Adrenoceptor activity of muscarinic toxins identified from mamba venoms

Abstract

BACKGROUND AND PURPOSE

Muscarinic toxins (MTs) are snake venom peptides named for their ability to interfere with ligand binding to muscarinic acetylcholine receptors (mAChRs). Recent data infer that these toxins may have other G-protein-coupled receptor targets than the mAChRs. The purpose of this study was to systematically investigate the interactions of MTs with the adrenoceptor family members.

EXPERIMENTAL APPROACH

We studied the interaction of four common MTs, MT1, MT3, MT7 and MTα, with cloned receptors expressed in insect cells by radioligand binding. Toxins showing modest to high-affinity interactions with adrenoceptors were additionally tested for effects on functional receptor responses by way of inhibition of agonist-induced Ca²⁺ increases.

KEY RESULTS

All MTs behaved non-competitively in radioligand displacement binding. MT1 displayed higher binding affinity for the human α_2B-adrenoceptor (IC₅₀ = 2.3 nM) as compared with muscarinic receptors (IC₅₀≥ 100 nM). MT3 appeared to have a broad spectrum of targets showing high-affinity binding (IC₅₀ = 1–10 nM) to M₄ mAChR, α_1A-, α_1D- and α_2A-adrenoceptors and lower affinity binding (IC₅₀≥ 25 nM) to α_1B- and α_2C-adrenoceptors and M₁ mAChR. MT7 did not detectably bind to other receptors than M₁, and MTα was specific for the α_2B-adrenoceptor. None of the toxins showed effects on β₁- or β₂-adrenoceptors.

CONCLUSIONS AND IMPLICATIONS

Some of the MTs previously found to interact predominantly with mAChRs were shown to bind with high affinity to selected adrenoceptor subtypes. This renders these peptide toxins useful for engineering selective ligands to target various adrenoceptors.

Introduction

G-protein-coupled receptors (GPCRs) constitute one of the largest known gene superfamilies with up to 750 members in humans (Vassilatis et al., 2003). Because of this receptor diversity and the vast amount of physiological processes these receptors control or modulate, they are excellent targets for therapeutic interventions. Amongst the GPCRs, many families or subfamilies of receptors activated by the same endogenous substance exist; for example the classical neurotransmitter acetylcholine activates all five subtypes of muscarinic acetylcholine receptors (mAChRs) with approximately equal potency (Caulfield and Birdsall, 1998). A classical enigma has been how to pharmacologically and therapeutically differentiate between such closely related subtypes within certain subfamilies of GPCRs, that is subtype-selective exogenous ligands have been very difficult to find from natural sources, or to synthesize chemically.

Many animals such as snakes, scorpions and marine snails have a diverse array of peptides in their venoms. These peptides have probably evolved to capture and immobilize preys or to be used in defense. Thus, many of the toxins interact with voltage-gated ion channels (Bosmans and Tytgat, 2007; Catterall et al., 2007) and ligand-gated ion channels (Nirthanan and Gwee, 2004), ensuring a rapid paralysing action when used to envenom victims. Regarding toxins acting on GPCRs, the actual benefit of these for the animal is more difficult to predict. However, as shown on several occasions with ion channel-specific toxins, GPCR toxins may provide us with highly receptor subtype-selective tools and drug candidates. Amongst the first GPCR-acting toxins to be found were the muscarinic toxins (MTs) from the Dendroaspis genus of snakes (Adem et al., 1988). The first two isolated toxins, MT1 and MT2, displaced radiolabelled quinuclidinyl benzilate from muscarinic receptors expressed in rat cortex in a way that suggested these toxins hold potential as subtype-selective ligands. Later studies showed that the toxins selectively targeted M₁ and M₄ of the mAChR family (Kornisiuk et al., 1995a, b). The subtype-selectivity increased interest in these peptides and soon afterwards additional toxins MT3–MT7 were isolated from Dendroaspis angusticeps and MTα, MTβ, MTγ from Dendroaspis polylepis. All MTs are 65–66 amino acids long, and they all probably adopt a similar structure as α-neurotoxins with three loops or fingers held together by internal disulphide bonds (Ségalas et al., 1995; Fruchart-Gaillard et al., 2008).

MT3 and MT6, a probable isotoxin of MT3 for which the primary sequence has not been determined, show about 100-fold selectivity for M₄ receptors over M₁ receptors (Jolkkonen et al., 1994; Karlsson et al., 2000). MT7 (also called m1-toxin) appears to have absolute selectivity for the M₁ subtype with affinity values in the pico- to nanomolar range and lack of detectable binding to M₂–M₅ subtypes (Max et al., 1993a; Näsman et al., 2000; Mourier et al., 2003). It is not known precisely how this selectivity is obtained at the structural level. Mutagenesis studies of MT7 suggest that all three fingers of the toxin are involved in the binding to the receptor (Fruchart-Gaillard et al., 2008). Looking from the opposite side, the outer loops of the receptor, especially the second loop connecting transmembrane helices four and five, are important for toxin binding (Kukkonen et al., 2004). The outer loops are usually less well conserved between subtypes of receptors in the rhodopsin-like GPCRs and may represent the structural elements required for high subtype selectivity of exogenous ligands.

