Stable expression of a functional GluR6 homomeric glutamate receptor channel in mammalian cells.

TitleStable expression of a functional GluR6 homomeric glutamate receptor channel in mammalian cells.
Publication TypeJournal Article
Year of Publication1994
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue26
Pagination13018-22
Date Published1994
ISSN0027-8424
Abstract

This study demonstrates the stable expression of a functional ionotropic glutamate receptor in a mammalian cell line of non-neuronal origin. The kainate-selective glutamate receptor GluR6 was constitutively expressed under the control of a metallothionein promoter. Clones were isolated expressing approximately 3 pmol of receptor per mg of protein. Functionality of the recombinant GluR6 was demonstrated both by electrophysiology and by Ca2+ imaging. Application of kainate to the GluR6-transfected cells activated an inward current response at a holding potential of -60 mV. The kainate concentration needed to evoke 50% of the maximal response (EC50) was calculated to be 0.82 +/- 0.39 microM. The current-voltage relationship was found to be almost linear, with a reversal potential of -2.5 +/- 4.8 mV. Application of kainate also resulted in an increase in the intracellular Ca2+ concentration measured by Ca2+ imaging. The pharmacological profile of [3H]kainate binding to the recombinant GluR6 resembled the high-affinity [3H]kainate binding sites in rat brain, showing high affinity for domoate (Ki = 5.1 +/- 3.0 nM) and kainate (Kd = 12.9 +/- 2.4 nM). No decrease in GluR6 expression level was observed over > 75 passages of the transfected cells. When domoate, a slowly desensitizing GluR6 agonist, was included in the growth medium for 3 weeks, the number of GluR6 binding sites decreased by 30%, indicating the importance of complete channel closure for stable expression.

URLhttp://www.pnas.org/cgi/pmidlookup?view=long&pmid=7528929
Short TitleProc Natl Acad Sci U S A
X
Enter your linkblue username.
Enter your linkblue password.
Secure Login

This login is SSL protected

Loading