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

Charlotte Klitgaard Tygesen, Jesper S Rasmussen, S V Penelope Jones, Annette Hansen, Kate Hansen, Peter Høngaard Andersen

Publikation: Bidrag til tidsskriftTidsskriftsartikelForskningpeer review

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 ≃3 pmol of receptor per mg of protein. Functionality of the recombinant GluR6 was demonstrated both by electrophysiology and by Ca 2+ 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 (EC 50) was calculated to be 0.82 ± 0.39 μM. 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 Ca 2+ concentration measured by Ca 2+ 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 (K(i) = 5.1 ± 3.0 nM) and kainate (K(d) = 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.

OriginalsprogEngelsk
TidsskriftProceedings of the National Academy of Sciences of the United States of America
Vol/bind91
Udgave nummer26
Sider (fra-til)13018-13022
Antal sider5
ISSN0027-8424
DOI
StatusUdgivet - 20 dec. 1994
Udgivet eksterntJa

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