Apart from their unusually high selectivity among muscarinic subtypes, the true specificity of MTs for mAChRs has occasionally been questioned (Bradley, 2000). In a study using MT1 and MT2 to show functional effects in different tissue preparations, Harvey et al. (2002) found that these toxins also interfered with adrenoceptor ligand binding. In a search for novel therapeutic agents for adrenoceptors, two new peptide toxins were recently found from D. angusticeps venom that showed high-affinity binding to α-adrenoceptors (Quinton et al., 2010; Rouget et al., 2010). The toxin ρ-Da1a appeared selective for the α_1A-adrenoceptor and ρ-Da1b displayed selective inhibition of the α_2A-adrenoceptor. We have recently reported on the high-affinity binding of MTα to the α_2B-adrenoceptor (Koivula et al., 2010). Using synthetic MTα, we found this toxin had no muscarinic receptor activity in contrast to what has previously been reported for the venom-derived toxin (Jolkkonen et al., 1995b). In this study, we have continued our screening of known MTs for adrenoceptor activity, and we now report that MT1 and MT3 display prominent adrenoceptor activity in addition to muscarinic receptor activity, whereas MT7 and MTα seem restricted to their single target receptors.

Methods

Test systems used

Spodoptera frugiperda Sf9 cells were grown in suspension at 27^○C in Grace's insect medium (Invitrogen, Paisley, UK) supplemented with 8% fetal bovine serum (Invitrogen), 50 µg·mL⁻¹ streptomycin (Invitrogen), 50 U·mL⁻¹ penicillin (Invitrogen) and 0.02% Pluronic F68 (Sigma-Aldrich, Helsinki, Finland). Human erythroleukaemia (HEL) cells were grown in RPMI 1640 medium (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin-streptomycin stock (Invitrogen) and 2 mM glutamine (Invitrogen). Cells were maintained at 37^○C in 95% air: 5% CO₂ atmosphere.

The human α₁-adrenoceptor cDNAs were kind gifts from Dr K Minneman (Emory University School of Medicine, Atlanta, GA). Baculovirus-mediated expression of the human α_1A- and α_1B-adrenoceptors (Koivula et al., 2010), α₂-adrenoceptors (Oker-Blom et al., 1993; Jansson et al., 1995) and mAChRs (Kukkonen et al., 1996; Näsman et al., 2000) in Sf9 cells has been described previously. In this study, new baculovirus vectors for all of these receptors and the α_1D-adrenoceptor were made to include a Rous sarcoma virus promoter, which we have found to enhance the expression of receptors in this system as compared with the polyhedrin promoter (the details of this will be described elsewhere). The cDNA for the human β₁-adrenoceptor was a kind gift from Dr S Uhlén (Institute of Medicine, University of Bergen, Bergen, Norway), and human β₂-adrenoceptor cDNA was purchased from Missouri S&T cDNA Resource Center (Rolla, MO). The β-adrenoceptor cDNAs were subcloned into FastBac1 and baculovirus generated with the Bac-to-Bac system (Invitrogen).

Sf9 cells aimed for membrane preparations were infected with receptor virus in batches of 80 mL (2 × 10⁶ cells·mL⁻¹) in suspension cultures for 48–72 h, whereafter harvested by centrifugation, washed once with cold PBS and stored as pellets at −70°C. For the functional assay, cells were seeded on tissue culture dishes and infected with receptor virus for 26–28 h. In the case of co-expression of G₁₁αi5 (Kukkonen et al., 2004) and receptor, which was required for the G_i-coupled M₄ mAChR and α_2A- and α_2C-adrenoceptors in measurements of intracellular Ca²⁺ increases, the cells were first infected with the chimeric G-protein construct for 30 min and then with the receptor virus for another 30 min before exchange to fresh medium.

Membrane preparations and buffer compositions

Crude membrane fractions of infected Sf9 cells and HEL cells were obtained by resuspending the thawed cell pellets in the buffer solution (same solutions as used for the binding experiments; see below) and homogenizing using a T25 Ultra-Turrax homogenizer (IKA, Staufen, Germany). The solutions were then subjected to low speed centrifugation and the supernatants centrifuged 30 000×g for 45 min at 4^○C. The pelleted membranes were washed once in buffer, resuspended in buffer (1–3 mg protein·mL⁻¹) and stored at −70°C. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with BSA as standards. The buffers used in the preparations and in the binding assays were 20 mM Tris, 1 mM EDTA, 5 mM MgCl₂, pH 7.4 for α₁- and β-adrenoceptors; 25 mM NaPO₄, pH 7.4 for α₂-adrenoceptors; 100 mM NaCl, 20 mM HEPES, 10 mM MgCl₂, 1 mM EDTA, pH 7.4 for mAChRs.

Adult female Sprague–Dawley rats were killed by carbon dioxide inhalation, and the kidneys and whole brain excised and immediately frozen at −70°C. After being thawed, the tissues were cut into pieces in buffer and homogenized with a Potter–Elvehjem glass homogenizer. For the brain tissue, we used a buffer containing 0.32 M sucrose, 10 mM HEPES, 2 mM EDTA, pH 7.4 and for the kidney a buffer containing 50 mM Tris, 1 mM EDTA, pH 7.4, both buffers supplemented with a protease inhibitor cocktail (Complete™, Roche Diagnostics GmbH, Mannheim, Germany). Low-speed centrifugations of the homogenates were done at 1000×g for 10 min, after which the supernatants were centrifuged 30 000×g for 45 min. The pellets were resuspended in 50 mM Tris, 1 mM EDTA, pH 7.4 at 3–5 mg protein·mL⁻¹ and stored at −70°C.

