Histamine H3 Receptor Isoforms: Insights from Alternative Splicing to Functional Complexity

Abstract

Alternative splicing significantly enhances the diversity of the G protein-coupled receptor (GPCR) family, including the histamine H₃ receptor (H₃R). This post-transcriptional modification generates multiple H₃R isoforms with potentially distinct pharmacological and physiological profiles. H₃R is primarily involved in the presynaptic inhibition of neurotransmitter release in the central nervous system. Despite the approval of pitolisant for narcolepsy (Wakix^®) and daytime sleepiness in adults with obstructive sleep apnea (Ozawade^®) and ongoing clinical trials for other H₃R antagonists/inverse agonists, the functional significance of the numerous H₃R isoforms remains largely enigmatic. Recent publicly available RNA sequencing data have confirmed the expression of multiple H₃R isoforms in the brain, with some isoforms exhibiting unique tissue-specific distribution patterns hinting at isoform-specific functions and interactions within neural circuits. In this review, we discuss the complexity of H₃R isoforms with a focus on their potential roles in central nervous system (CNS) function. Comparative analysis across species highlights evolutionary conservation and divergence in H₃R splicing, suggesting species-specific regulatory mechanisms. Understanding the functionality of H₃R isoforms is crucial for the development of targeted therapeutics. This knowledge will inform the design of more precise pharmacological interventions, potentially enhancing therapeutic efficacy and reducing adverse effects in the treatment of neurological and psychiatric disorders.

Keywords: RNA sequencing; dimerization; histamine H3 receptor; isoform; signaling.

Figure 1

HRH3 gene structure and identified H₃R isoforms in seven species. (A) Genome sequences of human chromosome 20 (NC_000020.11), monkey chromosome 10 (NC_041763.1), mouse chromosome 2 (NC_000068.8), rat chromosome 3 (NC_086021.1), guinea pig (NW_026947488.1), Siberian hamster (JANBXA010000429), and zebrafish chromosome 7 (NC_007118.7) were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/nuccore/; accessed on 1 April 2024)). Exons 1–4 (E1–E4) and introns 1–3 (I1–I3) are depicted as boxes (on scale) and dashed lines (not on scale), respectively. The colors indicate the H₃R protein domains that are encoded by the four exons, and, within exon 3, the pseudo intron encoding for ICL3 segments. The six ICL3 segments (A-F) have been defined based on identified human splice variants: A = R²²⁷–L²³³; B = D²³⁴–Q²⁶³; C = K²⁶⁴–H²⁷³; D = R²⁷⁴–S³⁰⁵; E = S³⁰⁶–Q³⁴²; F = S³⁴³–D³⁵³. Hatched red boxes in exon 4 indicate genomic sequences that encode for the extended C-tail but for which transcripts have so far not been detected. (B) Schematic protein sequence alignment of human (h), monkey (mk), mouse (m), rat (r), guinea pig (gp), Siberian hamster (sh) and zebra fish (zf) H₃R isoforms identified so far with exons and different segments (A–F) colored according to the DNA coding sequencing. The snake plot on the background indicates the structural domains of the 7TM GPCRs, with the N- and C-terminal tail, transmembrane (TM) domains, and intracellular loop 3 (ICL3) indicated. The H₃R isoforms that conserve the prototypical 7TM GPCR folding are indicated in black with reference H₃R-445 orthologs in bold, whereas isoforms that do not conserve this 7TM folding due to sequence deletions elsewhere in the protein and/or alternative sequences are depicted in grey and italics.

Figure 2

H₃R isoform expression in the CNS of a human, mouse, and rat. Isoform transcripts in the CNS detected by RNA sequencing and extracted from publicly available Adult GTEx [21], Splice-O-Mat [22], and HPA databases [44], in situ hybridization [54] or RT-PCR [13,20,48,49], and protein level by [¹²⁵I]iodoproxyfan binding [54]. TPM (transcripts per million) represents the relative transcript abundant. Splice-O-Mat: https://tools.hornlab.org/Splice-O-Mat/ (accession date: 23 April 2024); Adult GTEx: https://gtexportal.org/home/transcriptPage (version 8, accession date: 23 April 2024); HPA (mouse): https://www.proteinatlas.org/about/download (version 23.0, accession date: 23 April 2024).

Figure 3

H₃R-mediated intracellular signaling pathway and its regulation. H₃R activation triggers signaling cascades via Gα_i/o or Gβγ subunits of heterotrimeric G_i/o protein to mediate intracellular response. Recruitment of β-arrestin1/2 to the GRK-phosphorylated H₃R prevents further G protein coupling and directs the receptor towards internalization to prevent overstimulation.

Figure 4

hH₃R-365 has a shorter TM6 and ICL3 than hH₃R-445. Comparison between cryo-EM structure of (A) hH₃R-445 in complex with histamine (depicted as space filling molecule in light pink) (PDB: 8YUU) and predicted structures of (B) hH₃R-365 (Q8WY01) by AlphaFold (https://alphafold.ebi.ac.uk; accessed on 5 April 2024) show the shorter ICL3 and TM6 in hH₃R-365. The arginine at position 6.31 × 31 is present in both hH₃R-445 (R³⁵⁴) and hH₃R-365 (* indicated as R³⁵⁴ according to its number in the reference isoform 445) is depicted in green, whereas aspartate at position 6.30 × 30 in hH₃R-445 is depicted in red. The 80-amino-acid deletion of segment DEF in H₃R-365 places histidine 273 (indicated in magenta) at the end of segment C next to arginine at position 6.31 × 31. The AlphaFold confidence scores are indicated in blue (very high predicted local distance difference test (pLDDT) > 90), cyan (high 90 > pLDDT > 70), yellow (low > 70 > pLDDT > 50), and orange (very low pLDDT < 50) [82].

Figure 5

Presynaptic and postsynaptic H₃R. Depolarization during an action potential opens voltage-gated calcium channels in the presynaptic terminal of an axon. The Ca²⁺ influx subsequently triggers calcium/calmodulin-dependent kinase II (CaMKII) to stimulate histamine synthesis by phosphorylating histidine decarboxylase (HDC) and triggering histamine release in the synaptic cleft. Histamine activates postsynaptic H₁R, H₂R and H₃R, thereby modulating various neurological processes. Additionally, histamine activates presynaptic H₃ autoreceptors to inhibit histamine synthesis and release by promoting the closure of VGCC and reducing the phosphorylation of HDC. These negative feedback loops are indicated in blue.