Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes.
Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.
Eguchi
Y
Y
Department of Applied Biological Chemistry, Faculty of Agriculture, Kinki University, Nara, Japan.
Ihara
M
M
Ochi
E
E
Shibata
Y
Y
Matsuda
K
K
Fushiki
S
S
Sugama
H
H
Hamasaki
Y
Y
Niwa
H
H
Wada
M
M
Ozoe
F
F
Ozoe
Y
Y
D000818:Animals; D000977:Antiparasitic Agents / Q000494:pharmacology; D065101:Chloride Channel Agonists; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D015870:Gene Expression; D005971:Glutamates / Q000494:pharmacology; D001556:Hexachlorocyclohexane / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D009117:Muscidae / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D010852:Picrotoxin / Q000031:analogs & derivatives / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D018075:RNA, Complementary / Q000378:metabolism; D054830:Sesterterpenes; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology
D000977:Antiparasitic Agents; D065101:Chloride Channel Agonists; D018118:Chloride Channels; D018076:DNA, Complementary; D018755:GABA Agonists; D018756:GABA Antagonists; D005971:Glutamates; D001556:Hexachlorocyclohexane; D010852:Picrotoxin; D011720:Pyrazoles; D018075:RNA, Complementary; D054830:Sesterterpenes; D005680:gamma-Aminobutyric Acid; C109777:glutamate-gated chloride channels; C022961:picrotoxinin; C082360:fipronil