Adenovirus-mediated expression of AMPA-type glutamate receptor channels in PC12 cells.
The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor channels are expressed ubiquitously in brain neurons and mediate fast excitatory neurotransmission. They are composed of four subunits, GluR1, GluR2, GluR3 and/or GluR4. We constructed recombinant adenoviruses encoding rat AMPA receptor subunit cDNAs, GluR1 (AxCAGluR1) and silently mutated GluR2 (AxCAGluR2X) with modified chicken beta-actin promoter and cytomegalovirus immediate-early enhancer. Using these adenoviral vectors, we transferred the GluR1 and GluR2 genes into PC12 cells that possess no functional AMPA receptor channels. PC12 cells infected with these viruses expressed GluR1 and GluR2 RNAs. Immunoblot analysis indicated that the expressed GluR1 and GluR2 proteins were equivalent to those of the rat brain. Functional expression of the AMPA receptor channels was examined using the whole-cell patch clamp technique. In AxCAGluR1-infected cells, the current-voltage (I-V) relationship of response to kainate, a non-desensitizing agonist of AMPA receptors, exhibited a strong inward rectification, indicating the formation of functional GluR1-homomeric channels. In cells infected with both AxCAGluR1 and AxCAGluR2X, the I-V relationship of kainate responses exhibited an outward rectification, indicating the formation of heteromeric GluR1/R2 channels. Immunocytochemical analysis revealed that the AMPA receptor subunit genes were transferred in more than 95% of the infected PC12 cells.
Sudo
M
M
Department of Physiology, Gunma University School of Medicine, Gunma-ken, Maebaeshi, Japan. msudo@sb.gunma-u.ac.jp
Tsuzuki
K
K
Okado
H
H
Miwa
A
A
Ozawa
S
S
D000256:Adenoviridae; D000257:Adenoviridae Infections / Q000378:metabolism*; D000818:Animals; D018076:DNA, Complementary / Q000032:analysis*; D015151:Immunoblotting; D007150:Immunohistochemistry; D016716:PC12 Cells; D012367:RNA, Viral / Q000096:biosynthesis; D051381:Rats; D018091:Receptors, AMPA / Q000032:analysis*; D014779:Virus Replication
D018076:DNA, Complementary; D012367:RNA, Viral; D018091:Receptors, AMPA