Stable expression of a functional GluR6 homomeric glutamate receptor channel in mammalian cells.
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.[1]References
- Stable expression of a functional GluR6 homomeric glutamate receptor channel in mammalian cells. Tygesen, C.K., Rasmussen, J.S., Jones, S.V., Hansen, A., Hansen, K., Andersen, P.H. Proc. Natl. Acad. Sci. U.S.A. (1994) [Pubmed]
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