Ion-permeability properties of ionotropic glutamate receptors and their use as detectors in capillary electrophoresis
The aims of the thesis were to characterize ion-permeability properties of AMPA receptors expressed in interneurons from rat olfactory bulb and Xenopus oocytes injected by total RNA from human epileptic temporal lobe and, to develop a method using patch clamped cells as detector systems in capillary electrophoresis. The Ca2+-permeability of AMPA receptors was studied in interneurons from rat olfactory bulb. The interneurons were either acutely isolated or from primary cultures. The ion-permeability were studied by means of patch clamp. Kainate (KA) was used to activate AMPA mediated currents. The data from current-to-voltage relationships were fitted with the constant field equation or the Eyring rate theory. Functional AMPA receptors were expressed in Xenopus oocytes by injection of total RNA from human epileptic temporal lobe.The expressed AMPA receptors, activated by KA, were studied with two-electrode voltage clamp, in order to evaluate their Ca2+-permeability properties. Interneurons from the rat olfactory bulb were used as the detectors in a capillary elctrophoresis- patch clamp (CE-PC) analysis of biomolecules that activate or inhibit ligand-gated ion-channels. In CE-PC detection of antagonists, it is necessary to deliver constantly an agonist to the cell surface of the detector. This was performed by adding the specific agonist to the electrolyte solution contained in the electrophoresis capillary. In interneurons from the olfactory bulb, the current responses to kainate showed a linear/outwardly rectifying current-to-voltage relationship. Increasing the Ca2+ concentration from 1 mM to 60 mM gave a leftward the average reversal potential from +7 mV to -32 mV. The permeability properties of the AMPA receptors expressed by interneurons from rat olfactory bulb were well predicted by the Eyring rate theory. The model gave a pCa2+/pK+- permeability ratio of 0.06 for acutely isolate interneurons and 0.14 for interneurons in primary culture. The constant field equation gave corresponding low permeability ratios of 0.18 and 0.40 for acutely isolated cells and cells in primary culture, respectively. Thus it was concluded that interneurons in the olfactory bulb mainly express AMPA receptors with low permeability to Ca2+. Administration of kainate to Xenopus oocytes injected with total RNA from human epileptic temporal lobe gave non-desensitizing AMPA receptor mediated currents. The current-to-voltage relationhip showed an inward rectification and when the concentration of divalent cations were increased, there was a shift in the reversal potential from -11 mV (2mM Ca2+) to 12 mV (60 mM Ba2+). This yielded a pBa2+/pK+ permeability ratio of 1.6 when the constant field equation was used. It was concluded that the AMPA receptors from the human epileptic temporal lobe had a pronounced permeability to Ca2+. Mixtures of *-aminobutyrate, L-glutamate, N-methyl-D-aspartate (NMDA) and kainate (KA) were separated and detected by CE-PC. Also a mixture of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate (DiCl-HQC) were separated and detected by KA activated patch-clamp interneuron from rat olfactory bulb. Mg2+ that reversibly blocks the NMDA receptor in a voltage dependent way, was detected by the same detection system when activated by electrophoretically delivered NMDA and the co-agonist glycine. We have developed a CE-PC detection system that uses cell membranes from cells. The CE-PC system allows us to identify biologically active analytes in complex mixtures or samples. A broad spectrum of information including response characteristics, current-to-voltage relationships, dose-response measurements, kinetics and ion-channel conductance levels were extracted from the recordeD currents evoked by electrophoretically separated receptor agonists.The present method also offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor of interest.
Göteborgs universitet/University of Gothenburg
Institute of Anatomy and Cell Biology
Institutionen för anatomi och cellbiologi
Date of defence
Jardemark, Kent 1962-