Pharmacological detection of AMPA receptor heterogeneity by use of two allosteric potentiators in rat hippocampal cultures.

1. In order to examine whether a recently developed allosteric potentiator for AMPA receptors, 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difluoro-phenoxyaceta mide (PEPA), can be utilized as an indicator of AMPA receptor heterogeneity, the action of PEPA upon the increase of intracellular free calcium ion concentration ([Ca2+]i) elicited by AMPA was investigated in rat hippocampal cultures, and the action was compared with that of cyclothiazide, a well characterized allosteric modulator of AMPA receptors. 2. PEPA dose-dependently potentiated AMPA-induced increase of [Ca2+]i. In 90% (72 out of 80) of the cells in which cyclothiazide acts, PEPA potentiated the increased [Ca2+]i induced by AMPA with pronounced cell-to-cell variation in rat hippocampal cultures. 3. The ratio of the potentiation by PEPA to the potentiation by cyclothiazide (P/C ratio) also varied with cells between 0 and 2.15. It was found that the cultured hippocampal cells consisted of multiple populations with different P/C ratios. Among them two populations exhibited characteristic P/C ratios; low (0 to 0.15; 27 out of 80 cells, 34%) and high (> or = 2.00; 1 out of 80 cells, 1%) P/C ratios. The P/C ratios of the other populations were between 0.25 and 1.20, and these cells constituted 65% (52 out of 80 cells) of the cells tested. 4. Reverse transcriptase-polymerase chain reaction analysis suggested that GluR2-flip, GluR1-flip, GluR2-flop, and GluR1-flop were abundantly expressed (in this rank order) in the cultures used. 5. In Xenopus oocytes expressing GluR1, GluR3, or these subunits plus GluR2, the potentiation of AMPA response by PEPA and by cyclothiazide varied with subunit and splice-variant combinations, and the P/C ratio was between 0.19 and 2.20. Oocytes with low P/C ratios (0.19 to 0.50) and low sensitivity to PEPA potentiation (1.9 fold to 6.41 fold) were those expressing flip variants predominantly, and oocytes with high P/C ratios (1.8 to 2.2) were those expressing flop variants predominantly. Oocytes with intermediate P/C ratios (0.51 to 1.20) were those expressing various combinations of flip and flop variants, and it was impossible to specify the relative abundance of flip and flop variants in these cells. Therefore, the P/C ratio can be used to infer subunit/splice variant expression only when the ratio is low or high. 6. These results suggest that the potentiation by PEPA alone reveals cell-to-cell heterogeneity of AMPA receptors, but a comparison of the actions of PEPA and cyclothiazide further facilitates the detection of the heterogeneity.