Developmental expression of chick cortical GABA(A) receptor alpha1 subunits in vivo and in vitro.
In order to examine the expression of the GABA(A) receptor alpha1 subunit during chick cortical development in vivo and in vitro, we have utilized a polyclonal antibody (RP4) directed against an alpha1(331-381) fusion protein. This antibody exhibits a high titer for precipitation of [3H]flunitrazepam binding sites in chick cortical extracts, no significant cross-reactivity with GABA(A) receptor beta2- or beta4-subunit fusion proteins, and a robust reaction with a single 51-kDa polypeptide on immunoblots of cortical membranes. This indicates monospecificity of the RP4 antiserum for the GABA(A) receptor alpha1 subunit. The alpha1-subunit antibody also showed strong immunocytochemical reactions with neurons in the embryonic mediodorsal cortex and Purkinje cells of the chick cerebellum. The ontogeny of the alpha1 subunit in chick cortex and in derived neuronal cultures was examined by quantitative Western blotting. The level of the alpha1 polypeptide increased from day 2 to day 6 in culture, acquiring 50% of the maximum expression at day 4. Expression of the cortical GABA(A) receptor alpha1 subunit increased in vivo from embryonic day 8 (E8) to day 7 post-hatching, reaching 50% of adult levels at E16. Levels of the corresponding alpha1-subunit mRNA, analyzed from E8 to E20 by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), showed a corresponding incline. These findings correlated well with previous developmental studies of GABA(A) receptor ligand binding sites both in vivo and in vitro. The parallel increase of the alpha1 subunit transcript and polypeptide with [3H]flunitrazepam binding sites suggests that this subunit may be an important component of GABA(A) receptors early in cortical ontogeny. This was investigated further by quantitative immunoprecipitation. At saturation, the RP4 antiserum consistently precipitated 50-65% of the central [3H]flunitrazepam binding sites in the developing cortex from E12 through P7, despite a 5-fold increase in the binding level. The data suggest that during cortical development the fraction of GABA(A) receptors containing alpha1 subunits remains relatively constant. Furthermore, the alpha1 polypeptide appears to be a major component of GABA(A) receptor oligomers at all stages of cortical maturation.
Miranda
J D
JD
Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
Liu
S C
SC
Diaz
M E
ME
Barnes
E M
EM
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000502:physiology; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000737:chemistry / Q000254:growth & development; D002642:Chick Embryo; D005260:Female; D005445:Flunitrazepam / Q000378:metabolism / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D011963:Receptors, GABA-A / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000276:immunology; D014316:Tritium
D000906:Antibodies; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D014316:Tritium; D005445:Flunitrazepam