A single-point mutation (Ala280Val) in the third intracellular loop alters the signalling properties of the human histamine H₃ receptor stably expressed in CHO-K1 cells.

BACKGROUND AND PURPOSE: An alanine to valine exchange at amino acid position 280 (A280V) in the third intracellular loop of the human histamine H₃ receptor was first identified in a patient suffering from Shy-Drager syndrome and later reported as a risk factor for migraine. Here, we have compared the pharmacological and signalling properties of wild-type (hH₃ R(WT)) and A280V mutant (hH₃ R(A280V)) receptors stably expressed in CHO-K1 cells.

EXPERIMENTAL APPROACH: The hH₃ R(A280V) cDNA was created by overlapping extension PCR amplification. Receptor expression and affinity were assessed by radioligand (N-α-[methyl-³H]-histamine) binding to cell membranes, and receptor function by the inhibition of forskolin-induced cAMP accumulation and stimulation of ERK1/2 phosphorylation in intact cells, as well as stimulation of [³⁵S]-GTPγS binding to cell membranes.

KEY RESULTS: Both receptors were expressed at similar levels with no significant differences in their affinities for H₃ receptor ligands. Upon activation the hH₃ RWT was significantly more efficacious to inhibit forskolin-induced cAMP accumulation and to stimulate [³⁵S]-GTPγS binding, with no difference in pEC50 estimates. The hH₃ RWT was also more efficacious to stimulate ERK1/2 phosphorylation, but this difference was not significant. The inverse agonist ciproxifan was more efficacious at hH3 RWT to reduce [³⁵S]-GTPγS binding but, for both receptors, failed to enhance forskolin-induced cAMP accumulation.

CONCLUSIONS AND IMPLICATIONS: The A280V mutation reduces the signalling efficacy of the human H₃ receptor. This effect may be relevant to the pathophysiology of disorders associated with the mutation.