The AMPA receptor subunit GluR-B in its Q/R site-unedited form is not essential for brain development and function

Abstract

Calcium permeability of L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in excitatory neurons of the mammalian brain is prevented by coassembly of the GluR-B subunit, which carries an arginine (R) residue at a critical site of the channel pore. The codon for this arginine is created by site-selective adenosine deamination of an exonic glutamine (Q) codon at the pre-mRNA level. Thus, central neurons can potentially control the calcium permeability of AMPARs by the level of GluR-B gene expression as well as by the extent of Q/R-site editing, which in postnatal brain, positions the R codon into >99% of GluR-B mRNA. To study whether the small amount of unedited GluR-B is of functional relevance, we have generated mice carrying GluR-B alleles with an exonic arginine codon. We report that these mutants manifest no obvious deficiencies, indicating that AMPAR-mediated calcium influx into central neurons can be solely regulated by the levels of Q/R site-edited GluR-B relative to other AMPAR subunits. Notably, a targeted GluR-B gene mutant with 30% reduced GluR-B levels had 2-fold higher AMPAR-mediated calcium permeability in hippocampal pyramidal cells with no sign of cytotoxicity. This constitutes proof in vivo that elevated calcium influx through AMPARs need not generate pathophysiological consequences.

Figure 1

GluR-B alleles having an exonic arginine codon for the inner pore segment of AMPAR channels. (A) Schematic representation of the GluR-B subunit and of GluR-B gene segments corresponding to different alleles: GluR-B⁺ (wild-type allele), GluR-B^Rneo (targeted allele), and GluR-B^R (targeted allele after Cre-mediated removal of the floxed neo cassette). In the GluR-B protein (hatched), the N and C termini and the Q/R site are indicated; black boxes mark membrane insertion domains M1–M4. The GluR-B gene segments show numbered exons (boxes), the exon-complementary, cis-acting ECS element in intron 11, the position of the Q/R site, the location of the exonic arginine (R) codon for the Q/R site, as well as the locations of primers MH53 (a) and RSP36 (b) used for genotyping. The loxP sites are shown as filled arrowheads. The floxed PGK-neo marker in the GluR-B^Rneo allele was inserted between intronic NcoI (No) and KpnI (Kp) sites. • in the GluR-B^Rneo allele border the targeting vector (BsrGI-SalI fragment). (B) Genotyping by PCR. The expected sizes of amplicons for wild-type and GluR-B^R alleles are 494 and 599 bp, respectively. Genotypes are indicated on top of gel, size markers are given in kbp on left of gel.

Figure 2

Expression of GluR-B^R and GluR-B^Rneo alleles and increased Ca²⁺ permeability in hippocampal CA1 neurons of GluR-B^Rneo/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-B^Rneo/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 V_rev = −54.5 ± mV and in control solution, V_rev = −4.9 ± mV. Current–voltage relations for wild-type and GluR-B^R/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 V_rev = 2.7 ± mV and V_rev = 0.3 ± mV, respectively, and were indistinguishable from GluR-B^R/R and wild-type mice.

Figure 3

Histochemistry of adult mutant and wild-type mouse brains. (A) Nissl-stained hemibrain slice. (B–E) Representative hippocampal images of immunohistochemical stainings for marker genes. (B), GluR-A; (C), MAP-2; (D), parvalbumin; (E), calbindin. Mouse genotypes are indicated below images.