Identification of a domain which affects kinetics and antagonistic potency of clozapine at 5-HT3 receptors.

The widely used atypical antipsychotic clozapine is a potent
competitive antagonist at 5-HT(3) receptors which may contribute to
its unique psychopharmacological profile. Clozapine binds to
5-HT(3) receptors of various species. However, the structural
requirements of the respective binding site for clozapine remain to
be determined. Differences in the primary sequences within the
5-HT(3A) receptor gene in schizophrenic patients may result in an
alteration of the antipsychotic potency and/or the side effect
profile of clozapine. To determine these structural requirements we
constructed chimeras with different 5-HT(3A) receptor sequences of
murine and human origin and expressed these mutants in human
embryonic kidney (HEK) 293 cells. Clozapine antagonises recombinant
mouse 5-HT(3A) receptors with higher potency compared to
recombinant human 5-HT(3A) receptors. 5-HT activation curves and
clozapine inhibition curves yielded the parameters EC(50) and
IC(50) for all receptors tested in the range of 0.6 - 2.7 microM
and 1.5 - 83.3 nM, respectively. The use of the Cheng-Prusoff
equation to calculate the dissociation constant K(b) values for
clozapine revealed that an extracellular sequence (length 86 aa)
close to the transmembrane domain M1 strongly determines the
binding affinity of clozapine. K(b) values of clozapine were
significantly lower (0.3-1.1 nM) for receptors containing the
murine sequence and higher when compared with receptors containing
the respective human sequence (5.8-13.4 nM). Thus, individual
differences in the primary sequence of 5-HT(3) receptors may be
crucial for the antipsychotic potency and/or the side effect
profile of clozapine.