Seldeslachts A et al. (2023), Histamine Receptors: Ex Vivo Functional Studies...

XB-ART-60495

Membranes (Basel) 2023 Dec 02;1312:. doi: 10.3390/membranes13120897.

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Histamine Receptors: Ex Vivo Functional Studies Enabling the Discovery of Hits and Pathways.

Seldeslachts A , Peigneur S , Tytgat J .

Abstract
Histamine receptors (HRs) are G-protein-coupled receptors involved in diverse responses triggered by histamine release during inflammation or by encounters with venomous creatures. Four histamine receptors (H1R-H4R) have been cloned and extensively characterized. These receptors are distributed throughout the body and their activation is associated with clinical manifestations such as urticaria (H1R), gastric acid stimulation (H2R), regulation of neurotransmitters in neuronal diseases (H3R), and immune responses (H4R). Despite significant homologous overlap between H3R and H4R, much remains unknown about their precise roles. Even though some drugs have been developed for H1R, H2R, and H3R, not a single H4R antagonist has been approved for clinical use. To enhance our understanding and advance innovative therapeutic targeting of H1R, H2R, H3R, and H4R, we established a robust ex vivo functional platform. This platform features the successful heterologous expression of H1R-H4R in Xenopus laevis oocytes, utilizing an electrophysiological readout. Our findings contribute to a deeper understanding of the function and pharmacological properties of the histamine receptors. Researchers can benefit from the utility of this platform when investigating the effects of histamine receptors and exploring potential therapeutic targets. In doing so, it broadens the horizon of drug discovery, offering new perspectives for therapeutic interventions.

PubMed ID: 38132901
PMC ID: PMC10744718
Article link: Membranes (Basel)
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: camp dtl kcnj12 kcnj3 rgs4 rho
GO keywords: histamine receptor activity

Article Images: [+] show captions

	Figure 1. The structure of G-protein coupled receptors (GPCR). (Left) Extracellular N-terminus, seven transmembrane helices with three extracellular loops, three intracellular loops, and an intracellular C-terminus. (Right) Structure of a GPCR (dark green) in complex with heterotrimeric Gα (purple), Gβ (pink), and Gγ (light green) (PDB: 7L0Q).
	Figure 2. Histamine receptor—mediated downstream signaling cascades. Upon GPCR activation by histamine, the heterotrimeric G protein will dissociate into two subunits: Gα and Gβγ. Gα initiates a distinct intracellular signaling cascade: Gαq/11, Gαs, Gαi/o, and Gα12/13. Gαq/11 stimulates PLC, which will eventually regulate Ca2+ signaling and PKC activity. The elevated Ca2+ levels also act on the endogenous calcium-activated chloride channels (CaCC). Gαs will activate AC, which will result in an accumulation of intracellular cAMP and activation of PKA. Gαi/o will inactivate AC and thus reduce the cAMP levels. Gα12/13 will participate in the regulation of the Rho GTPase signaling pathways (GEF = guanine nucleotide exchange factors). The Gβγ subunit can interact with an effector channel, the G protein-coupled inward rectifying potassium channels (GIRK). The activation of the GIRK channel due to the GPCR signaling will cause a flow of potassium ions (K+) out of the cell. RGS4 accelerates the intrinsic GTPase activity of G proteins, facilitating the exchange of GTP for GDP.
	Figure 3. Activation of histamine 1 receptor (H1R) by histamine. (A) No effect of 1 µM histamine on non-injected oocytes. (B) A representative current trace of H1R-injected oocytes was measured with a ramp protocol from −120 to +70 mV from a holding potential of −20 mV. Upon 1 µM histamine application (blue), a shift in the reversal potential (Erev) from −50 mV to −20 mV was visible. (C) A representative current trace of H1R injected oocytes treated with 100 µM niflumic acid. No appreciable current enhancement can be seen by 1 µM histamine for oocytes treated with 100 µM niflumic acid. All experiments were repeated at least three times (n ≥ 3).
	Figure 4. Evaluation of the observations and function of the bio-assay. The current is visible on the y-axis, the time is visible on the x-axis, and the holding potential is −90 mV. (A) A representative current trace showed no significant currents induced by the application of HK and 1 µM histamine + HK (blue) in the non-injected oocytes. (B,C) A representative current trace shows that K+ currents were induced by the HK solution and represents a ‘basal’ K+ current (IK, basal), indicating a receptor-independent GIRK channel activity. No significant currents were induced by the application of 1 µM histamine + HK (blue) in the GIRK1/2 injected oocytes and IRK1 injected oocytes.
	Figure 5. The activation of H2R, H3R, and H4R by histamine in the H2R/H3R/H4R–GIRK1/2-RGS4 coupling system expressed in oocytes. The current is visible on the y-axis and the time is visible on the x-axis. (A) Oocytes co-injected with cRNAs of H2R, GIRK1/2 channels, and RGS4 proteins. (B) Oocytes co-injected with cRNAs of H3R, GIRK1/2 channels and RGS4 proteins. (C) Oocytes co-injected with cRNAs of H4R, GIRK1/2 channels, and RGS4 proteins. IK, basal was observed by exchanging the normal physiological solution (ND96, 2 mM KCl) for a solution containing a high potassium concentration (HK, 96 mM). IK, histamine was observed by exchanging HK to 1 µM histamine (blue) + HK. (D) Time to washout of 1 µM histamine of H2R, H3R, and H4R in the H2R/H3R/H4R-GIRK1/2–RGS4 coupling system expressed in oocytes. The graph represents the washout time in seconds versus the specific histamine receptor subtype. Differences were considered significant if the p value was smaller or equal to 0.05. For the difference between H2R and H3R, the * p value was 0.017 and for the difference between H2R and H4R, the ** p value was 0.0030. All cells were voltage-clamped at −90 mV, and experiments were repeated at least three times (n ≥ 3).
	Figure 6. The effect of PTX and a specific H4R antagonist on H4R injected oocytes. The current is visible on the y-axis and the time is visible on the x-axis. (A) No alteration of the current was observed after the addition of 1 µM histamine (blue) to H4R co-injected GIRK1/2–RGS4 injected oocytes after PTX treatment. Upon penetration of the A-protomer of PTX in the oocytes, ADP-ribosylation occurred at the cysteine residue of Gαi/o, leading to the inactivation of Gαi/o. Consequently, the suppressive impact of Gαi/o on adenylate cyclase (AC) activity diminished, causing an elevation in intracellular cAMP levels. (B) A representative trace of H4R-GIRK1/2–RGS4 showed that the effect of 1 µM histamine (blue) was blocked by JNJ7777120 (pink). All cells were voltage-clamped at −90 mV and experiments were repeated at least three times (n ≥ 3).
	Figure 7. Activation–response curve of H1R, H2R, H3R, and H4R. The percentage of activation of histamine in H1R (blue), H2R (orange), H3R (green), or H4R (pink) was plotted against the logarithm of the different concentrations tested. The corresponding EC50 value for H1R, H2R, H3R, and H4R yielded 8.4 ± 3.1 µM, 2.1 ± 1.1 μM, 0.024 ± 0.0012 µM, and 0.013 ± 0.0011 µM histamine, respectively. The visualized error bars represent the standard error of the mean (S.E.M). All experiments were repeated at least three times (n ≥ 3).

References [+] :

Albrecht, Expression and function of histamine and its receptors in atopic dermatitis. 2015, Pubmed