Radioligand binding assays

Prior to radioligand binding experiments, the membrane preparations were thawed on ice and diluted to appropriate volumes. To determine the K_d and B_max values of receptors with their respective radioligand, 15–50 µg of cell membranes were incubated with various concentrations of radioligand (0.1–30 nM [³H]-prazosin, 0.1–10 nM [³H]-MK-912, 0.1–30 nM [³H]-CGP-12177, 0.1–10 nM [³H]-NMS) in a total volume of 150 µL for 90 min with shaking at room temperature. Non-specific binding was determined in the presence of 10–100 µM phentolamine (α₁- and α₂-adrenoceptors), 10 µM propranolol (β-adrenoceptors) or 10 µM atropine (mAChRs). In displacement studies, the receptors were first pre-incubated for 30 min with different concentrations of toxins in 100 µL, after which radioligand in 50 µL was added to the receptors for another 60 min. The concentrations of toxins used in the calculations were always the concentrations present in the final 150 µL volumes. Unless otherwise stated, the concentration of radioligand was 1 nM for adrenoceptors and 0.5 nM for mAChRs in the displacement binding experiments.

To determine the radioligand dissociation rate, receptors were first incubated with 1.5 nM radioligand for 45 min, and thereafter dissociation was initiated by the addition of 1 µM phentolamine (α₁- and α₂-adrenoceptors) or 10 µM atropine (mAChRs). Samples were withdrawn at different time points and bound radioactivity determined. When the effect of MT was tested, the toxin was included in the dissociation initiator solution.

All reactions were terminated by rapid filtration through prewashed GF/B filters (PerkinElmer, Waltham, MA). Filter plates were then washed five times with cold wash buffer containing 180 mM NaCl, 25 mM MgCl₂ and 20 mM HEPES, pH 7.4 in a microplate harvester (PerkinElmer). Radioactivity bound to the filters was determined in a microplate scintillation counter (PerkinElmer).

Measurement of intracellular [Ca²⁺]

Infected Sf9 cells were detached from culture dishes and incubated for 20 min with 4 µM fura-2 acetoxymethyl ester in culture medium. After this, cells were diluted two times with culture medium and kept at room temperature in the dark. For fluorescence recordings, an aliquot of the cells was spun down and washed twice in assay buffer [129.7 mM NaCl, 5.44 mM KCl, 1.2 mM MgCl₂, 4.2 mM NaHCO₃, 7.3 mM NaH₂PO₄, 1 mM CaCl₂, 20 mM 2-(N-morpholino)ethanesulphonic acid (MES), 10 mM glucose, 63 mM sucrose, pH adjusted to 6.3]. After the final resuspension in assay buffer, the cells were transferred into a thermostatted (27^○C) cuvette with magnetic stirring in a Hitachi F-2000 fluorescence spectrophotometer. Recordings at 340 nm (excitation) and 505 nm (emission) were briefly interrupted for additions of agonists and toxins, and at the end calibrated using 0.04% Triton X-100 to obtain F_max and 10 mM EGTA to obtain F_min. Intracellular calcium concentrations were calculated according to the equation [Ca²⁺] = (F – F_min) / (F_max – F) ×K_d for the fura-2–Ca²⁺ complex (255 nM at 27^○C) (Shuttleworth and Thompson, 1991).

When MTs were included in the experiments, the toxins were allowed to bind to receptors for 2–3 min prior to agonist additions in the short-term incubation experiments. For longer times of toxin incubations (≥60 min), the cells were kept in culture medium with the toxin and then treated as above in the constant presence of the toxin.

HEL cells were detached from the 75 cm² culture bottles by gentle shaking, centrifuged, resuspended in culture medium and loaded with 4 µM fura-2 acetoxymethyl ester for 20 min.

After this, the cells were spun down and washed once in assay buffer (137 mM NaCl, 5 mM KCl, 0.44 mM KH₂PO₄, 4.2.mM NaHCO₃, 20 mM HEPES, 10 mM glucose, 1 mM CaCl₂, 1.2 mM MgCl₂, pH adjusted to 7.4), resuspended in assay buffer, divided into aliquots and kept at 37^○C in the dark. Fluorescence measurements were done as described for Sf9 cells except for the temperature, which was 37^○C in the cuvette holder. The K_d of 224 nM was used in the calculation of the Ca²⁺ concentration.

Data analysis

All data analyses were done using GraphPad Prism (GraphPad Software, San Diego, CA). Non-linear curve fitting was used to determine K_d and B_max values. IC₅₀ values in displacement studies were determined using the equation

where B is the bound radioligand and [I] is the concentration of toxin (inhibitor) in the sample.

In functional inhibition experiments with toxins, the apparent inhibitory constants were obtained by curve-fitting to the equation:

where Δ[Ca²⁺] is the cellular increase in Ca²⁺ concentration, [A] the concentration of agonist, respectively, and EC₅₀ the [A] producing half-maximal stimulation. In cases where the toxin also caused depression of the maximum response (insurmountable inhibition), the maximum response (Δ[Ca²⁺]_max) was fitted accordingly. The apparent K_i values were derived from the shift according to:

where [I] is the concentration of the inhibitor. Each K_i value was derived using several toxin concentrations and thus the apparent pK_i values reported correspond to the pA₂ values from Schild analysis (for inhibitors displaying pure surmountable inhibition).

