Abstract
The kinetic mechanism of super high affinity [3H]oxotremorine M binding to porcine m2 muscarinic receptors expressed in Chinese hamster ovary cells was examined. In cell lines expressing low receptor numbers (10(4) binding sites/cell) and in high expression (10(6) sites/cell) cell lines treated with cholate, [3H]oxotremorine M association and dissociation kinetics were monophasic. The reciprocal relaxation time for the association reaction was independent of [3H]oxotremorine M concentration and equaled the dissociation rate constant consistent with a special case for a mechanism involving a protein conformational change followed by ligand binding. Membranes from high expression cell lines and porcine atrial membranes showed complex kinetic behavior. Two kinetic phases were observed for [3H]oxotremorine M association, and both reciprocal relaxation times were independent of ligand concentration. The number of kinetic phases and their relative amplitudes seen in dissociation experiments were dependent on whether dissociation was initiated by dilution or by addition of unlabeled ligand(s) as well as on the fractional saturation of the receptor. Computer simulations of the data led to a model consistent with the existence of asymmetric receptor dimers as well as monomers and the ligand-dependent interconversion of fully occupied dimers and monomers.
Highlights
A Kinetic Model for Oxotremorine M Binding to Recombinant Porcine m2 Muscarinic Receptors Expressedin Chinese Hamster Ovary Cells*
Computer simulationsof the data Agonist binding kinetics araelso complex since displacement led to a model consistent with the existence of asymofmtheet-antagonist N-methyl-4-piperidinyl benzilate with the ric receptor dimerass well as monomers and the liganda-gonists carbamylcholine and oxotremorine could not be exdependent interconversionof fully occupied dimers andplained by competition of agonists and antagonists for free monomers
Preincubating membranes with 100 nM GTP@ for 35 min reduced equilibrium super high affinity Oxo M binding by 65%, but failed to affect either the reciprocal relaxation times or the relative amplitudes of [3H]0xoM association
Summary
The abbreviationsused are: mAChRs, muscarinic acetylcholinere- all guanine nucleotides were from Boehringer Mannheim. Research Biochemicals Inc. CHO cell lines expressing lo or lo pm2 cine m2 subtype of the muscarinic receptor; CHO, Chinese hamster receptor-binding siteskell [18] were grown to near confluency in ovary; Z-QNB, (-)-quinuclidinyl benzilateG;TPyS, guanosine5‘-O-(thio- 100-mm tissue culture dishes in a 1:l mixture of Dulbecco’s modified triphosphate). CHO cell lines expressing lo or lo pm2 cine m2 subtype of the muscarinic receptor; CHO, Chinese hamster receptor-binding siteskell [18] were grown to near confluency in ovary; Z-QNB, (-)-quinuclidinyl benzilateG;TPyS, guanosine5‘-O-(thio- 100-mm tissue culture dishes in a 1:l mixture of Dulbecco’s modified triphosphate) Muscarinic Agonist Binding Kinetics poxanthine, and thymidine plus 10%(v/v) calf serum.
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