Thiel U et al. (2015), Identification of amino acids involved in hista...

XB-ART-50850

Front Pharmacol 2015 May 26;6:106. doi: 10.3389/fphar.2015.00106.

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Identification of amino acids involved in histamine potentiation of GABA A receptors.

Thiel U , Platt SJ , Wolf S , Hatt H , Gisselmann G .

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Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of GABAA receptors (GABAARs) and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABAARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABAARs. We expressed GABAARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues β2(N265) and β2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues α1(R120), β2(Y157), β2(D163), β3(V175), and β3(Q185). We showed that the amino acid residues β2(Y157) and β3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas α1(R120) and β2(D163) form a potential histamine interaction site in GABAARs.

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Species referenced: Xenopus laevis
Genes referenced: gabarap

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	FIGURE 1. GABA potentiated by histamine in wt receptors. GABA-induced (EC10-30) currents are potentiated by 3 mM histamine. (A–C) Xenopus oocytes expressing the abg isoforms: α1β2γ2L, α1β3γ2L, and α2β3γ2L were voltage clamped, and GABA at a concentration of 10 μM was bath-applied with or without 3 mM histamine. (D) Average potentiation of GABA-induced currents calculated from measurements as shown in (A–C). Bars two to four show the mean enhancement ± SEM, as a percentage of the amplitude of GABA alone (gray line), for the αβγ isoforms: α1β2γ2L, α1β3γ2L, and α2β3γ2L, (n = 9–12). Significant data were marked with ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001.
	FIGURE 2. Effect of 3 mM histamine on the GABA-induced current of wt or mutated GABAARs. The bars show the increase of the GABA-induced (typically EC10-30) currents by 3 mM histamine of the GABAAR subunit combination (A) α1β2γ2L and (B) α2β3γ2L. Significant data were marked with ∗p ≤ 0.05 and ∗∗p ≤ 0.01.
	FIGURE 3. Histamine action on GABA-induced currents of GABAARs with the point mutated β2(Y157F) subunit. (A,D) Original traces of the histamine (3 mM) action on the GABA-induced currents of the GABAARs α1β2(Y157F)γ2L and α1β2(Y157F). (B,C,E,F) Bar diagrams show the action of various histamine concentrations on the GABA-induced currents of the GABAARs α1β2(Y157F)γ2L, α1β2(Y157F), α1β2γ2L, and α1β2. Significant data were marked with ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001.
	FIGURE 4. Histamine action on GABA-induced currents of GABAARs with point mutated subunits α1(R120A) and β2(D163A). (A–C) Original traces of the modulatory effect of 3 mM histamine on GABA-induced currents from GABAARs α1(R120A)β2γ2L, α1β2(D163A)γ2L, and α1(R120A)β2(D163A)γ2L. (D–F) Bar diagrams show the effect of 0.1 to 10 mM histamine on the GABA-induced (typically EC10-30) current. Significant data were marked with ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001.
	FIGURE 5. Histamine action on the GABA-induced currents of GABAARs with point mutated β3(V175A). (A) Original trace of the effect of 3 mM histamine on the GABA-induced currents of the GABAAR α2β3(V175A)γ2L. (B) Bar diagram show the effect of various histamine concentrations up to 10 mM on the GABA-induced currents of the GABAAR α2β3(V175A)γ2L. Significant data were marked with ∗∗p ≤ 0.01.
	FIGURE 6. Histamine action on the GABA-induced currents of GABAARs with the point mutated β3(Q185A) subunit. (A) Original trace of the histamine (3 mM) action on the GABA-induced (EC11) currents of the GABAAR α2β3(Q185A)γ2L. (B,C) Bar diagrams that show the action of different histamine concentrations on the GABA-induced currents of the GABAARs α2β3(Q185A)γ2L and α2β3γ2L. Significant data were marked with ∗p ≤ 0.05 and ∗∗p ≤ 0.01.
	FIGURE 7. Overview of positions and functions of mutated residues. Structural model based on PDB ID 4COF (Miller and Aricescu, 2014). Backbone displayed as cartoon, van der Waals surface as surface, selected residues as sticks, or balls and sticks, respectively. Extracellular domains visible as sheets, transmembrane domains as helical domains. Putative binding positions of small organic molecules according to TRIDOCK analysis (te Heesen et al., 2007) as orange spheres and histamine in magenta. Y157 is actively taking part in formation of the interface between the two subdomains. D163(β) and R120(α) form a putative histamine binding site at the interface between subdomains α and β. Q185 at the interface between transmembrane and extracellular domains is part of a hydrogen bonding network with the highly conserved residues E52 and R216. The V175 side chain is buried within the extracellular subdomains, and thus important for their correct folding. None of the investigated residues except D163(β) and R120(α) show a putative small molecule binding site.

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