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

Background and Purpose

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 (hH₃R_WT) and A280V mutant (hH₃R_A280V) receptors stably expressed in CHO-K1 cells.

Experimental Approach

The hH₃R_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.

Key Results

Both receptors were expressed at similar levels with no significant differences in their affinities for H₃ receptor ligands. Upon activation the hH₃R_WT was significantly more efficacious to inhibit forskolin-induced cAMP accumulation and to stimulate [³⁵S]-GTPγS binding, with no difference in pEC₅₀ estimates. The hH₃R_WT was also more efficacious to stimulate ERK1/2 phosphorylation, but this difference was not significant. The inverse agonist ciproxifan was more efficacious at hH₃R_WT to reduce [³⁵S]-GTPγS binding but, for both receptors, failed to enhance forskolin-induced cAMP accumulation.

Conclusions and Implications

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.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1

Introduction

In the mammalian brain histamine regulates several functions by acting at four different GPCRs, H₁–H₄ (Haas et al., 2008; receptor nomenclature follows Alexander et al., 2011). Histamine H₃ receptors are primarily expressed on nerve terminals, where they regulate the synthesis and release of histamine as well as the release of other neuroactive substances namely ACh, dopamine, noradrenaline, 5-HT, glutamate, GABA and substance P (Feuerstein, 2008). There is also evidence for post-synaptic H₃ receptors in the striatum, cerebral cortex and hippocampus (Pillot et al., 2002).

Histamine H₃ receptors couple to Gα_i/o proteins and thus trigger several signalling pathways that include inhibition of adenylyl cyclase, inhibition of voltage-operated Ca²⁺ channels, activation of phospholipase A₂ (PLA₂), modulation of the MAPK pathway and activation of the Akt/glycogen synthase kinase-3β axis (Bongers et al., 2007)

The originally cloned human H₃ (hH₃) receptors cDNA encoded a 445 amino acid protein (Lovenberg et al., 1999) and in 2002, Wiedemann et al. reported a variation in the genomic sequence of the H₃ receptors of a patient suffering from Shy–Drager syndrome, currently classified as multiple system atrophy with orthostatic hypotension (Jecmenica-Lukic et al., 2012). The identified variation in the hH₃ receptors was a cytosine to thymine transition at nucleotide position 839 with respect to the start codon, which results in an alanine to valine exchange at amino acid position 280 in the third intracellular loop (Wiedemann et al., 2002; see Supporting Information Figure S1). Further, a recent report by Millán-Guerrero et al. (2011) indicated that in a Mexican population (147 cases and 186 controls) the A280V mutation was associated with increased risk of migraine.

In this work we therefore set out to compare the pharmacological and signalling properties of wild-type (hH₃R_WT) and A280V mutant (hH₃R_A280V) receptors by stably expressing them in CHO-K1 cells. A preliminary account of this work has been presented in abstract form to the European Histamine Research Society (Flores-Clemente et al., 2012).

Methods

Generation of the A280V mutant hH₃ receptor

The hH₃R_A280V cDNA was created by overlapping extension PCR amplification essentially as described by Horton et al. (1993). Amplifications were carried out in 25 μL incubations containing 0.8 mM dNTPs, 0.4 μM each primer, 0.5 U Pfu DNA polymerase (Fermentas, Glen Burnie, MD, USA) and either 100 ng of hH₃R_WT for initial amplifications or gel purified fragments from the amplifications as templates. The mutated DNA fragment was reintroduced into hH₃R_WT backbone after digestion of unique restriction sites BstEII (position 405 relative to start codon) and NotI (vector polylinker). The sequence of the amplified fragment was verified both by restriction analysis and automated sequencing performed at FESI-UNAM., Mexico.

Cell culture and transfection

CHO-K1 cells were cultured in DMEM Nutrient F-12 (DMEM-F12) mix supplemented with 10% FBS and 1% antibiotic/antimycotic solution (Gibco Life Technologies, Grand Island, NY, USA). For stable transfection, CHO-K1 cells (70–80% confluence) were transfected with cDNA constructs using Lipofectamine 2000 (Invitrogen Life Technologies, Grand Island, NY, USA), selected in 600 μg·mL⁻¹ geneticin (G-418), tested for receptor expression by radioligand binding and maintained in the presence of 400 μg·mL⁻¹ geneticin.

