Báo cáo khoa học: Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors potx

Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors Dmitry Yu. Mordvintsev 1 , Yakov L. Polyak 1, *, Dmitry I. Rodionov 1, , Jan Jakubik 2 , Vladimir Dolezal 2 , Evert Karlsson 3 , Victor I. Tsetlin 1 and Yuri N. Utkin 1 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia 2 Institute of Physiology, Prague, Czech Republic 3 Institute of Biochemistry and Organic Chemistry, Uppsala, Sweden Introduction Two main classes of acetylcholine (ACh) receptors (AChRs) are involved in cholinergic transmission. The nicotinic AChRs (nAChRs) are activated by nicotine and belong to the ligand-gated ion channel superfam- ily, whereas muscarinic AChRs (mAChRs) are activated by muscarine and belong to the family of Keywords muscarinic acetylcholine receptor; nicotinic acetylcholine receptor; snake venom; weak neurotoxin Correspondence Y. N. Utkin, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, ul. Miklukho-Maklaya 16 ⁄ 10 Moscow, 117997 Russia Fax: +7 495 335 57 33 Tel: +007 495 336 65 22 E-mail utkin@mx.ibch.ru Present addresses *Max-Planck-Institute fur Kohlenforschung, Mulheim an der Ruhr, Germany McGill University, Montreal, Canada (Received 16 December 2008, revised 25 May 2009, accepted 7 July 2009) doi:10.1111/j.1742-4658.2009.07203.x Iodinated [ 125 I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ-specific binding was monitored. Relatively high levels of [ 125 I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [ 125 I]WTX-binding sites. Specific [ 125 I]WTX binding was partially inhibited by both a-cobratoxin, a blocker of the a7 and muscle-type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a K d of 2.0 ± 0.8 lm and was inhibited by a-cobratoxin but not by a short-chain a-neurotoxin antagonist of the muscle-type nAChR, suggesting a specific interaction with the a7-type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 lm did not inhibit binding of [ 3 H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [ 3 H]N-methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G-protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G-proteins with the M 1 ,M 4 , and M 5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs. Abbreviations ACh, acetylcholine; AChR, acetylcholine receptor; Bgt, a-bungarotoxin; CHO, Chinese hamster ovary; CTX, a-cobratoxin; mAChR, muscarinic acetylcholine receptor; MT1, muscarinic toxin 1; MT2, muscarinic toxin 2; MT3, muscarinic toxin 3; MT7, muscarinic toxin 7; nAChR, nicotinic acetylcholine receptor; NMS, N-methylscopolamine; NTII, neurotoxin II of Naja oxiana; QNB, quinuclidinyl benzilate; SEM, standard error of the mean; WTX, weak toxin from Naja kaouthia. FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS 5065 G-protein-coupled receptors. Both receptor families comprise multiple subtypes, and whereas nAChRs pre- dominantly control fast synaptic neurotransmission, mAChRs control the slower processes of smooth mus- cle contraction and secretion. Both AChR types are activated by ACh, but their antagonists are generally type-specific. For example, snake venoms contain toxins of the three-finger toxin family [1], which specifically block either mAChRs or nAChRs. So-called muscarinic toxins isolated from mamba venoms specifically interact with different sub- types of mAChRs [2], whereas a-neurotoxins present in Elapidae venoms specifically block muscle-type and a7 nAChRs [3,4]. Elapidae venoms also contain so-called weak or nonconventional toxins [5]. It was recently shown [6–10] that these toxins also interact in vitro with muscle-type and a7 nAChRs, but with lower affinity than a-neurotoxins. Weak toxins are members of the three-finger toxin family, which contain a triple-fingered structural fold stabilized by five disulfide bridges [5]. Weak toxins possess an additional disulfide in loop I that is also present in the endogenous nAChR modula- tors lynx1 and SLURPs [11,12]. We previously reported that weak toxin from Naja kaouthia (WTX) produces hemodynamic and behavioral effects in rodents that can be explained by its interaction with mAChR [13,14]. We now report the in vivo distribution of radiolabeled WTX in mouse organs and specific binding to rat adrenal membranes. This interaction was inhibited by both nicotinic and muscarinic ligands, suggesting that WTX interacts with both nAChR and