Paulo Kofuji

Ethanol sensitivity of the GABAA receptor expressed in xenopus oocytes requires 8 amino acids contained in the γ2L subunit

Expression of brain mRNA or cRNAs in Xenopus oocytes was used to determine what subunits of the GABAA receptor are required for modulation by barbiturates, benzodiazepines, and ethanol. Mouse brain mRNA was hybridized with antisense oligonucleotides complementary to sequences unique to specific subunits and injected into oocytes. Antisense oligonucleotides to the alpha 1, beta 1, gamma 1, gamma 2S + 2L, gamma 2L, or gamma 3 subunits did not alter GABA action or enhancement by pentobarbital. Action of diazepam was prevented by antisense oligonucleotides to gamma 2S + 2L and reduced by antisense sequences to gamma 2L, but was not affected by the other oligonucleotides. Ethanol enhancement of GABA action was prevented only by antisense oligonucleotides to gamma 2L (which differs from gamma 2S by the addition of 8 amino acids). Expression of either the alpha 1 beta 1 gamma 2S or the alpha 1 beta 1 gamma 2L subunit cRNA combination in oocytes resulted in GABA responses that were enhanced by diazepam or pentobarbital, but only the combination containing the gamma 2L subunit was affected by ethanol.

Generation of two forms of the gamma-aminobutyric acidA receptor gamma 2-subunit in mice by alternative splicing.

Abstract: γ‐Aminobutyric acidA (GABAA) receptors are multisubunit ligand‐gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (α, β, γ, and δ). The γ2‐subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine γ2‐subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids, LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the γ2‐subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight‐amino‐acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.

Characterization of recombinant GABAA receptors produced in transfected cells from murine α1, β1, and γ2 subunit cDNAs

In order to explore the structural basis of GABAA receptor function, we have expressed murine α1, β1, and γ2 subunit cDNAs by transient transfection of human 293 cells. Expression of GABAA receptors was measured by ligand binding assay and by electrophysiological analysis. As in other species, expression of the α1 and β1 subunits produced a receptor that was insensitive to modulation by benzodiazepines as measured by electrophysiological analysis; however, a small number of flunitrazepam binding sites were detectable. The coexpression of the γ2 subunit was found to be essential for this modulation, and also resulted in a dramatic (14-fold) increase in the number of binding sites for flunitrazepam. On the coexpression of all 3 subunit cDNAs, a receptor was produced that exhibited a similar number of binding sites for flunitrazepam and muscimol.

The alpha 1, alpha 2, and alpha 3 subunits of GABAA receptors: comparison in seizure-prone and -resistant mice and during development.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, opens chloride channels through actions on GABAA receptors. We now report base and amino acid sequences of the α1, α2, and α3 subunits from GABAA receptors of audiogenic seizure-prone (DBA/2J) and -resistant (C57BL/6J) inbred strains of mice. Inbreeding had fixed different alleles of the α1 subunit in the two strains, giving five base differences in the cDNAs. None of these affected amino acid sequence, but one did create a NsiI restriction site potentially useful in mapping genomic DNA. No base or amino acid sequence differences between the strains were detected for the other two subunits. Northern blots revealed no apparent strain differences in message levels for these three subunits in whole brains of the mice at 3 weeks of age, the peak of seizure susceptibility in DBA/2J, but did reveal distinct regional and developmental patterns of expression among the subunits in mouse brain.

The Molecular Biology of the Na+‐Ca2+ Exchanger and Its Functional Roles in Heart, Smooth Muscle Cells, Neurons, Glia, Lymphocytes, and Nonexcitable Cellsa

The Na+-Ca2+ exchanger, first identified in 1968 and 1969 in heart muscle,-2 has since been identified in virtually every tissue examined3-j and in species ranging from h ~ m a n , ~ . ~ to dog, to squid and to fruit f l ~ . ~ , ~ This brief commentary is an attempt to review several important findings, identify and provide commentary on critical outstanding questions and suggest lines of work that should prove fruitful in the future. The questions raised by recent work fall into two categories. (1) Questions about how the known or suspected functions of the Nat-Ca2+ exchanger are brought about by the Naf-Ca2+ exchanger protein. (2) Questions about why different tissues have different primary sequences of the Nat-Ca2+ exchanger. Included in this second class of questions are those that center on why there are several genes encoding for the Naf-CaZt exchanger and how or why evolutionary changes may have occurred.

GABAA receptor β1, β2, and β3 subunits: comparisons in DBA/2J and C57BL/6J mice

Abstract GABAA receptors link binding of GABA (γ-aminobutyric acid) to inhibitory chloride flux in the brain. They are the site of action of several important classes of drugs, and have been implicated in animal models of epilepsy and in the actions of alcohol. We compare the sequence and expression of the β1, β2 and β3 subunits of GABAA receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ markedly in seizure susceptibility and in a variety of behaviors related to alcohol. Only the β3 3 subunit had strain differences in cDNA nucleotide sequence, which did not affect amino acid sequence but which did create restriction fragment length polymorphisms (RFLPs) potentially useful in gene mapping. We have also tested mouse β1 and β2 subunits for internal alternative splicing, detecting none.

Strain comparisons and developmental profile of the delta subunit of the murine GABAA receptor.

GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunits postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunits first putative transmembrane region.

Alternative splicing of the Na+-Ca2+ exchanger gene, NCX1

We describe an analysis of the NCX1 gene and show that various tissues express different alternatively spliced forms of the gene. Alternative splicing has been confirmed by the genomic analysis of the Na(+)-Ca2+ exchanger gene. We also describe the Drosophila Na(+)-Ca2+ exchanger as having many of the same structural characteristics of the mammalian exchangers and this locus as possibly undergoing alternative splicing in the same region that has been described in the NCX1 gene. The general structure of the exchangers is similar to that of the alpha-subunit of the (Na(+)+ K+)-A Pase. Finally, sequence comparison of the various molecules demonstrates that structural characteristics of these molecules are more strongly conserved than the primary sequence of these products.