Effects of chlorotrifluoroethylene oligomer fatty acids on recombinant GABA receptors expressed in Xenopus oocytes.
GABA-activated Cl- current was expressed in Xenopus oocytes after injecting cRNA that had been transcribed in vitro from complementary DNA (cDNA) coding for a single GABA rho i-subunit cloned from human retina. The expressed current was insensitive to 100 microM bicuculline, but was activated by the GABA analogue trans-4-aminocrontonic acid (TACA). Anion-selective permeability of the expressed rho 1-subunit was determined by isotonically replacing the extracellular Cl- with different anions. The anion permeability was very similar to the native GABAA receptor/channel following a sequence of SCN- > I- > NO3- > Br- > or = Cl-. Halogenated fatty acids, such as chlorotrifluoroethylene (CTFE) and perfluorinated oligomer acids inhibited the GABA-induced current in oocytes expressing the human retinal GABA rho 1-subunit or rat brain GABAA receptor alpha 1,beta 2,gamma 2 subunits. The inhibitory effect of halogenated fatty acids demonstrated a carbon chain length-dependent manner of: C10 > C8 > C6 > C4. Perfluorinated C8-oligomer acid (PFOA) was less effective at blocking this channel than the C8-CTFE oligomer acid. Radiolabeled GABA binding assay indicated that CTFE oligomer acids do not interfere at the GABA binding site of the receptor. Furthermore, the C8-CTFE oligomer fatty acid did not compete with picrotoxin for binding sites within the pore of the channel. These studies demonstrated that the heterologous expression system is useful for studying the molecular interaction between potential neurotoxic agents and neuroreceptors. Our results provide detailed information that should contribute to our understanding of the structure and function of retinal GABA receptors.
DelRaso
N J
NJ
Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA.
Huang
Y
Y
Lu
L
L
D000818:Animals; D017402:Chlorofluorocarbons / Q000494:pharmacology*; D004553:Electric Conductivity; D005227:Fatty Acids / Q000494:pharmacology*; D005260:Female; D006801:Humans; D009865:Oocytes; D018408:Patch-Clamp Techniques; D010852:Picrotoxin / Q000494:pharmacology; D018079:Receptors, GABA / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014982:Xenopus laevis
D017402:Chlorofluorocarbons; D005227:Fatty Acids; D010852:Picrotoxin; D018079:Receptors, GABA; D011993:Recombinant Fusion Proteins; C016304:chlorotrifluoroethylene