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 (hH₃R_WT) and A280V mutant (hH₃R_A280V) receptors stably expressed in CHO-K1 cells.

Experimental Approach

The hH₃R_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 hH₃R_WT was significantly more efficacious to inhibit forskolin-induced cAMP accumulation and to stimulate [³⁵S]-GTPγS binding, with no difference in pEC₅₀ estimates. The hH₃R_WT was also more efficacious to stimulate ERK1/2 phosphorylation, but this difference was not significant. The inverse agonist ciproxifan was more efficacious at hH₃R_WT 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.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1