Figure 2.
Expression of GluR-BR and GluR-BRneo alleles and increased Ca2+ permeability in hippocampal CA1 neurons of GluR-BRneo/Rneo mice. (A) Aligned sequence chromatograms of RT-PCR products encompassing exon 11 sequences of GluR-B mRNA from homozygous and heterozygous mice. The GluR-B alleles are listed on the left. The Q/R site is indicated by an arrow. The positions for the five silent mutations in the mutant alleles are boxed. The automatic sequence-analysis software attributes N to those nucleotide positions where the different allelic sequences in the RT-PCR product from GluR-B+/R mice have comparable peak heights but differ in sequence. The differential oligonucleotide-hybridization analysis shows that in GluR-B+/R mice, 50.1 ± 0.3% (n = 3) of GluR-B mRNA is from the mutant allele. In RT-PCR amplicons from heterozygous GluR-B+/Rneo mice, peak heights corresponding to the sequence of the mutant allele are significantly lower from those of the wild-type allele. (B) Immunoblot analysis of GluR-B protein in brains from wild-type and homozygous mutant mice. Genotypes are indicated on top of gel, size markers in kDa are shown on the right. The GluR-B antibody recognizes a single band corresponding to a polypeptide of about 110 kDa. (C) Current–voltage relations for glutamate-evoked AMPAR currents recorded in nucleated patches pulled from CA1 pyramidal cells and dentate gyrus (DG) basket cells of GluR-BRneo/Rneo mice in control (•, Normal Rat Ringer) and high Ca2+ (○, 30 mM Ca2+) solutions. For the pyramidal cells, the reversal potential in high-Ca2+ solution was Vrev = −54.5 ± mV and in control solution, Vrev = −4.9 ± mV. Current–voltage relations for wild-type and GluR-BR/R mice were identical, with the averaged Ca2+ reversal potential in high-Ca2+ solution (−69 ± mV, n = 5) indicated by arrow. For the DG basket cells, the reversal potentials in high Ca2+ and control extracellular solutions were Vrev = 2.7 ± mV and Vrev = 0.3 ± mV, respectively, and were indistinguishable from GluR-BR/R and wild-type mice.