N-α-(methyl-³H)-histamine [(³H)-NMHA] binding assays

Cells grown in plastic Petri dishes (100 mm diameter) were scrapped and homogenized in ice-cold hypotonic buffer (10 mM Tris-HCl, 1 mM EGTA, pH 7.4) and lysates centrifuged (42,000× g, 20 min at 4°C). Pellets were re-suspended in incubation buffer (50 mM Tris-HCl, 5 mM MgCl₂, pH 7.4) and binding determinations (20 μg protein aliquots) were carried out and analysed as described in detail elsewhere (Osorio-Espinoza et al., 2011). Protein contents were determined by the bicinchoninic acid assay (BCA; Pierce, Rockford, IL, USA), using BSA as standard.

Measurement of cAMP accumulation in intact cells

Cells, grown in 24-well plates, were incubated (37°C) in 250 μL Krebs–Ringer–HEPES (KRH) buffer (composition (mM): NaCl 113, NaHCO₃ 25, KCl 3, MgCl₂ 1, KH₂PO₄ 1, CaCl₂ 1.8, d-glucose 11, HEPES 20; pH 7.4 with NaOH) containing 1 mM IBMX. After 15 min, forskolin was added in a 10 μL volume and incubations continued for a further 15 min. Final forskolin concentrations were 10 or 3 μM for the test of agonists or inverse agonists, respectively, and where required H₃ receptor agonists were added 5 min before forskolin.

Incubations were terminated with 25 μL ice-cold 1 M HCl. After neutralization with 25 μL 1 M NaOH and 100 μL 1 M Tris-HCl (pH 7.4), endogenous cAMP was determined by a competition assay for which 50 μL samples were incubated in 125 μL of incubation buffer (composition (mM): 50 Tris-HCl, 100 NaCl, 5 EDTA, 5 mg·mL⁻¹ BSA, pH 7.0 at 4°C) containing the PKA regulatory subunit (0.5 UI per sample) and [³H]cAMP (10 nM). After 2.5 h at 4°C, incubations were terminated by filtration over GF/B filters pre-soaked in 0.3% polyethylenimine followed by three subsequent washes with 1 mL ice-cold deionized water. Retained radioactivity was determined by liquid scintillation and the amount of endogenous cAMP present in each sample was calculated by using a standard cAMP curve (10⁻¹²–10⁻⁵ M).

[³⁵S]-GTPγS binding assay

Assays were carried out essentially as described by Gardner et al. (1996). Briefly, cells were scraped and lysed in ice-cold hypotonic buffer (20 mM HEPES, 2 mM EDTA, pH 7.4) containing a protease inhibitor cocktail (1:100 v : v; Sigma Aldrich, Mexico City, Mexico), followed by centrifugation at 42,000× g (20 min, 4°C). The pellet (membranes) was re-suspended in 10 mL HEPES buffer (20 mM HEPES, 100 mM NaCl, pH 7.4) containing adenosine deaminase (10 UI·mL⁻¹) and incubated for 30 min at 30°C. Ice-cold HEPES buffer (20 mL) was added and the membrane suspension was centrifuged as described earlier. The resulting pellet was re-suspended and incubated (20 μg protein aliquots) in the absence and presence of drugs under test in 500 μL HEPES buffer containing 10 mM MgCl₂, 0.1 mM dithiothreitol, 10 μM GDP, 1 mg·mL⁻¹ BSA, 0.2 mM EDTA, 10 mg·mL⁻¹ saponin and 50 pM [³⁵S]-GTPγS. Non-specific binding was determined in the presence of 10 μM unlabelled GTPγS. After 30 min at 30°C, the reaction was terminated by rapid filtration through GF/B filters with four washes of 1 mL ice-cold HEPES buffer. Radioactivity bound to the filters was quantified by liquid scintillation counting.

Detection of H₃ receptor-induced phosphorylation of ERK1/2 (p44/p42-ERK) by Western blotting

Cells, incubated overnight in DMEM-F12 medium containing 0.1% FBS, were washed with KRH buffer and incubated at 37°C with the H₃ receptor agonist (R)-α-methylhistamine (RAMH). After rinsing twice with pre-warmed KRH solution and once with ice-cold solution, cells were scraped into lysis buffer [20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.1% Triton X-100, 10% deoxycholate, 10% SDS, 1% glycerol, 150 mM NaCl and 1% (v : v) protease inhibitor cocktail]. The samples were cleared by centrifugation and samples (30 μg protein) were resolved by SDS-PAGE (10% gel) and then transferred onto a PVDF membrane.

