Binding of [3H]hemicholinium-3 to the high-affinity choline transporter in electric organ synaptosomal membranes.
Sodium-dependent binding of [3H]hemicholinium-3 was observed to be 10-fold higher with presynaptic membranes from the electric organ than with electroplaque membranes and this binding site copurified with synaptosomal membranes. The KD for specific [3H]hemicholinium-3 binding was found to be 31 +/- 4 nM and the Bmax, 5.0 +/- 0.2 pmol/mg protein; a Ki of 16 nM was estimated for hemicholinium-3 as a competitive inhibitor of high-affinity choline transport in electric organ synaptosomes. Choline and choline analogues were equally potent as inhibitors of [3H]choline uptake and [3H]hemicholinium-3 binding. Tubocurarine and oxotremorine also inhibited uptake and binding, but carbachol was without effect in both tests. These findings suggest that [3H]hemicholinium binds to the high-affinity choline transporter present at the cholinergic nerve terminal membrane. A comparison of maximal velocities for choline transport and the maximal number of hemicholinium-3 binding sites indicated that the high-affinity choline transporter has an apparent turnover number of about 3s-1 at 20 degrees C under resting conditions. The high transport rates observed in electric organ synaptosomes are likely due to the high density of high-affinity choline transporters in this tissue, estimated on the basis of [3H]hemicholinium-3 binding to be of the order of 100/micron2 of synaptosomal membrane.
O'Regan
S
S
Département de Neurochimie, Laboratoire de Neurobiologie Cellulaire et Moleculaire-C.N.R.S., Gif sur Yvette, France.
D000605:Amino Alcohols / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002794:Choline / Q000378:metabolism; D003472:Curare / Q000494:pharmacology; D004557:Electric Organ / Q000378:metabolism*; D006426:Hemicholinium 3 / Q000378:metabolism* / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013570:Synaptic Membranes / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014101:Torpedo / Q000378:metabolism*
D000605:Amino Alcohols; D011950:Receptors, Cholinergic; C057694:4-trimethylamino-1-butanol; D006426:Hemicholinium 3; D010095:Oxotremorine; D003472:Curare; D012964:Sodium; D002794:Choline