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(Investigative Ophthalmology and Visual Science. 2003;44:3124-3129.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0669
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Kainate-Triggered Currents in Xenopus Oocytes Injected with Chick Retinal Membrane Fragments: Effect of Guanine Nucleotides

Javier S. Burgos,1 Jordi Aleu,2 Ana Barat,1 Carles Solsona,2 Jordi Marsal,2 and Galo Ramírez1

1From the Center for Molecular Biology (CSIC-UAM), Autonomous University of Madrid, Madrid, Spain; and the 2Cellular and Molecular Biology Laboratory, Department of Cell Biology and Pathology, School of Medicine, Bellvitge Hospital, University of Barcelona, Barcelona, Spain.

PURPOSE. To electrophysiologically characterize {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors in chick retinal membrane fragments, incorporated into Xenopus oocytes by direct microinjection.

METHODS. A 6-day retinal membrane suspension was injected into Xenopus oocytes by use of an electronic nanoliter injector. Fifteen to 40 hours after injection, the oocytes were assayed for kainate-elicited inward currents, under voltage-clamp conditions (membrane potential held at -70 mV). The structural incorporation of the retinal membrane fragments into the oocyte membrane was verified by specific immunofluorescent staining.

RESULTS. Chick retinal membrane fragments were efficiently grafted onto Xenopus oocytes after microinjection, with 22.9% ± 7.6% of the oocyte membrane being stained with anti-chick retina antibody. Part of the retinal material was seen as patches of relatively uniform size (292.1 ± 72.3 µm2). Bath-applied kainate induced dose-dependent (EC50: 64 ± 7 µM), nondesensitizing inward currents (15–90 nA) in the chimeric Xenopus oocytes. Sham-injected oocytes did not respond to kainate. Kainate-driven currents were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 1-(4-aminopropyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), but not by {gamma}-D-glutamylaminomethyl sulfonic acid (GAMS) or aminophosphonoheptanoate (AP7), suggesting the involvement of AMPA receptors in the observed responses. Guanine nucleotides (GNs) also blocked kainate currents in a concentration-dependent manner.

CONCLUSIONS. An alternative oocyte microinjection technique to analyze the electrophysiological properties of glutamate receptors in chick retinal membranes is described. The results show the functional activity of putative AMPA-preferring receptors from chick retina and confirm, in the chick retinal model, the antagonistic behavior of guanine nucleotides toward glutamate receptors and their potential role as neuroprotective agents under excitotoxic conditions.






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