Blots were blocked (1 h at 22°C) with Tris-buffered saline containing Tween-20 (0.1% v : v; T-BST) and 5% non-fat dry milk, before incubation overnight at 4°C with primary antibodies (rabbit anti phospho-ERK1/2 or anti total-ERK1/2, Cell Signaling, Danvers, MA, USA; 1:1000 dilution in T-BST containing 1.5% BSA). After rinsing with T-BST, membranes were incubated for 1 h at 22°C with a secondary antibody (goat anti-rabbit IgG coupled to HRP; 1:5000 in T-BST/1.5% BSA). Membranes were rinsed with T-BST, developed with chemiluminescence (Enhanced ChemiLuminescent Substrate; Biorad, Hercules, CA, USA) and quantified by densitometry analysis with the Kodak Image Station 4000R (Eastman Kodak, Rochester, NY, USA) and ImageJ software (National Institutes of Health, USA). Target bands were expressed quantitatively by normalization to the intensity of the non-phosphorylated ERK1/2 signal on the same lane. Protein loading was also controlled by actin immunodetection.

Data analysis

Results are presented as the means ± s.e.m. of the indicated number of experiments. Statistical comparisons were performed with Student's unpaired t-test or one-way ANOVA followed by Dunnett's test, as appropriate (Prism 5.0 software; GraphPad Software, San Diego, CA, USA). Differences were considered significant at P < 0.05.

Materials

The following drugs and reagents were purchased from Sigma Aldrich: A-331440 (4′-[3-[(3(R)-dimethylamino-1-pyrrolidinyl]propoxy]-[1,1-biphenyl]-4′-carbonitrile dihydrochloride), adenosine deaminase (from bovine spleen), cAMP, ciproxifan hydrochloride, clobenpropit dihydrobromide, DTT, GDP, IBMX, geneticin, GTPγS, histamine dihydrochloride, PKA regulatory subunit (from bovine heart), (R)-α-methylhistamine dihydrochloride and saponin. N-α-[methyl-³H]-histamine (85.4 Ci·mmol⁻¹), [³⁵S]GTPγS (1250 Ci·mmol⁻¹) and [³H]-cAMP ([2,8-³H]-adenosine 3′,5′-cyclic phosphate, 34 Ci·mmol⁻¹) were from Perkin Elmer (Boston, MA, USA).

Results

Binding characteristics of hH₃ receptors stably expressed in CHO-K1 cells

Specific [³H]-NMHA binding to cell membranes, yielded maximum binding (B_max) 203 ± 34 and 176 ± 26 fmol·mg·protein⁻¹ (mean ± SEM; four experiments, Figure 1A) for hH₃R_WT and hH₃R_A280V, respectively, with no statistical difference (P = 0.25, Student's t-test). Estimates for the equilibrium dissociation constant (K_d) were 0.86 ± 0.23 and 0.83 ± 0.24 nM respectively (P = 0.46).

Figure 1.

Binding of N-α-[methyl-³H]histamine ([³H]-NMHA) to membranes from CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells. (A) Saturation binding. Cell membranes were incubated with the indicated concentrations of [³H]-NMHA and specific receptor binding was determined by subtracting the binding in the presence of 10 μM histamine from total binding. Points are means ± SEM from triplicate determinations from a single experiment, which was repeated a further three times. The line drawn is the best fit to a hyperbola. Best-fit values for the equilibrium dissociation constant (K_d) and maximum binding (B_max) are given in the text. (B,C) Inhibition by H₃ receptor ligands. Membranes from CHO-K1-hH₃R_WT (B) or CHO-K1-hH₃R_A280V cells (C) were incubated with 1.5 nM [³H]-NMHA and the indicated drug concentrations. Values are expressed as percentage of control specific binding and are averages ± ranges of duplicates from a single experiment, repeated a further two to four times with similar results. The line drawn is the best fit to a logistic equation for a one-site model. pK_i values calculated from the best-fit IC₅₀ estimates are compared in Table 1.

