Membrane depolarization regulates AMPA receptor subunit expression in cerebellar granule cells in culture |
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Authors: | Salvatore IncontroJorge Ramí rez-Franco,José Sá nchez-PrietoMagdalena Torres |
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Affiliation: | Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain |
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Abstract: | The physiological responses of AMPA receptors can be modulated through the differential expression of their subunits and by modifying their number at the cell surface. Here we have studied the expression of AMPA receptor subunits (GluR1-4) mRNAs in cerebellar granule cells grown in depolarizing (25 mM K+) medium, and we have evaluated the effect of decreasing the [K+] in the culture medium for 24 h on both GluR1-4 expression (both mRNA and protein) and their presence at the plasma membrane. The expression of the four AMPAR subunits increases as the [K+] decreases, although the increase in GluR2 and GluR3 was only observed in the cell soma but not in the dendrites. Calcium entry through L-type calcium channel and CaMKIV activation are responsible for the reduction in the expression of AMPA receptor subunits in cells cultured in depolarizing conditions. Indeed, prolonged reduction of extracellular [K+] or blockage of L-type calcium channels enhanced both the surface insertion of the four AMPAR subunits and the AMPA response measured through intracellular calcium increase. These findings reveal a balanced increase in functional AMPA receptors at the surface of cells that can trigger strong increases in calcium in response to the persistent reduction of calcium entry. |
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Keywords: | AMPA, α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate AMPAR, AMPA receptor CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate CaMKIV, Ca2+/calmodulin-dependent protein kinase type IV CGC, cerebellar granule cell DAPI, 4&prime ,6-diamino-phenylindole dihydrochloride DIV, days in vitro FIC, freshly isolated cells GFAP, green fibrillary acidic protein IRD, near-infrared fluorescent dye KA, kainate NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate NMDAR, NMDA receptor ROI, region of interest TBE buffer, Tris borate EDTA buffer TES, N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid VSCCs, voltage-sensitive calcium channels |
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