Materials

Synthetic MTα, MT1 and MT3 were from Peptide Institute (Osaka, Japan). MT7 was recombinantly expressed and purified as previously described (Näsman et al., 2000). Noradrenaline bitartrate, carbachol, atropine hemisulphate and neuropeptide Y (NPY) were from Sigma-Aldrich. UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalineamine] and phentolamine hydrochloride were from RBI (Natick, MA). Propranolol was from Tocris Biosciences (Bristol, UK). Fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR). [³H]-MK-912 [(2S,12bS)1′,3′-dimethylspiro(1,3,4,5′,6,6′,7,12b-octahydro-2H-benzo[b]furo[2,3a]quinolizine)-2,4′-pyrimidin-2′-one, specific activity 79.1 Ci·mmol⁻¹], [³H]-prazosin [2-[4-(2-furoyl)piperazin-1-yl]-6,7-dimethoxyquinazolin-4-amine, specific activity 85.3 Ci·mmol⁻¹], [³H]-CGP-12177 [(−)-4-(3-tert-butylamino-2-hydroxypropoxy)-[5,7-H]benzimidazol-2-one, specific activity 30.0 Ci·mmol⁻¹] and [³H]-NMS (N-methyl scopolamine, specific activity 82.0 Ci·mmol⁻¹) were from PerkinElmer.

Results

Receptor expression

In order to study the effect of toxins on receptors, we expressed the adrenoceptors and muscarinic receptors in Sf9 cells (Table 1). The muscarinic receptors and the α₂-adrenoceptors have been used for many years with this expression system for the characterization of MT action (Näsman et al., 2000; Koivula et al., 2010). The α_1A- and α_1B- adrenoceptors were recently expressed and characterized functionally by measuring the noradrenaline-induced Ca²⁺ responses (Koivula et al., 2010). In the present study, we used radiolabelled prazosin to further characterize the α₁-adrenoceptors. We also managed to obtain reasonable expression of the α_1D-adrenoceptors by manipulating with the transcriptional promoters in the virus constructs. The K_d and B_max values for different receptors determined with their respective radioligand are listed in Table 1. All K_d values are in general agreement with previously reported affinity values obtained in other expression systems (Uhlen et al., 1998; Williams et al., 1999; Baker, 2005). MK-912, which is selective for the α_2C subtype, also displays high affinities for the α_2A- and α_2B-adrenoceptors and is thus very useful when studying heterologously expressed receptors.

Table 1.

Radioligand binding data for expressed receptors

Receptor	3H-ligand Kd (pM)	Bmax (fmol mg-1 protein)
Adrenoceptor
	[3H]-prazosin
α1A	234 ± 31	2191 ± 53
α1B	181 ± 28	1946 ± 59
α1D	148 ± 31	438 ± 18
	[3H]-MK-912
α2A	876 ± 128	5190 ± 141
α2B	599 ± 134	10343 ± 409
α2C	86 ± 32	1101 ± 186
	[3H]-CGP-12177
β1	664 ± 189	2910 ± 249
β2	738 ± 204	2385 ± 204
mAChR	[3H]-NMS
M1	460 ± 78	3162 ± 115
M4	190 ± 34	888 ± 35

Saturation binding experiments were performed using crude membrane preparations of infected Sf9 cells. Data are mean values ± SD from one batch of membranes analysed in two separate experiments.

The binding of the toxins used here to mAChRs has previously been analysed in many different laboratories (reviewed in Bradley, 2000; Karlsson et al., 2000; Servent and Fruchart-Gaillard, 2009). The M₁ and M₄ subtypes were included in this study, because MT1 and MT3 have not been tested previously in the experimental setup of our laboratory, and these receptor subtypes also served as positive controls for MT1, MT3 and MT7.

Radioligand displacement studies

MT1 is one of the best-studied MTs, with selective binding to the M₁ and M₄ subtypes of mAChRs (Kornisiuk et al., 1995a, b; Mourier et al., 2003). In displacement studies with the α_2B-adrenoceptor, MT1 inhibited the [³H]-MK-912 binding with an IC₅₀ of 2.3 nM (Figure 1, Table 2). The toxin binding behaved non-competitively as judged from the inability to completely displace bound radioligand. A similar IC₅₀ value was obtained with MTα (Table 2), in agreement with the IC₅₀ value (3.2 nM) we reported previously in competition with [³H]-RX821002 (K_d = 3.3 nM) (Koivula et al., 2010). In accordance with its role as a muscarinic toxin, MT1 also displaced [³H]-NMS from M₁ and M₄ mAChRs (Table 2). Interference with [³H]-prazosin binding could be detected on each α₁ subtype with the highest affinity determined for the α_1A-adrenoceptor. For receptors binding with low affinities, we chose to indicate % inhibition obtained with 1 µM toxin since the inhibition curves did not reach saturation at the concentrations used (Table 2). The usual pattern found for these titrations is exemplified by the α_2A-adrenoceptor in Figure 1.

Figure 1.

Cellular membranes with α_2A- or α_2B-adrenoceptors were incubated with different concentrations of MT1 and 1 nM [³H]-MK-912 for a total of 90 min, after which the samples were filtrated to remove unbound ligands and then subjected to scintillation counting. Data points are given as % of control binding and represent means ± SEM of three experiments each performed with triplicate samples. The pIC₅₀ value for the saturating inhibition was determined with computational fitting (Table 2). The data with the α_2A-adrenoceptor serve as an example of inhibitory potencies when given as % inhibition in Table 2.

Table 2.

MT inhibition profiles at different receptors

	pIC50 ±SEM (n = 3–4)
Receptor	MT1	MT3	MT7	MTα
α1A	6.98 ± 0.17	8.86 ± 0.14	NI	NI
α1B	<6.5 (48%)a	7.57 ± 0.22	NI	NI
α1D	<6 (28%)a	8.13 ± 0.08	NI	NI
α2A	<6.5 (47%)a	8.49 ± 0.06	NI	NI
α2B	8.64 ± 0.10	<6.5 (39%)a	NI	8.62 ± 0.12
α2C	NI	7.29 ± 0.13	NI	NI
β1	NI	NI	NI	NI
β2	NI	NI	NI	NI
M1	6.85 ± 0.06	6.71 ± 0.14	9.36 ± 0.06b	NI
M4	6.54 ± 0.09	8.79 ± 0.06	NI	NI

The inhibitory potencies of different MTs were determined in radioligand displacement experiments.