mAChR. Experiments using hetero- logous expression of mAChRs revealed that the mech- anism of WTX interaction with mAChRs is allosteric. This is the first report that WTX interacts with both nAChRs and mAChRs. Furthermore, the binding mechanisms were found to be different: we report that WTX binds to the orthosteric site (the site where the endogenous ligand ACh binds) of nAChR, whereas its interaction with mAChR is allosteric. Results Distribution of WTX binding in mouse organs Mice were injected intraperitoneally with radiolabeled WTX, and organ-specific radioactivity was monitored 5–30 min postinjection. Relatively high levels of radio- activity were detected in adrenal gland, muscle, dia- phragm, and spleen, whereas only low levels were detected in heart, lung, brain, and spinal cord. The accumulation of radioactive WTX in muscles and dia- phragm is consistent with WTX binding to resident muscle-type nAChRs. On the other hand, retention in spleen, which does not contain nAChRs, indicates that WTX can bind to other target sites. Notably, high lev- els of WTX were retained in the adrenal gland. Although a-bungarotoxin (Bgt)-binding sites in adrenomedullary chromaffin cells were previously dem- onstrated by autoradiography [15], nAChR density in the adrenal gland was not investigated. WTX binding to rat adrenal membranes To identify WTX-binding sites in the adrenal gland, we studied the interaction with rat adrenal membranes. [ 125 I]WTX bound specifically to these membranes (Fig. 1) with relatively low affinity (K d of 0.3–0.6 lm, B max of 138 ± 32 pmolÆmg protein )1 ). Binding was only partially inhibited by a-cobratoxin (CTX), a selective antagonist of the a7 and muscle nAChRs, and residual binding had a K d of 50 ± 20 nm and a B max of 30 ± 8 pmolÆmg protein )1 [Fig. 1, calculated from Eqn (1); see Experimental procedures]. On the basis of these parameters, the affinity of WTX for the CTX-dependent site was calculated using Eqn (2) (Experimental procedures), yielding a K d of 2.0 ± 0.8 lm. This value is in good agreement with IC 50 values obtained in competition experiments. To investigate the levels of nonspecific binding of [ 125 I]WTX, we performed competition studies with dif- ferent concentrations of native WTX. Binding was almost completely inhibited at high WTX concentra- tions (Fig. 2). High concentrations of another weak toxin (weak toxin from the cobra Naja oxiana; Uni- ProtKB accession number P85520) also completely Fig. 1. Curve 1 shows binding of [ 125 I]WTX to rat adrenal gland membranes; curve 2 shows binding in the presence of 100 l M CTX. Nonspecific binding was determined in the presence of 100 l M WTX. Weak toxin binds two acetylcholine receptor types D. Yu. Mordvintsev et al. 5066 FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS inhibited [ 125 I]WTX binding to adrenal membranes (not shown). The binding curve in competition experi- ments with native WTX was indicative of two types of [ 125 I]WTX-binding site (Fig. 2) with calculated IC 50 values of 92 nm and 26 lm. Low molecular weight AChR ligands also competed with [ 125 I]WTX for binding to adrenal membranes. The ganglionic nicotinic blocker hexamethonium inhibited binding by about 60% at a concentration of 1.5 mm. The same effect was observed for the nicotinic agonist choline, and 1 mm carbamoylcholine, a nonselective agonist of mAChRs and nAChRs, almost completely inhibited [ 125 I]WTX binding. The mAChR-selective ligands atropine and oxotremorine M inhibited binding by about 40% at 1 mm (data not shown). To further characterize [ 125 I]WTX binding to adre- nal membranes, we performed competition experiments using CTX and the muscle-type nAChR-selective short neurotoxin II of N. oxiana (NTII). NTII had no effect on [ 125 I]WTX binding, and CTX, even at high concen- trations ($ 100 lm), inhibited binding only partially ($ 60% of total specific binding). In contrast, WTX completely inhibited [ 125 I]CTX binding to adrenal membranes, with an IC 50 of about 13 lm (Fig. 3). It was previously shown that WTX inhibits binding of [ 125 I]Bgt to Torpedo nAChR with an IC 50 of 2.2 lm [6]. We directly analyzed the binding of [ 125 I]WTX to Torpedo membranes, and found that this was inhibited by native WTX with an IC 50 of 2.95 lm (data not shown), in good agreement with the previously reported IC 50 value. Taken together, these data show that WTX