The presynaptic H₃ Histamine receptor subtype is expressed almost exclusively in the CNS where it acts as both an autoreceptor and a heteroreceptor. Pharmacological heterogeneity of H₃ receptors within and across species has long been recognised (Hancock et al., 2003). In all species tested so far the full length H₃ receptor encodes a polypeptide of 445 amino acids (M_r 47,000). Shorter isoforms have also been identified with deletions predominantly in the third intracellular loop domain (M_r 35,000-45,000). These splicing events yield potentially different polypeptides in rodents and man (Chazot & Shenton, 2004). The aim of our work is to develop antibodies specific to these different H₃ isoforms and use them to investigate the structure, distribution and pharmacological properties of cloned and native H₃ receptors. Our laboratory has developed the first anti-H₃ receptor antibodies (Chazot et al., 2001). Recently, we have developed a new anti-H₃ 445 antibody, which may prove to be the first immunological probe specific for the human H₃ 445 isoform.

Immunohistochemical (IHC) studies performed as described in Chazot et al., 2001, with the new anti-H₃ antibody, using wild-type and H₃-/- mice (Johnson and Johnson, USA) showed a profound reduction in immunoreactivity in the H₃ (-/-) brains compared to the wild type, demonstrating the specificity of the new probe. Pronounced specific (ie. absent from H₃-/- brain) decoration was observed of cortical laminae II, striatum, and substantia nigra; but reduced cortical laminae V, and little or no hippocampal labelling. The distinct labelling pattern indicates that this antibody recognises a subset of H₃ receptors in the mouse. A lipofectaminePLUS transfection method was used to transiently express epitope-tagged FLAG-hH₃ 445, FLAG-hH₃ 365 and FLAG-HIS-hH₃ 329 isoforms in HEK 293 cells, confirmed using an anti-FLAG antibody in immunoblots of transfected cell homogenates. Using the same immunoblotting protocol, the new anti-H₃ antibody was strongly reactive against hH₃ 445, with little or no reactivity against the hH₃ 365 and hH₃ 329 isoforms, respectively. All three isoforms migrated as monomeric, as well as larger putative dimeric, trimeric and tetrameric structures. As with the native preparations, following cross-linking, higher di-sulphide linked oligomers were also observed. HEK 293 cells cotransfected with both the hH₃ 445 and hH 3 329 isoforms were solubilised with 1% (v/v) Triton X100 and subjected to nickel affinity chromatography. A major species (approx. M_r 74,000) was purified, which was recognised by both the new specific anti-H₃ 445 antibody and the anti-FLAG antibody. These results remain to be confirmed, but are consistent with the expression of a H₃ 445 /H₃ 329 heterodimer. These data have implications for our present understanding of H₃ receptor structure and for future drug development.

Chazot, P.L. and Shenton (2004) F.C. Curr. Anaesth.Crit. Care 15, p23-28.
Chazot, P.L. et al. (2001) Neuroreport 12, p259-262.
Hancock, A.A. et al . (2003) Life Sciences 73, p3043-3072.

This work is funded by the Wellcome Trust. We thank Dr Coge (Servier, France) and Prof LB Hough (NY, USA) for the H 3 receptor clones and H 3 (-/-) brains, respectively .