For both receptors, a set of agonists (histamine, imetit, immepip and RAMH) and antagonists/inverse agonists (A-331440, ciproxifan, clobenpropit and thioperamide) inhibited [³H]-NMHA binding in a concentration-dependent manner (Figure 1B,C). Estimates for the inhibition constants (-log Ki, pKi; Cheng and Prusoff, 1973) are shown in Table 1. There was no significant difference for any of the ligands tested between the native (wild type) and mutant receptors. Further, the affinities of both receptors were in good accord with values previously reported for the expression of cloned hH₃ receptors in CHO-K1 cells (Ligneau et al., 2000; Cogé et al., 2001; Table 1).

Table 1.

Affinities of hH₃R_WT or hH₃R_A280V for selective ligands

	pKi values
		This study	Ligneau et al., 2000	Cogé et al., 2001
	hH3RWT	hH3RA280V
Agonists
Histamine	8.30 ± 0.10	8.38 ± 0.12 (P = 0.623, n = 4)	7.89	8.24 ± 0.01
Immepip	9.51 ± 0.03	9.58 ± 0.01 (P = 0.091, n = 3)	ND	9.60 ± 0.15
Imetit	9.23 ± 0.19	9.29 ± 0.05 (P = 0.775, n = 3)	ND	9.37 ± 0.22
RAMH	8.92 ± 0.11	9.05 ± 0.10 (P = 0.431, n = 3)	8.56	8.73 ± 0.09
Antagonists/inverse agonists
A-331440	7.75 ± 0.04	7.77 ± 0.08 (P = 0.834, n = 3)	ND	ND
Ciproxifan	7.17 ± 0.04	7.21 ± 0.10 (P = 0.729, n = 3)	7.33	7.26 ± 0.02
Clobenpropit	8.79 ± 0.08	8.64 ± 0.12 (P = 0.329, n = 5)	8.62	9.27 ± 0.05
Thioperamide	7.34 ± 0.13	7.37 ± 0.05 (P = 0.840, n = 3)	7.22	7.70 ± 0.07

Values are pK_i estimates (-Log₁₀ of the inhibition constant, K_i) and correspond to means ± SEM from the indicated number of determinations. Figures in parentheses are P values (Student's t-test) for the comparison between hH₃R_WT and hH₃R_A280V cells. Determinations reported in Ligneau et al. (2000) and Cogé et al. (2001) were performed with the hH₃ receptor stably expressed in CHO-K1 cells. Values from Ligneau et al. (2000) were transformed to pK_i estimates from the K_i values provided. ND, not determined.

Agonist-induced inhibition of forskolin-induced cAMP accumulation

Forskolin-stimulated cAMP accumulation in a similar manner for CHO-K1-hH₃R_WT and CHO-K1-hH₃R_A280V cells (Supporting Information Figure S2), and the effect of 10 μM of forskolin was significantly reduced by the H₃ receptor agonist RAMH (Figure 2A). However, maximum inhibition was significantly lower in cells expressing the mutant receptor (Table 2). The estimates for half-maximal effective concentrations (pEC₅₀) showed a tendency to a lower potency for CHO-K1-hH₃R_A280V cells, but the difference was not statistically significant, as shown in Table 2.

Figure 2.

Effect of H₃ receptor activation on forskolin-induced cAMP accumulation and [³⁵S]-GTPγS binding. (A) Forskolin-induced cAMP accumulation. CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells were pre-incubated (15 min) with 1 mM IBMX and then exposed for 15 min to forskolin (10 μM). Where required the H₃ receptor agonist RAMH was added 5 min before forskolin. Values are expressed as percentage of the response to forskolin, after basal subtraction, and correspond to the means ± SEM from four replicates from a representative experiment, repeated a further four times with similar results. The line drawn is the best fit to a logistic equation. Estimates for maximum effect (E_max) and pEC₅₀ are compared in Table 2. Raw data from a representative experiment are presented in Supporting Information Figure S2. (B). [³⁵S]-GTPγS binding. Membranes from CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells were incubated (30 min) with 50 pM [³⁵S]-GTPγS in the presence and absence of the indicated concentrations of RAMH. Values are expressed as percentage of basal [³⁵S]-GTPγS binding after subtraction of non-specific binding and correspond to the means ± SEM from three replicates from a representative experiment, repeated a further seven times with similar results. The line drawn is the best fit to a logistic equation. Estimates for maximum stimulation and pEC₅₀ are compared in Table 2. Basal [³⁵S]-GTPγS binding was 4993 ± 226 and 5179 ± 43 dpm for membranes from CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells, respectively. Raw data from a representative experiment are presented in Supporting Information Figure S3.