^a% values in parentheses indicate inhibition with 1 µM toxin.

^bAnalysis of the dissociation kinetic of [³H]-NMS in relation to MT7 concentrations resulted in pIC_{50, diss} = 8.31 ± 0.04.

NI, no inhibition. The threshold for inhibition was set to 25% at 1 µM concentration for MT1, MT3 and MTα and 0.3 µM for MT7.

The synthetic MT3 exhibited much higher affinity for the M₄ subtype than for the M₁ mAChR, as previously found for the venomous MT3 (Jolkkonen et al., 1994; Liang et al., 1996). Surprising was the high-affinity displacement we found with several adrenoceptor subtypes. MT3 was equally potent at the α_1A and only slightly less potent at α_1D and α_2A as compared with the M₄ subtype (Figure 2, Table 2). Relatively high affinity was also found for the α_1B and α_2C subtypes (Table 2). In all displacements obtaining saturating inhibitions with MT3, the non-competitive nature of binding was prevailing. The non-competitivity was also tested by using different radioligand concentrations. As exemplified in Figure 3 for the α_1A-adrenoceptor and MT3, there were no obvious shifts in the IC₅₀ values, which would be expected if the ligands competed for the same binding site. MT3 behaved similarly at the α_2A-adrenoceptor (Figure 3C) and at the α_1D- and α_2C-adrenoceptors when loaded with different radioligand concentrations (0.3 and 1 nM; data not shown). For the M4 receptor, the inhibition curve shifted as for competitive interactions when going from 0.5 to 1.5 nM radioligand, but no further shift was seen with 5 nM (Figure 3C).

Figure 2.

Different receptors expressed in membranes of Sf9 cells were subjected to MT3 displacement binding of [³H]-prazosin for α₁-adrenoceptors (A), [³H]-MK-912 for α₂-adrenoceptors (B) and [³H]-NMS for mAChRs (C). Sample and data processing were as in Figure 1. Data points are means ± SEM of three experiments performed in triplicate.

Figure 3.

The inhibitory potency of MT3 was tested on α_1A-adrenoceptor membranes in the presence of different concentrations of [³H]-prazosin (A). Sample and data processing were as in Figure 1. All data points are from one experiment performed using the same membrane preparation, and plotted as means of triplicate samples. (B) The control binding for α_1A-adrenoceptor expressed in fmol·(mg of protein)⁻¹ (means ± SD, n = 3) with different concentrations of [³H]-prazosin. (C) pIC₅₀ values plotted as a function of radioligand concentrations for α_1A, α_2A and M₄ mAChR. Values are means ± SEM (n = 3) from fitted curves.

Allosteric effects on orthosteric ligand dissociation have sometimes been implicated in MT action (Max et al., 1993b; Mourier et al., 2003; Olianas et al., 2004). Because MT3 bound to several receptors with high affinity, we aimed to look for potential differences in the interaction of MT3 with its multiple targets. MT3 did not, however, show any effect on the dissociation rates of radioligands from α_1A, α_2A or M₄ receptors (Table 3), indicating that the toxin does not allosterically alter antagonist dissociation or obstruct the exit of the antagonist by steric hindrance at the entrance of the ligand binding cavity.

Table 3.

Dissociation rate constants for the radioligands [³H]-prazosin (α_1A), [³H]-MK-912 (α_2A) and [³H]-NMS (M₄)

	[3H]-Ligand dissociation rate (min−1)
Receptor	Control	30 nM MT3	300 nM MT3
α1A	0.0400 ± 0.0024	0.0441 ± 0.0024	0.0403 ± 0.0050
α2A	0.0264 ± 0.0024	0.0273 ± 0.0026	0.0328 ± 0.0089
M4	0.0271 ± 0.0054	0.0268 ± 0.0051	0.0287 ± 0.0068

Data are given as means ± SD from two experiments.

Functional inhibition

In general, the MTs behave as antagonists of receptor responses. MT2 could be an exception to this rule, based on the toxin-induced cytosolic Ca²⁺ increases reported with CHO cells expressing different mAChRs (Bradley et al., 2003). MT1 has also been implicated in agonistic actions using functional output from different tissue preparations (Jerusalinsky et al., 1995; Jolkkonen et al., 1995a; Harvey et al., 2002). We tested the effect of MT1 on the α_2B-adrenoceptor and M₁ mAChR by monitoring the receptor-activated Ca²⁺ response in Sf9 cells. In the concentration range of 10–300 nM, acute application of MT1 did not elicit any response from either receptor (data not shown). Instead, the responses to the agonists noradrenaline and carbachol, respectively, were inhibited. In our previous work, we have noticed that it may be necessary to incubate receptors with toxin for a considerable time (>30 min) before stimulating with agonist to reveal the true antagonistic potencies of toxins (Koivula et al., 2010). We therefore pre-incubated cells for ≥60 min with different concentrations of MT1 and measured the agonist responses (Figure 4 and Table 4). MT1 behaved very similarly to MTα on the α_2B-adrenoceptor (Koivula et al., 2010), with a pronounced insurmountable inhibition of the response. On the M₁ receptor, MT1 inhibited the response surmountably with an apparent K_i of 35 ± 11 nM (mean ± SD, n = 4). A low concentration of the M₁-selective MT7 was included in the same assay for comparison with the more complex inhibition pattern produced by this toxin on the M₁ receptor (Figure 4B).

