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.
Tygesen
C K
CK
Department of Molecular Pharmacology, Novo Nordisk A/S, Bagsvaerd, Denmark.
Rasmussen
J S
JS
Jones
S V
SV
Hansen
A
A
Hansen
K
K
Andersen
P H
PH
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D004594:Electrophysiology; D007608:Kainic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D051381:Rats; D017470:Receptors, Glutamate / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D017931:DNA Primers; D017470:Receptors, Glutamate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D007608:Kainic Acid; D002118:Calcium