Table 2.

Effect of receptor activation on forskolin-induced cAMP accumulation and [³⁵S]-GTPγS binding

	hH3RWT	hH3RA280V	P
cAMP accumulation
Emax (%)	−93.9 ± 2.6	−57.2 ± 8.4	0.003
pEC50	8.22 ± 0.35	7.75 ± 0.05	0.224
[35S]-GTPγS binding
Emax (% basal)	129.2 ± 2.6	112.6 ± 2.7	<0.001
pEC50	8.50 ± 0.14	9.00 ± 0.32	0.174
ERK1/2 phosphorylation
Emax (% basal)	302 ± 63	217 ± 31	0.155
pEC50	7.94 ± 0.17	7.49 ± 0.19	0.128

Values are means ± SEM from five (cAMP accumulation), eight ([³⁵S]-GTPγS binding) or four (ERK1/2 phosphorylation) experiments. Statistical comparisons were carried out with Student's t-test.

Agonist-induced [³⁵S]-GTPγS binding

The ability of GPCRs to activate G-proteins is also studied by determining [³⁵S]-GTPγS binding to cell membranes. Maximum stimulation evoked by the agonist RAMH was significantly higher in membranes from CHO-K1-hH₃R_WT cells (Figure 2B and Table 2, and Supporting Information Figure S3A), with no significant difference in the pEC₅₀ estimates.

Agonist-induced ERK1/2 phosphorylation

Activation of transfected H₃ receptors leads to ERK1/2 phosphorylation (Bongers et al., 2007), and in both CHO-K1-hH₃R_WT and CHOK1-hH₃R_A280V cells, incubation (1–15 min) with the agonist RAMH resulted in increased ERK1/2 phosphorylation over basal levels that reached a peak between 2.5 and 5 min to decay steadily afterwards (Supporting Information Figure S4A). At 5 min incubations, the effect was concentration-dependent (Supporting Information Figure S4B), with a tendency for higher efficacy in CHOK1-hH₃R_WT cells, although the effect was not significant (Table 2).

Effect of inverse agonists on [³⁵S]-GTPγS binding and forskolin-induced cAMP accumulation

Native (wild type) and cloned H₃ receptors may exhibit spontaneous or constitutive activity (Arrang et al., 2007), and for hH₃ receptors expressed in CHO cells such an activity is apparent in cells expressing 250–400 fmol mg⁻¹ protein of the hH₃ receptor (Rouleau et al., 2002). Because the reduced efficacy of hH₃R_A280V observed in our experiments may rely on higher intrinsic activity, the effect of antagonists/inverse agonists was tested in functional assays.

For both native and mutant receptors, the H₃ receptor antagonist/inverse agonist ciproxifan (Leurs et al., 2005) reduced [³⁵S]-GTPγS binding to cell membranes. The effect was modest (Figures 3A and Supporting Information Figure S3B), but significantly higher for membranes from CHO-K1-hH₃R_WT cells (−12.03 ± 0.29 vs. −5.85 ± 0.60% of basal for CHOK1-hH₃R_A280V membranes; P < 0.001, n = 4, Student's t-test), without a significant effect in agonist potency (pEC₅₀ values 9.06 ± 0.45 and 9.09 ± 0.73, respectively; P = 0.964). The errors associated to pEC₅₀ values are large, most likely due to the small window for the ciproxifan effect which hampers the estimate accuracy.

Figure 3.

