STABLE EXPRESSION OF A FUNCTIONAL GLUR6 HOMOMERIC GLUTAMATE-RECEPTOR CHANNEL IN MAMMALIAN-CELLS

This study demonstrates the stable expression of a functional ionotrop ic glutamate receptor in a mammalian cell line of non-neuronal origin. The kainate-selective glutamate receptor GluR6 was constitutively exp ressed under the control of a metallothionein promoter. Clones were is olated expressing approximate to 3 pmol of receptor per mg of protein. Functionality of the recombinant GluR6 was demonstrated both by elect rophysiology and by Ca2+ imaging. Application of kainate to the GluR6- transfected cells activated an inward current response at a holding po tential of -60 mV. The kainate concentration needed to evoke 50% of th e maximal response (EC(50)) was calculated to be 0.82 +/- 0.39 mu M. T he current-voltage relationship was found to be almost linear, with a reversal potential of -2.5 +/- 4.8 mV. Application of kainate also res ulted in an increase in the intracellular Ca2+ concentration measured by Ca2+ imaging. The pharmacological profile of [H-3]kainate binding t o the recombinant GluR6 resembled the high-affinity [H-3]kainate bindi ng 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 ex pression level was observed over > 75 passages of the transfected cell s. When domoate, a slowly desensitizing GluR6 agonist, was included in the growth medium for 3 weeks, the number of GluR6 binding sites decr eased by 30%, indicating the importance of complete channel closure fo r stable expression.