Figure 4.

Sf9 cells expressing the α_2B-adrenoceptor (A) or M₁ mAChR (B) were loaded with fura-2 and subjected to fluorescence recordings to measure intracellular [Ca²⁺] levels. Different concentrations of toxins were added to aliquots of cells and incubated for ≥60 min. Control cells were treated similarly with vehicle. Noradrenaline (NA) and carbachol (CCh) were used to stimulate the α_2B and M₁ receptors, respectively. Data points (means ± SD, n = 3–5) are given as % of control maxima. The absolute response maxima varied somewhat between days of experimentation. For α_2B-AR, the response maxima were in the range 570–743 nM and for M₁ in the range 214–254 nM. Data for MT7 were included for comparison with the effect of a toxin with high affinity binding to the M₁ receptor.

Table 4.

Apparent affinity constants for MT inhibition of receptor-stimulated Ca²⁺ increases

Receptor	Apparent pKi
MT1
α2B	8.28 ± 0.38
M1	7.47 ± 0.13
MT3
α1A (high)	9.19 ± 0.13
α1A (low)	8.74 ± 0.23
α1D	8.28 ± 0.05
α2A	8.20 ± 0.32
α2C	7.92 ± 0.11
M4	8.50 ± 0.18

Analyses were done with two or three different concentrations of toxin and the values represent means ± SD from three to six experiments.

MT3 is a well-characterized mAChR antagonist with a pA₂ value of 8.33 derived from functional inhibition of cloned M₄ receptors (Olianas et al., 1999). To further explore its action on the adrenoceptors, we performed functional Ca²⁺ assays with the different high-affinity targets (Figure 5). For these, we analysed the responses both after short (2–3 min) and long (≥60 min) pre-incubations with MT3. Potential responses due to toxin applications were monitored in the short-term experiments to reveal agonistic effects, but none was found. The responses with control cells without toxin did not generally change during the time windows used, and if so, the experiments were discarded. In the long-term incubations, we did not observe further inhibition of any receptor from 60 min up to 120 min (data not shown), indicating that the binding of toxin reached equilibrium within 60 min.

Figure 5.

Cellular [Ca²⁺] responses were measured in Sf9 cells expressing the α_1A (A and B; high receptor expression in A and low in B), M₄ mAChR (C), α_2A (D), α_1D (E) and α_2C (F). Different concentrations of MT3 were added to aliquots of cells and pre-incubated for 2–3 min (short) or ≥60 min (long) before being stimulated with agonist. Control cells were treated similarly with vehicle. Data points (means ± SD, n = 3–6) are given as % of control maxima. The ranges for the response maxima were (in nM): 595–855 (A), 526–572 (B), 576–728 (C), 526–803 (D), 662–978 (E), 299–528 (F).

Short pre-incubations with toxins may not be adequate to observe an effect on receptor responses, as shown in Figure 5. This slow binding was most pronounced for the α_2A-adrenoceptor (Figure 5D), where there was no obvious effect even with 100 nM MT3, despite the high affinity determined in radioligand binding experiments. The inhibitory potency of MT3 became obvious though with a long pre-incubation time. Another important observation we made, as regards interpretation of toxin binding modes from functional receptor analyses, was the effect of MT3 on the α_1A-adrenoceptor. In Figure 5A the analyses of responses using cells expressing a relatively high density of receptors (1350 ± 160 fmol mg^-1 protein of homogenized cells, mean ± SD, n = 3) are plotted. Long pre-incubations with 30 nM MT3 did not show any insurmountable effect. In Figure 5B, the same type of experiments were performed with cells expressing the same α_1A-adrenoceptor cDNA, but at a much lower density (59 ± 10 fmol mg^-1 protein of homogenized cells, mean ± SD, n = 3). With these cells, the response was strongly depressed with 30 nM MT3. Our interpretation of these data is that a receptor reserve exists in the high-expressing cells, and therefore, the non-occupied receptor population can still elicit a full response. The almost 100-fold difference in the potency for noradrenaline with control cells from these two expression levels points towards a similar interpretation. However, using lower concentrations of MT3 to assess the potency of the toxin with the low-expressing α_1A-adrenoceptor gave a similar apparent pK_i value as with the high-expressing cell batch (Table 4). Overall, the relative potencies (pK_i values) of the toxin, determined from the inhibited responses of the different receptors, followed quite closely the relative potencies (pIC₅₀ values) found in the displacement binding (Tables 2 and 4).

Native receptor inhibition by MTs

The HEL cell line has been used by several laboratories to study the intracellular Ca²⁺ dynamics elicited through α₂-adrenoceptors (Michel et al., 1989; Musgrave and Seifert, 1995; Kukkonen et al., 1997). Pharmacological data indicate that the α_2A subtype is endogenously expressed in these cells. To confirm the effect of MT3 on endogenously expressed α_2A-adrenoceptors, we pre-incubated the cells with 100 nM MT3 for 60 min and measured the responses. This concentration of MT3 totally blocked the response to 100 nM UK14,304, an α₂-adrenoceptor agonist (Figure 6A). The response to NPY, through the activation of endogenous NPY receptors, did not change with MT3. In the radioligand displacement assay, MT3 inhibited [³H]-MK-912 binding with a pIC₅₀ = 8.65 ± 0.20, confirming its potency also for the native human α_2A-adrenoceptor (Figure 6B).

Figure 6.

