Details

Autor(en) / Beteiligte

• Tygesen, Charlotte K.
• Rasmussen, Jesper S.
• S. V. Penelope Jones
• Hansen, Annette
• Hansen, Kate
• Andersen, Peter H.

Titel

Stable Expression of a Functional GluR6 Homomeric Glutamate Receptor Channel in Mammalian Cells

Ist Teil von

• Proceedings of the National Academy of Sciences - PNAS, 1994-12, Vol.91 (26), p.13018-13022

Ort / Verlag

United States: National Academy of the Sciences of the United States of America

Erscheinungsjahr

1994

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 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 μM. The currentvoltage 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.