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.

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.

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.

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.

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).

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.