Functional properties of glycine receptors expressed in primary cultures of mouse cerebellar granule cells.
Expression of the glycine receptor was investigated in membranes prepared from primary cultures of mouse cerebellar granule cells and postnatal mouse cerebellum using the antagonist [3H]strychnine for ligand binding. Scatchard analysis of the binding data obtained from P17 cerebellum showed a single population of binding sites (K(D) approximately 6 nM) and [3H]strychnine binding to membranes prepared from cultured neurons and P17 cerebellum was found to have the same sensitivity to the glycinergic agonists glycine, beta-alanine and taurine. The development of [3H]strychnine binding sites in cultured cerebellar granule cells and cerebellum showed opposing profiles. [3H]strychnine binding to primary cultures increased significantly during the culture period whereas during development in vivo the number of binding sites decreased over time and was hardly detectable in the adult cerebellum. Release of preloaded D-[3H]aspartate evoked by 40 mM K+ from granule cells cultured for seven days was inhibited by glycine by about 50%. Beginning after seven days in culture the ability of glycine to inhibit transmitter release declined to no inhibition after 17 days in culture. Experiments with the non-competitive antagonist, picrotoxinin, showed no blocking effect of 150 microM picrotoxinin on the glycine-induced inhibition of transmitter release. This contrasted with the inhibitory effect of 100 microM picrotoxinin in whole-cell patch-clamp recordings on responses to 500 microM glycine (56% block). Furthermore, it was demonstrated that the amplitude of the glycine activated peak current had the same size after six to seven days and after 16-17 days in culture. Northern blot analysis, and co-injection of messenger RNA plus antisense oligonucleotides into Xenopus oocytes revealed glycine receptor alpha2 and beta messenger RNAs in the cultured granule cells. These findings suggest that granule cells in culture express glycine receptor isoforms containing alpha2 picrotoxinin-sensitive and alpha2/beta picrotoxinin-insensitive receptors.
Elster
L
L
PharmaBiotec Research Center, Department of Biological Sciences, Royal Danish School of Pharmacy, Copenhagen.
Banke
T
T
Kristiansen
U
U
Schousboe
A
A
Wahl
P
P
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D005260:Female; D005998:Glycine / Q000494:pharmacology*; D007539:Isonicotinic Acids / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010852:Picrotoxin / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay; D018009:Receptors, Glycine / Q000819:agonists / Q000096:biosynthesis / Q000502:physiology*; D054830:Sesterterpenes; D013331:Strychnine / Q000378:metabolism / Q000494:pharmacology; D013654:Taurine / Q000494:pharmacology; D013997:Time Factors; D014316:Tritium; D014982:Xenopus laevis; D015091:beta-Alanine / Q000494:pharmacology
D005998:Glycine; D007539:Isonicotinic Acids; D010852:Picrotoxin; D012333:RNA, Messenger; D018009:Receptors, Glycine; D054830:Sesterterpenes; D013331:Strychnine; D013654:Taurine; D014316:Tritium; D015091:beta-Alanine; C022961:picrotoxinin; C015300:isoguvacine