Effect of the H₃ receptor inverse agonists on [³⁵S]-GTPγS binding and forskolin-induced cAMP accumulation. (A) [³⁵S]-GTPγS binding. Membranes from CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells were incubated (30 min) with 50 pM [³⁵S]-GTPγS in the presence and absence of the indicated concentrations of ciproxifan. Values are expressed as percentage of basal [³⁵S]-GTPγS binding after subtraction of non-specific binding and correspond to the means ± SEM from three replicates from a representative experiment, repeated a further three times with similar results. The line drawn is the best fit to a logistic equation. Best-fit values for maximum effect (E_max) and pEC₅₀ are given in the text. Basal [³⁵S]-GTPγS binding was 3527 ± 54 and 4487 ± 77 dpm for membranes from CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells respectively. Raw data from a representative experiment are presented in Supporting Information Figure S3. (B,C) Forskolin-induced cAMP accumulation. CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells were pre-incubated (15 min) with 1 mM IBMX and then exposed for 15 min to forskolin (3 μM). Where required, H₃ receptor inverse agonists (0.1 and 1 μM) were added 5 min before forskolin. Values are expressed as percentage of the response to forskolin, after basal subtraction, and correspond to the means ± SEM from three or four experiments for 0.1 or 1 μM of the inverse agonists, respectively. None of the values in the presence of H₃ receptor inverse agonists was significantly different from control values (P > 0.05, one-way ANOVA and post hoc Dunnett's test).

Forskolin-stimulated cAMP accumulation increased in a concentration-dependent manner (Supporting Information Figure S2), but the slope was significantly higher between 1 and 10 μM than between 10 and 30 μM (2.05 ± 0.21 and 0.53 ± 0.13, respectively; P < 0.001, Student's t-test). For testing receptor constitutive activity, the forskolin concentration was therefore set at 3 μM in order to reliably assess the possible enhancing action of inverse agonists.

In contrast to [³⁵S]-GTPγS binding to cell membranes, for both CHO-K1-hH₃R_WT and CHOK1-hH₃R_A280V cells ciproxifan had no significant effect on forskolin-induced cAMP accumulation over a wide range of concentrations (10⁻¹¹–10⁻⁶ M, three experiments; data not shown). Other H₃ receptor inverse agonists (clobenpropit, thioperamide and A-331440) also failed to modify cAMP accumulation at concentrations of 0.1 and 1 μM, as shown in Figure 3C (panels B and C).

Discussion and conclusions

The main finding of this work is that the A280V mutation on the hH₃ receptor modifies its signalling efficacy without affecting the binding characteristics.

Effect of the A280V mutation on H₃ receptor binding and functional characteristics

Binding determinations indicated that the A280V mutation did not affect hH₃ receptor expression or its affinity for a series of selective ligands. The latter result was not unexpected because the mutation is not located on any of the receptor regions primarily involved in ligand binding, namely trans-membrane domains 3, 5 and 6 (Uveges et al., 2002; Ishikawa et al., 2010; Kim et al., 2011). In contrast, the mutant receptor was less efficacious to inhibit forskolin-stimulated cAMP accumulation and to stimulate [³⁵S]-GTPγS binding, indicating that the mutation did affect the signalling properties of the receptor.

There was also a trend for lower efficacy for the mutant receptor for the stimulation of ERK1/2 phosphorylation, but the effect did not reach statistical significance. Receptor-induced ERK1/2 phosphorylation can be due either to Gβγ subunits or β-arrestins bound to activated receptors acting as a signalling scaffold (Gutkind, 2000; DeWire et al., 2007), but the fast response induced by hH₃R_WT and hH₃R_A280V indicates that the effect is more likely to rely on βγ complexes than on β-arrestin recruitment because the latter is a delayed response with a peak at 30 min incubations (Rosethorne and Charlton, 2011). The lack of difference in the efficacy of hH₃R_WT and hH₃R_A280V for the stimulation of ERK1/2 phosphorylation may reflect the distinct sensitivity of the pathways to the signalling molecules activated upon receptor stimulation.

Native and cloned H₃ receptors show constitutive activity (Arrang et al., 2007), and in CHO cells spontaneous activity of the hH₃ receptor, evaluated by ciproxifan-induced inhibition of basal [³⁵S]-GTPγS binding is apparent from a density of 250 fmol receptor per mg protein (Rouleau et al., 2002). Our [³⁵S]-GTPγS binding data indicate that both receptors do have constitutive activity when expressed in CHO-K1 cells, although to a modest extent. However, H₃ receptor inverse agonists failed to enhance forskolin-induced cAMP accumulation. This discrepancy may rely on the activated G proteins detected by [³⁵S]-GTPγS binding not yielding the concentration required for a significant inhibitory effect upon adenylyl cyclases stimulated by forskolin.

