Effect of mutations in the β1-thyroid hormone receptor on the inhibition of T3 binding by desethylamiodarone

Desethylamiodarone (DEA) acts as a competitive inhibitor of triiodothyronine (T-3) binding to the alpha(1)-thyroid hormone receptor (TR alpha(1)) but as a non-competitive inhibitor with respect to TR beta(1), To gain insight into the position of the binding site of desethylamiodarone on TR beta(1) we investigated the naturally occurring mutants Y321C, R429Q, P453A, P453T and the artificial mutants L421R and E457A in the ligand binding domain of human TR beta(1), The IC50 values (in mu M) of DEA for P453A (50 +/- 11) and P453T (55 +/- 16) mutant TR beta(1) are not different from that for the wild type TR beta 1 (56 +/- 15), but the IC50 values of R429Q (32 +/- 7; P < 0.001) and E457A (17 +/- 3; P < 0.001) are significantly lower than of the wild type. Scatchard plots and Langmuir analyses indicate a non-competitive nature of the inhibition by DEA of T-3 binding to all four mutant TR beta(1)s tested. Mutants P453A and P453T do not influence overall electrostatic potential, and also do not influence the affinity for DEA compared to wild type. Mutant E457A causes a change from a negatively charged amino acid to a hydrophobic amino acid, enhancing the affinity for DEA, Mutant R429Q, located in helix 11, causes an electrostatic potential change from positive to uncharged, also resulting in greater affinity for DEA, We therefore postulate that amino acids R429 and E457 are at or close to the binding site for DEA, and that DEA does not bind in the T-3 binding pocket itself, in line with the non-competitive nature of the inhibition of T-3 binding to TR beta(1) by DEA, (C) 1999 Federation of European Biochemical Societies.