Molecular cloning of a GABA receptor subunit from Laodelphax striatella (Fallén) and patch clamp analysis of the homo-oligomeric receptors expressed in a Drosophila cell line.
A cDNA encoding a gamma-aminobutyric acid (GABA) receptor subunit was cloned from the small brown planthopper Laodelphax striatella. The L. striatella GABA receptor subunit was found to have high amino acid sequence similarity to the bd-type splice variant of the Drosophila GABA receptor Rdl subunit and several other GABA receptor subunits, with identities of over 70%. The cDNA was inserted into the expression vector pAc5.1-lac-Hygro. Clonal cell lines stably expressing homo-oligomeric L. striatella GABA receptors were generated by transfecting the vector into D.mel-2 cells. Expression of functional GABA receptors in the cell lines was demonstrated by whole-cell patch clamp recordings. GABA induced inward currents with an EC(50) value of 29 microM and a Hill coefficient of 1.7. The GABA-evoked responses reversed close to the Nernst equilibrium potential for chloride ions. The amplitudes of agonist-induced currents were found to be in the order muscimol (100 microM) >/= GABA (100 microM) > isoguvacine (100 microM) > cis-4-aminocrotonic acid (CACA) (100 microM) > 5-(4-piperidyl)-3-isoxazolol (4-PIOL) (1 mM). Antagonists such as fipronil (100 nM), 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) (100 nM), dieldrin (100 nM) and SR95531 (gabazine) (1 microM) suppressed GABA-induced currents. The functional expression of a GABA receptor from an agricultural pest presents a unique opportunity to discover new molecules active at this important target site.
Narusuye
K
K
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane, Japan.
Nakao
T
T
Abe
R
R
Nagatomi
Y
Y
Hirase
K
K
Ozoe
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004331:Drosophila melanogaster; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D017344:Genes, Insect; D006430:Hemiptera / Q000235:genetics* / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D018079:Receptors, GABA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D017931:DNA Primers; D018755:GABA Agonists; D018756:GABA Antagonists; D019476:Insect Proteins; D021122:Protein Subunits; D018079:Receptors, GABA; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid