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Zusammenfassung:
1. The properties of glutamate receptor (GluR) channels in outside-out patches from the dendrites and somata of rat cerebellar Purkinje cells in brain slice were studied using fast agonist application techniques. Dendritic patches were isolated 40-130 micronm from the soma. 2. Outside-out patches from both dendrites and somata of Purkinje cells responded to application of glutamate with a current which desensitized rapidly and nearly completely. Currents evoked by glutamate application were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), were mimicked by L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and were modulated by cyclothiazide. Kainate produced small, non-desensitizing currents. No currents were observed in response to aspartate application. Responses characteristic of NMDA receptor activation were not observed. These findings indicate that glutamate-activated currents were mediated by the AMPA subtype of GluR. 3. Deactivation of the GluR channels following 1 ms pulses of glutamate occurred with a time constant of 1.23 +/- 0.07 ms in dendritic and 1.12 +/- 0.04 ms in somatic patches. Desensitization occurred with a time constant of 5.37 +/- 0.26 ms in dendritic and 5.29 +/- 0.29 ms in somatic patches. The time constant of recovery from desensitization caused by a 1 ms application of 1 mM glutamate was 36 ms in dendritic patches and 33 ms in somatic patches. 4. Half-maximal activation of the GluR channels was achieved at a glutamate concentration of 432 microM. Deactivation kinetics were not dependent on the glutamate concentration, while desensitization became slower at lower glutamate concentrations. 5. Pre-equilibration of patches with low concentrations of glutamate reduced the peak current activated by 1 mM glutamate. The IC50 for this effect was 8.7 microM. Equilibrium desensitization did not affect the kinetics of the current activated by 1 mM glutamate. 6. The current-voltage relationship of the peak current was linear in normal Na(+)-rich external solution, with a reversal potential near 0 mV. In Ca(2+) -rich external solution, the reversal potentials were -51.4 +/- 2.9 and -51.5 +/- 2.8 mV for dendritic and somatic patches, respectively, indicating that these glutamate channels have a low permeability to Ca2+ (PCa/PCa = 0.053). 7. The mean single-channel conductance of the GluR channels measured using non-stationary fluctuation analysis was approximately 8 pS in dendritic and somatic patches, and the maximum open probability was at least 0.7 with 5 mM glutamate. 8. GluR channel kinetics in patches excised from the soma of neonatal (postnatal day 4; P4) Purkinje cells, before the development of the dendritic arborization of the Purkinje cell, were similar to those in patches excised from more mature (P12-18) Purkinje cells. 9. Dendritic and somatic GluR channels in Purkinje cells appear to be functionally identical, are AMPA-subtype receptors containing the GluR-B subunit, and have rapid kinetics and low permeability to Ca2+. A kinetic model was constructed which faithfully reproduces the gating characteristics of the GluR channels.