MT effects on native receptors. (A) HEL cells were assayed for α_2A-induced Ca²⁺ responses. After fura-2 loading, the cells were pre-incubated for 60 min with or without 100 nM MT3. Receptors were stimulated with either 100 nM UK-14,304 or 100 nM NPY. Data are means ± SD of two separate experiments for each column. (B) HEL cell membranes were incubated with 1 nM [³H]-MK-912 and various concentrations of MT3 and the bound radioligand concentrations determined. Data points are means ± SD of two experiments with duplicate samples. (C) Rat brain and kidney membranes were incubated with 1 nM [³H]-prazosin and various concentrations of MT3 and the bound radioligand concentrations determined. Data points are means ± SD of one experiment with triplicate samples. The effect of MT3 was confirmed in two additional experiments. (D) Titration of MT1 and MTα against 1 nM [³H]-MK-912 in rat kidney membranes. Data points are means ± SD of one experiment with triplicate samples. The effect of the MTs was confirmed in two additional experiments.

Because we could not find appropriate human cell lines to test native α₁- and α_2B-adrenoceptor binding, we used rat tissues to verify the toxin effects. Whole rat brain membranes were labelled with [³H]-prazosin and analysed for displacement by MT3. The pIC₅₀ for MT3 was 8.85 ± 0.07 with this preparation (Figure 6C). A similar pIC₅₀ (8.50 ± 0.18) was found for [³H]-prazosin-labelled kidney membranes (Figure 6C). To test MT binding on α_2B-adrenoceptors, we used rat kidney membranes. The kidney tissue is a known source of α_2B-adrenoceptors (Huang et al., 1996), and the [³H]-MK-912-labelled sites of this organ were potently displaced by MT1 (pIC₅₀ = 9.01 ± 0.11) and by MTα (pIC₅₀ = 8.76 ± 0.08) (Figure 6D).

Discussion

The present study confirms the specificity of MT7 for the M₁ mAChR and the specificity of synthetic MTα for the α_2B-adrenoceptor, with reservations for the activity of venomous MTα, which has not been thoroughly investigated (Koivula et al., 2010). In contrast to these toxins, MT1 and MT3 exhibited a somewhat unexpectedly broad range of receptor targets among the adrenoceptors. Some of the α-adrenoceptors were found to bind these toxins with high affinities, and MT1 actually bound more tightly to the α_2B-adrenoceptor than to the previously identified muscarinic targets. No activity of the tested toxins was detected on the β-adrenoceptors. This could indicate that the β-adrenoceptors are structurally very different from the α-adrenoceptors in terms of MT binding. A hint of such a structural difference could emanate from the folding of the second extracellular receptor loop, which in β-adrenoceptors contains two disulphide bridges that constrain the loop folding more than in the α-adrenoceptors that contain only one such bridge (Cherezov et al., 2007). Nevertheless, the structurally related β-cardiotoxin found from the king cobra shows β-adrenoceptor activity (Rajagopalan et al., 2007), suggesting that snake venoms could be a source of subtype-selective β-adrenoceptor ligands as well.

The primary sequences of MT1 and MTα are very similar (Table 5). Of the four different residues, three are clustered near the tip of the middle finger, that is I³¹VP³³ in MT1 and L³¹NH³³ in MTα. The tip of the middle finger, in general a basic residue, R³⁴ or K³⁴ in MTs, is believed to reach down into the orthosteric binding cleft and to interact with transmembrane helix residues of the receptor (Ségalas et al., 1995). The adjacent residues on both sides of the finger would thus be in such positions that they could interact with the outer surface of the receptor. Our hypothesis was that the LNH cluster of MTα could be an α_2B recognition motif. However, since MT1 was found to bind equally well to the α_2B-adrenoceptor as MTα, such a restricted motif probably does not exist. On the other hand, the LNH sequence of MTα appears to prevent this toxin from binding to the muscarinic receptors and thereby fulfills a specific function from a pharmacological point of view. The fourth difference in the sequences between MT1 and MTα, R⁵⁷ and H⁵⁷ is located adjacent to the disulphide bridge making up the third finger of the toxin. It is likely that this substitution does not play a significant role in binding selection because MT4, which differs from MT1 only at position H⁵⁷, binds to M₁ and M₄ receptors with similar affinities as MT1 (Vandermeers et al., 1995; Karlsson et al., 2000). It is therefore also likely that MT4 is an antagonist with higher affinity for the α_2B-adrenoceptor than for muscarinic receptors. The influence of side chain differences at residue 57 could be rigorously tested with MT4 when synthesized.

Table 5.

Sequence alignment of selected mamba toxins

	1	15	30	45	60	% Identity
MT1	LTCVTSKSIFGITTE	NCPDGQNLCFKKWYY	IVPRYSDITWGCAAT	CPKPTNVRETIRCCE	TDKCNE	100
MT4	LTCVTSKSIFGITTE	NCPDGQNLCFKKWYY	IVPRYSDITWGCAAT	CPKPTNVRETIHCCE	TDKCNE	98
MTα	LTCVTSKSIFGITTE	NCPDGQNLCFKKWYY	LNHRYSDITWGCAAT	CPKPTNVRETIHCCE	TDKCNE	94
ρ-Da1b	LTCVTKDTIFGITTQ	NCPAGQNLCFIRRHY	INHRYTEITRGCTAT	CPKPTNVRETIHCCN	TDKCNE	74
ρ-Da1a	LTCVTSKSIFGITTE	DCPDGQNLCFKRRHY	VVPKIYDSTRGCAAT	CPIPENY-DSIHCCK	TDKCNE	73
MT3	LTCVTKNTIFGITTE	NCPAGQNLCFKRWHY	VIPRYTEITRGCAAT	CPIPENY-DSIHCCK	TDKCNE	71
MT7	LTCVKSNSIWFPTSE	DCPDGQNLCFKRWQY	ISPRMYDFTRGCAAT	CPKAE-YRDVINCCG	TDKCNK	64

