Rapid desensitization of α1β1 GABAA receptors expressed in Sf9 cells under optimized conditions
Tóm tắt
α1 and β1 subunits of human GABAA receptors were expressed in Sf9 cells using the Sf9-baculovirus system. Better expression was obtained by manipulating the system. Cell growth phase at the time of infection determined the practical range of virus titre, the period postinfection during which cells were useful for signal detection and the maximal current obtained. Cells in the early exponential phase were relatively insensitive to multiplicity of infection (MOI) whereas cells in the midto late-exponential phase were highly dependent on MOI and they responded with the largest Cl−} current generated by GABA. Channels activated by GABA were chloride-selective. Half the maximum peak whole-cell current was obtained with 11 μm GABA. The time course of Cl−} currents activated by saturating GABA concentrations in cells infected with α1β1recombinant viruses was examined employing a rapid perfusion system which allowed whole-cell solution exchange in less than 1 msec. The current decay could be fitted by 3 to 4 exponentials for the first 8 sec. The initial fast current decrease had a time constant of about 23 msec. No voltage dependence of time constants was detected but the whole-cell IV relation showed outward rectification. Currents were depressed by bicuculline, penicillin and picrotoxin and potentiated by pentobarbitone.
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