There is no direct evidence from this study as to the molecular mechanism responsible for the effect of the A280V mutation on H₃ receptor function, although the change in agonist efficacy but not in potency suggests that the mutation is hampering the receptor ability to activate G-proteins. The A280V mutation is located in the receptor third intracellular loop (Figure S1) which plays a key role in determining receptor/G-protein coupling (Wess, 1998). Activated receptors catalyse GDP release from G-protein heterotrimers and this is the rate-limiting step in G-protein activation and, consequently, the activation of downstream signalling proteins (Oldham and Hamm, 2007). Because both alanine and valine are neutral amino acids, the A280V mutation confers no change in charge, but in size (71.1 and 99.1 daltons, and 0.067 and 0.105 nm³, respectively; Zamyatnin, 1972), and one possible explanation for the reduced efficacy of the mutant receptor is a steric reduction in the access of the C-terminus of the Gα_i/o protein to sites in the cytoplasmic face of the receptor trans-membrane helices required for G-protein stimulation (Oldham and Hamm, 2007).

Possible implications of the A280V mutation for the pathophysiology of the Shy–Drager syndrome and migraine

This study originated from reports that the A280V mutation may be involved in the pathophysiology of two neurological disorders, the Shy–Drager syndrome (Wiedemann et al., 2002) and migraine (Millán-Guerrero et al., 2011).

The Shy–Drager syndrome is now recognized as a form of multiple system atrophy, a sporadic and progressive neurological disease characterized by autonomic dysfunction, Parkinsonism and ataxia in any combination (Jecmenica-Lukic et al., 2012). In the patient in whom the A280V mutation was first identified, a reduction in both noradrenaline plasma level and urinary excretion of the amine and its metabolites was found, and a link between the H₃ receptor mutation and altered noradrenaline release was suggested (Wiedemann et al., 2002). Because H₃ receptor activation reduces depolarization-evoked noradrenaline release from sympathetic nerve terminals (Silver et al., 2002), the decrease in noradrenaline plasma levels cannot be directly explained by the reduced efficacy of the mutant receptor reported herein, which would instead lead to increased noradrenaline release from peripheral neurones, in analogy with the enhanced release from sympathetic nerve endings observed in mice lacking H₃ receptors (Koyama et al., 2003). Further, orthostatic hypotension has been mainly related to dysfunction of the catecholaminergic rostral ventrolateral medulla nuclei (Colosimo, 2011), and one possibility to explain the alterations in arterial pressure is that the reduced signalling efficacy of mutant H₃ auto-receptors enhances histamine release resulting in augmented activation of H₃ hetero-receptors located on central noradrenergic/adrenergic neurones (Pillot et al., 2002) and thus in inhibition of neuronal firing. Alternatively, reduced receptor efficacy might lead to diminished H₃ receptor-mediated inhibition of neurotransmitter release and increased auto-receptor activation in cathecolaminergic neurones resulting in decreased neuronal activity. In turn, reduced activity of the rostral ventrolateral medulla nuclei would result in decreased sympathetic vasomotor tone (Guyenet, 2006).

Migraine is a neurovascular disorder involving activation of the trigemino-vascular system with the primary dysfunction located in brainstem centres regulating vascular tone and pain sensation (Cutrer, 2010). Its frequency is significantly higher in patients with allergic (histamine-driven) disease (Ku et al., 2006; Aamodt et al., 2007) and plasma histamine levels are significantly elevated in migraine patients (Heatley et al., 1982). Recently, Millán-Guerrero et al. (2011) reported that the A280V mutation in the hH₃ receptor was significantly associated with increased risk of migraine with V allele frequencies of 6.46% and 2.68% for cases and controls, respectively (odds ratio 2.67), and frequencies of the V/V and V/A genotypes 12.92% and 3.22% in migraine patients and controls (odds ratio 4.45). This group also reported that S.C. injections of low doses of histamine or the H₃ receptor agonist NAMH (twice a week over 12 weeks) reduced pain intensity, frequency and length of attacks as well as painkiller use in migraine patients, relating these actions to the activation of H₃ receptors controlling the release of histamine and other neurotransmitters (Millán-Guerrero et al., 2006; 2009).