MT1 antagonized the responses of α_2B in the functional assay. Characteristic for the toxins that binds with high affinity to the receptors in this type of assays is the insurmountability of inhibition. This could relate to the non-competitive characteristic of the toxins seen in equilibrium binding experiments, but more likely it is a consequence of the slow dissociation from the receptor that generates this type of behaviour. In general, the affinity of the toxin for a certain receptor, and thus also the dissociation rate, seems to determine whether the inhibition of fast, transient responses will behave surmountably or insurmountably. MT7 inhibits the M₁ receptor insurmountably in the same functional assay, but when part of the toxin-binding domains is exchanged the affinity drops and the inhibition becomes increasingly surmountable (Kukkonen et al., 2004). Another factor that needs to be taken into account is the receptor expression level and the existence of potential receptor reserves, which is not only an artefact of recombinant expression but a phenomenon also apparent in physiological systems (Kenakin, 1984; Chong and Peachell, 1999). Here, we demonstrate with the α_1A-adrenoceptor that the inhibitory profile of a toxin antagonist is strongly affected by a receptor reserve. Such receptor reserves have also been implicated in the inhibition profile of another α₁-adrenoceptor toxin, ρ-TIA from a marine snail, on contractile responses of rat vas deferens (Sharpe et al., 2003).

There have been some uncertainties regarding the effect of MT1 on the M₁ receptor. Some early reports measuring the effect of MT1 administration either in vivo or on tissue responses suggested an agonistic property of the toxin (Jerusalinsky et al., 1995; Jolkkonen et al., 1995a). In contrast, muscarinic receptor-stimulated adenylyl cyclase activity in rat frontal cortex was antagonized by MT1 without signs of agonism (Onali and Olianas, 1998). Using up to 20 µM of synthetic MT1, Servent and colleagues could not detect Ca²⁺ mobilization in M₁-transfected cells (Servent and Fruchart-Gaillard, 2009). In the present study, we used much lower concentrations of MT1 together with carbachol to assess potential allosteric enhancements of the response. However, MT1 showed only antagonistic behaviour with a similar affinity to that found previously for venomous MT1 (Mourier et al., 2003). It has also been found that MT1 inhibits the noradrenaline-stimulated contraction of rabbit vas deferens (Harvey et al., 2002), indicating that the anti-adrenoceptor activity of MT1 might be responsible for some of the effects seen on tissue responses.

MT3 has been well characterized on mAChRs due to its high affinity for the M₄ receptor and the high selectivity for this subtype (Jolkkonen et al., 1994; Olianas et al., 1996; Olianas et al., 1999). We found that this toxin was rather promiscuous among the α-adrenoceptors, binding with high affinity to α_1A, α_1D and α_2A, and with slightly lower affinity to α_1B and α_2C. All of the high-affinity binding was found to be non-competitive, as judged by an inability to displace all radioligand. This also included the M₄ receptor where about 13% of the radiolabelled ligand remained bound at a saturating MT3 concentration (1 µM). It has previously been suggested that MT3 would bind competitively with orthosteric ligands at the M₄ receptor; this was based on complete displacement of radioligand and shifts of the K_d values for [³H]-NMS measured in saturation binding in the presence of the toxin (Olianas et al., 1996).

Varying the radioligand concentration in the MT3 displacement experiments also revealed an initial shift in the MT3 potency in our work. However, this competitive shift saturated at higher radioligand concentrations, indicating that the reciprocal binding of the two ligands is more complex than a simple competition. A similar lack of potency shift has also been observed previously for MT3 on the M₄ receptor (Liang et al., 1996).

One of our aims was to determine whether MT3 binds in different ways to the different high-affinity targets. In the kinetic experiments measuring the toxin's influence on antagonist dissociation rates, we could not find any effect of MT3 on either receptor. In experiments analysing the functional antagonism in the same cell background, MT3 displayed quite similar inhibition profiles for all the receptors tested, with pronounced suppressions of the maximal responses. The use of short and long pre-incubations revealed that there might be some differences in the binding to different receptors. For example, the α_2A-adrenoceptor appeared insensitive to MT3 with short-term incubations, suggesting that some structural features in this receptor protein need to be re-arranged by the toxin in order to bind or to exert an effect on agonist action. There have been some reports on isomerization phenomena in the binding of MTs to muscarinic receptors (Toomela et al., 1994; Jolkkonen et al., 2001). It is possible that the toxins induce changes in the receptor conformation in order to engage more binding contacts and strengthen the binding, or alternatively to simply coerce the receptor into an inactive state in the case of antagonists.

To conclude, peptide toxins from venomous animals have mostly evolved to target specific molecules. Their specificity is probably a cause of the multiple contact sites, which together make up the total binding affinity and also preclude interactions with imperfectly matched homologous proteins. As described here, at least the MT3 toxin seems to be an exception to this specificity and should be regarded as a multi-target toxin, with cross-over activity among adrenoceptors and mAChRs. However, if the MT3 toxin uses different receptor epitopes for binding to the different receptors and by doing so also exposes structurally distinct contact points to the receptors, it might be possible to restrict the selectivity of MT3 by mutagenesis to single receptor subtypes. This will be a challenging task for the future.

Glossary

Abbreviations

CCh

carbachol

HEL

human erythroleukaemia

mAChR

muscarinic acetylcholine receptor

MT

muscarinic toxin

NA

noradrenaline

NPY

neuropeptide Y

Conflicts of interest

None.