title: 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.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  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
  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.
authors.affiliation: Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados (Cinvestav) del IPN, México.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Flores-Clemente
authors.forename: Angélica
authors.initials: A
authors.lastname: Osorio-Espinoza
authors.forename: Juan
authors.initials: J
authors.lastname: Escamilla-Sánchez
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.forename: Juan-Manuel
authors.initials: JM
authors.lastname: Arias
authors.forename: José-Antonio
authors.initials: JA
authors.lastname: Arias-Montaño
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D018100:Receptors, Histamine H3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D018076:DNA, Complementary; D007093:Imidazoles; D008024:Ligands; D018100:Receptors, Histamine H3; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C115705:ciproxifan; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: CHO-K1 cells; H3 receptor; histamine; migraine; neuromodulation; orthostatic hypotension