Kinetic properties of alpha 1 beta 1 gamma-aminobutyric acidA receptor channels expressed in Chinese hamster ovary cells: regulation by pentobarbital and picrotoxin.
Single-channel recordings from excised outside-out patches were obtained from Chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (GABA) type A (GABAA) receptor channel alpha 1 and beta 1 subunit cDNAs. The predominant or main conductance level recorded had a 17-pS chord conductance. There were minor contributions made from 25-pS and 11-pS conductance levels. Average open duration, burst duration, and openings/burst did not change as the GABA concentration was increased from 5 to 25 microM. However, opening frequency increased from 11.0 to 19.5 openings/sec. Pentobarbital increased average channel open duration without increasing opening frequency, whereas picrotoxin slightly reduced average channel open duration and reduced opening frequency. Open duration frequency distributions were fitted best with the sum of two exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had at least two open states. The time constants and relative proportions of the two components did not vary when GABA concentration was increased from 5 to 25 microM. Closed duration distributions of closures between main conductance level openings were fitted best with multiple exponential functions, suggesting that the alpha 1 beta 1 GABAA receptor channel had several closed states. Burst duration frequency distributions were fitted best with two exponential functions whose time constants and relative proportions did not change with GABA concentration. A gating kinetic scheme for the alpha 1 beta 1 GABAA receptor channel was proposed that consisted of a single binding site for GABA and at least two open and five closed states. The kinetic properties of the alpha 1 beta 1 main conductance level differed from those of the spinal cord neuron (native) 27-pS main conductance level and the 19-pS subconductance level. The native main conductance and subconductance levels were characterized by longer openings and at least three open states. Based on the aforementioned observations, it appears that different subunit combinations produce receptor channels with different kinetic properties, but the basic mechanism of regulation by pentobarbital and picrotoxin may be similar for the different receptor channels. Also, it is unlikely that the 19-pS substate of the native GABAA receptor is produced by an alpha 1 beta 1 dimer.
Porter
N M
NM
Department of Neurology, University of Michigan Medical Center, Ann Arbor 48104.
Angelotti
T P
TP
Twyman
R E
RE
MacDonald
R L
RL
D000818:Animals; D016466:CHO Cells; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects* / Q000378:metabolism; D008954:Models, Biological; D010424:Pentobarbital / Q000494:pharmacology*; D010852:Picrotoxin / Q000494:pharmacology*; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D010424:Pentobarbital; D010852:Picrotoxin; D011963:Receptors, GABA-A; D011994:Recombinant Proteins