Alterations in the release of glutamate and neuroactive peptides have also been linked to migraine. Higher levels of glutamate have been found in plasma and in saliva samples in migraine patients when compared with healthy controls (Tajti et al., 2011), and trigeminal nociceptive fibres release the vasoactive neuropeptides, substance P, CGRP and neurokinin A (Cutrer, 2010). Pre-synaptic H₃ receptors reduce substance P and neurokinin release from sensory nerve endings as well as glutamate release in several areas of the rat brain (see Feuerstein, 2008 and Osorio-Espinoza et al., 2010). Thus, the reduced efficacy of the mutant hH₃ receptor reported herein may be also related to a diminished inhibitory action of endogenous histamine on the release of glutamate and neuroactive peptides, leading to increased susceptibility to migraine attacks.

In conclusion, we have here provided evidence that a single point mutation in the third intracellular loop of the human histamine H₃ receptor modifies its functional properties. This effect may have relevance for the pathophysiology of disorders associated with the mutation, namely migraine and multiple system atrophy with orthostatic hypotension, but further research on this issue is clearly required.

Glossary

NHMA

N-α-methyl histamine

RAMH

(R)-α-methyl histamine

Conflicts of interest

The authors disclose no conflict of interest.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

Figure S1 Location of the A280V mutation in the human H₃R₄₄₅. A cytosine to thymine transition at nucleotide position 839 with respect to the start codon results in an alanine to valine exchange at amino acid position 280 in the third intracellular loop (red circle).

Figure S2 Characteristics of forskolin-induced cAMP accumulation and hH₃ receptor-mediated inhibition. In all determinations cells were pre-incubated (15 min) with 1 mM IBMX and then exposed for 15 min to forskolin. (A,B) Concentration-response curve in CHO-K1 cells. (A) Representative experiment. Values are means ± SEM from four replicates. (B) Analysis of the concentration–response curve from four experiments. Values are expressed as pmol cAMP per sample after subtraction of basal accumulation, and correspond to the means ± SEM. Lines are data best-fit by linear regression analysis. (C,D) Representative determinations of H₃ receptor-mediated inhibition of forskolin-induced cAMP accumulation in CHO-K1-hH₃R_WT (C) or CHO-K1-hH₃R_A280V (D) cells. Cells were incubated for 15 min with forskolin (10 μM) in the absence and presence of the H₃ receptor agonist RAMH, added 5 min before forskolin. Values are means ± SEM from three to four replicates from representative determinations. The experiments were repeated a further 4 times with similar results.

Figure S3 Representative determinations for [³⁵S]-GTPγS binding. Membranes from CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells were incubated (30 min) with 50 pM [³⁵S]-GTPγS in the presence and absence of the indicated drug concentrations. (A) Stimulation by the agonist RAMH. (B) Inhibition by the inverse agonist ciproxifan. For both panels values are means ± SEM from four replicates from a representative experiment after subtraction of non-specific binding. The line drawn is the best fit to a logistic equation. The experiment was repeated a further four times with similar results.

Figure S4 Effect of H₃ receptor activation on ERK1/2 phosphorylation. ERK1/2 phosphorylation was analysed by Western blot. (A) Time course. CHO-K1-hH₃R_WT or CHO-K1-hH₃R_A280V cells were incubated for 1–15 min with the agonist RAMH (100 nM). A1) Representative blot. Similar results were obtained in two independent determinations for each cell line. P-ERK1/2, phosphorylated ERK1/2. A2) Densitometric analysis. Western blot target bands were quantified by densitometry analysis and normalized according to the intensity of the non-phosphorylated ERK1/2 signal on the same lane. Values are expressed as percentage of basal phosphorylation and correspond to the means from two determinations. (B) Concentration-response curves. Cells were incubated for 5 min with the indicated concentrations of the agonist RAMH. B1) Representative blot. Similar results were obtained in three other independent experiments. B2) Analysis from four determinations. Values (means ± SEM) are expressed as percentage of basal phosphorylation. The lines drawn are the best fit to a logistic equation. Estimates for maximum stimulation and EC₅₀ are given in the text.