Functional chloride channels by mammalian cell expression of rat glycine receptor subunit.
Cultured human cells were transfected with cloned rat glycine receptor (GlyR) 48 kd subunit cDNA. In these cells glycine elicited large chloride currents (up to 1.5 nA), which were blocked by nanomolar concentrations of strychnine. However, no corresponding high-affinity binding of [3H]strychnine was detected in membrane preparations of the transfected cells. Analysis by monoclonal antibodies specific for the 48 kd subunit revealed high expression levels of this membrane protein. After solubilization, the 48 kd subunit behaved as a macromolecular complex when analyzed by sucrose density centrifugation. Approximately 50% of the solubilized complex bound specifically to a 2-aminostrychnine affinity column, indicating the existence of low-affinity antagonist binding sites on most of the expressed GlyR protein. Thus, the 48 kd strychnine binding subunit efficiently assembles into high molecular weight complexes, resembling the native spinal cord GlyR. However, formation of functional receptor channels of high affinity for strychnine occurs with low efficiency.
Sontheimer
H
H
Institute for Neurobiology, University of Heidelberg, Federal Republic of Germany.
Becker
C M
CM
Pritchett
D B
DB
Schofield
P R
PR
Grenningloh
G
G
Kettenmann
H
H
Betz
H
H
Seeburg
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010852:Picrotoxin / Q000494:pharmacology; D051381:Rats; D018009:Receptors, Glycine; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000502:physiology; D013331:Strychnine / Q000494:pharmacology; D014162:Transfection
D002712:Chlorides; D004247:DNA; D005998:Glycine; D007473:Ion Channels; D010852:Picrotoxin; D018009:Receptors, Glycine; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D013331:Strychnine