GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP.
The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.
Luu
Tien
T
Division of Molecular Bioscience, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory 0200, Australia.
Gage
Peter W
PW
Tierney
M Louise
ML
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000502:physiology*; D003975:Diazepam / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005347:Fibroblasts / Q000378:metabolism; D018757:GABA Modulators / Q000494:pharmacology; D015640:Ion Channel Gating; D008565:Membrane Proteins / Q000096:biosynthesis / Q000502:physiology*; D051379:Mice; D008869:Microtubule-Associated Proteins; D008956:Models, Chemical; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D003598:Cytoskeletal Proteins; D018757:GABA Modulators; C480861:GABARAP protein, mouse; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D003975:Diazepam