A number of studies have demonstrated that a major portion of the ligand binding site of the Torpedo nicotinic acetylcholine receptor is near cysteines 192 and 193 of the α subunit. The role of conserved tyrosine and aspartate residues within this region in ligand binding and receptor activation was investigated using a combination of site-directed mutagenesis and expression in Xenopus oocytes. Wild-type receptors are half-maximally activated (K( 1/2 )) by 20 μM acetylcholine with a Hill coefficient, n, of 1.9. Substitution of αY190 and αY198 with phenylalanines (αY190F, αY198F) or αD200 with asparagine (αD200N) shifts the K( 1/2 ) to 408, 117, and 75 μM, respectively, with no effect on the Hill coefficient. To further study the effects of these mutations on activation, the responses of the receptors to the partial agonists phenyltrimethylammonium (PTMA) and tetramethylammonium (TMA) were examined. Wild-type receptors are half-maximally activated by 73 μM PTMA and 2 mM TMA. In contrast, αY190F, αY198F, and αD200N receptors are not activated by PTMA and TMA by concentrations of up to 500 μM or 5 mM, respectively. However, PTMA and TMA do act as competitive antagonists of the mutant receptors, an indication that the binding of these compounds is not abolished by these mutations. Comparison of the K(i) values for TMA and PTMA inhibition with the K( 1/2 ) values for TMA and PTMA activation of wild-type receptors indicates that the affinities of these compounds are similar in wild-type and mutant receptors. Therefore, αY190F, αY198F, and αD200N mutations do not significantly alter the affinity of the ligand binding site; rather, these mutations appear to interfere with the coupling of ligand binding to channel opening.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Jan 1 1992|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology