4 transcripts preferentially accumulate in different cerebellar cell types and their expression is differentially regulated during development.