Properties of AMPA Receptors Expressed in Rat Cerebellar Granule Cell Cultures: Ca2+ Influx Studies

Abstract: Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to ( RS )‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA), in terms of AMPA‐induced toxicity or 45 Ca 2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45 Ca 2+ accumulation. 45 Ca 2+ influx was induced specifically by AMPA‐receptor stimulation, because it was blocked virtually completely by 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non‐NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA‐receptor stimulation contributed significantly to the overall 45 Ca 2+ influx. These included Ca 2+ uptake through NMDA‐receptor channels, voltage‐sensitive Ca 2+ channels, and via Na + /Ca 2+ exchange. However, nearly one‐fifth of the total 45 Ca 2+ influx remained unaccounted for and this estimate was similar to 45 Ca 2+ influx observed under Na + ‐free conditions. This observation suggested that a significant proportion of the Ca 2+ flux passes through the AMPA‐receptor channel proper, a view supported by Co 2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor‐subunit composition of cerebellar granule cells in vitro.