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 ⁴⁵Ca²⁺ 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 ⁴⁵Ca²⁺ accumulation. ⁴⁵Ca²⁺ 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 ⁴⁵Ca²⁺ influx. These included Ca²⁺ uptake through NMDA-receptor channels, voltage-sensitive Ca²⁺ channels, and via Na⁺/Ca²⁺ exchange. However, nearly one-fifth of the total ⁴⁵Ca²⁺ influx remained unaccounted for and this estimate was similar to ⁴⁵Ca²⁺ influx observed under Na⁺-free conditions. This observation suggested that a significant proportion of the Ca²⁺ flux passes through the AMPA-receptor channel proper, a view supported by Co²⁺ 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.