interacts with both muscle-type and a7-like nAChRs with micromolar affinity, in accord with an earlier report by Utkin et al. [6]. In view of our previous data pointing to mAChR involvement in the biological effects of WTX in vivo [13,14], we tested the influence of muscarinic ligands on [ 125 I]WTX binding to adrenal membranes. After sequential incubation of membranes with CTX, with high concentrations of atropine, and finally with [ 125 I]WTX, specific binding of [ 125 I]WTX was com- pletely inhibited. The same effect was observed if the membranes were incubated first with atropine and then with CTX followed by [ 125 I]WTX. Muscarinic toxin 1 (MT1) and muscarinic toxin 2 (MT2) from Dendroaspis angusticeps competed with [ 125 I]WTX for binding to adrenal membranes, whereas muscarinic toxin 3 (MT3) did not inhibit WTX bind- ing at concentrations up to 10 lm. This competition was characterized by IC 50 values of 31 ± 8 and 52±14nm for MT1 and MT2, respectively (Fig. 4). Fig. 2. Inhibition of [ 125 I]WTX binding to rat adrenal gland mem- branes by different concentrations of WTX. Fig. 3. Inhibition of [ 125 I]CTX binding to rat adrenal gland mem- branes by different concentrations of WTX. Fig. 4. Inhibition of [ 125 I]WTX binding to rat adrenal glands mem- branes by muscarinic toxins MT1 (s) and MT2 (d). D. Yu. Mordvintsev et al. Weak toxin binds two acetylcholine receptor types FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS 5067 Both toxins almost completely (96%) inhibited [ 3 H]atropine and [ 3 H]quinuclidinyl benzilate (QNB) binding to adrenal membranes. The competition with atropine had K i values of 80 ± 36 nm and 1.4 ± 0.3 lm for MT1 and MT2, respectively (data not shown). MT3 did not influence atropine binding at concentrations up to 10 lm. Blockade of WTX binding to adrenal membranes by muscarinic ligands demon- strates a specific interaction of WTX with mAChRs. WTX interaction with heterologously expressed mAChRs To characterize the WTX interaction with mAChR in more detail, we performed competition experiments using five human mAChR subtypes expressed in Chinese hamster ovary (CHO) cells. Radiolabeled competitors were [ 3 H]ACh and methyl-[ 3 H]-N-methyl- scopolamine ([ 3 H]NMS). Data obtained in equilibrium binding experiments were fitted to Eqn (2) (Experimen- tal procedures). The following equilibrium dissociation constants (K d ) were measured and used for fitting: for [ 3 H]NMS binding to the M 1 ,M 2 ,M 3 ,M 4 and M 5 receptors, K d values were, respectively, 240, 320, 235, 225 and 280 pm; for [ 3 H]ACh, the K d values were, respectively, 240, 320, 235, 225 and 280 pm. The calcu- lated WTX-binding parameters, i.e. equilibrium disso- ciation constants (K a ) and factors of cooperativity (a), are summarized in Table 1. These experiments demonstrate that WTX binds to all subtypes of mAChRs with affinities in the sub- micromolar range (Fig. 5, Table 1), although the toxin displays a slight preference for the M 2 and M 5 recep- tors. The cooperativity of binding between WTX and [ 3 H]NMS was either neutral (M 2 ,M 4 , and M 5 )or positive (M 1 and M 3 ), and the cooperativity of binding between WTX and [ 3 H]ACh was either neutral (M 3 )or negative (M 1 ,M 2 ,M 4 , and M 5 ). To further characterize the influence of WTX on sig- nal transduction via different mAChR subtypes, we investigated G-protein activation by ACh in the absence or presence of saturating concentrations (1 lm) of WTX, or by WTX itself (Fig. 6). WTX had no effect on [ 35 S]GTP[cS] binding in control experi- ments using membranes from nontransfected CHO cells. The results summarized in Table 2 show that WTX alone significantly stimulated [ 35 S]GTP[cS] bind- ing with submicromolar potency in transfected cells, although the maximal effect was always less than for ACh. However, at the M 2 ,M 4 and M 5 receptors, WTX potencies in stimulating [ 35 S]GTP[cS] binding represented 34%, 29% and 43% of the E max for ACh. ACh alone stimulated [ 35 S]GTP[cS] binding with Table 1. Parameters of WTX binding to membranes from CHO cells expressing mAChRs. The equilibrium dissociation constant (K a ) and factor of cooperativity (a) were obtained by fitting the data to Eqn (3) (see Experimental procedures). K a and a are expressed as negative logarithms of means ± SEMs from four independent experiments performed in quadruplicate. NE, no effect. [ 3 H]ACh [ 3 H]NMS pK a pa pK a pa M 1 5.83 ± 0.17* )0.081 ± 0.057 6.35 ± 0.02 0.59 ± 0.02 M 2 6.80 ± 0.06 )0.25 ± 0.04 7.13 ± 0.25* )0.08 ± 0.04 M 3 NE NE 6.51 ± 0.03 0.42 ± 0.02 M 4 6.67 ± 0.07 )0.18 ± 0.05 NE NE M 5 6.83 ± 0.05 )0.32 ± 0.03 N0045 NE *P< 0.05, pK a is significantly different from the rest by ANOVA and Tukey–Kramer post hoc test. Fig. 5. Effects of WTX on [ 3 H]NMS and [ 3 H]ACh binding to mem- branes from CHO cells expressing heterologous mAChR subunits. Binding of [ 3 H]ACh (A) and [ 3 H]NMS (B) in the presence of WTX (concentrations as indicated on the x-axis) was to membranes from CHO cells expressing M 1 (circles), M 2 (squares), M 3 (diamonds), M 4 (upwards-pointing triangles) and M 5 (leftwards-pointing trian- gles) receptors; binding levels are expressed as percentage binding in the absence of WTX. Data are means ± SEMs of quadruplicate measurements from three independent experiments. Weak toxin binds two acetylcholine receptor types D. Yu. Mordvintsev et al. 5068 FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS micromolar potency. WTX at 1 lm did not markedly change the [ 35 S]GTP[cS]-binding potency of ACh, with the exception of M 5 receptors, where it caused an approximately three-fold decrease, whereas ACh potency was reduced by 19%, 58% and 46% at the M 1 ,M 5 and M 4 receptors, respectively. In these experiments, there were significant differ- ences in both EC 50 and E max values, according to the individual mAChR subtype expressed. This suggests that the effects of WTX on GTP binding are due to an interaction with the mAChRs, and that WTX does not interact directly with the G-proteins themselves: signifi- cant differences in [ 35 S]GTP[cS] binding according to receptor subtype are not consistent with a direct effect of WTX on G-proteins. Discussion It was previously reported that WTX injection into mice and rats produces signs of intoxication consistent Fig. 6. Stimulation of [ 35 S]GTP[cS] binding to membranes from CHO cells expressing heterologous mAChRs. Binding of [ 35 S]GTP[cS] to membranes from CHO cells expressing the indicated mAChR subtype was performed in the presence of WTX (triangles), ACh (circles) or ACh and 1 l M WTX (squares) at the concentrations indicated on the x-axis. The extent of stimulation is expressed as fold increase over basal [ 35 S]GTP[cS] binding. Data are means ± SEMs of quadruplicate measurements from three independent experiments. D. Yu. Mordvintsev et al. Weak toxin binds two acetylcholine receptor types FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS 5069 with WTX interactions with both nAChRs and mAChRs [13,14]. To determine the organs affected by the toxin in vivo, we studied the distribution of [ 125 I]WTX following intraperitoneal injection into mice. High lev- els of WTX accumulated in the adrenal gland, and this organ was selected for further analysis. Neuronal nAChRs of various types (including a7), mAChRs, adrenoreceptors and some neuropeptide receptors are known to be expressed in the adrenal gland [15–19]; indeed, chromaffin cells are known to be modified postganglionic neurons. However, although a7-type receptor gene expression has been reported in this tissue [17], neither a7 immunoreactivity nor functional a7 receptors have been described. This may be explained by mRNA splicing variations [18] or by a7 complex formation with other subunits to generate a heteromeric a7-like receptor [19]. We then studied [ 125 I]WTX binding to membrane preparation. Binding activity was first tested with Torpedo membranes containing a muscle-type nAChR. Competition experiments with native WTX gave an IC 50 of 2.95 lm, in good agreement with an earlier value of 2.2 lm obtained from [ 125 I]Bgt competition studies [6]. In further experiments, rat adrenal mem- brane preparations were used. Saturable specific bind- ing of [ 125 I]WTX (Fig. 1) was almost completely inhibited by carbachol (not shown), a nonselective ago- nist of nAChRs and mAChRs. In contrast, binding was only partially inhibited by atropine (binding reduced by $ 40%), an antagonist of mAChRs, or by CTX (binding reduced by $ 60%), an antagonist of both muscle-type and a7 nAChRs. These data indicate that [ 125 I]WTX interacts with both mAChRs and nAChRs, in accordance with the two types of adrenal gland binding sites indicated by competition experi- ments using native WTX (Fig. 2). [ 125 I]WTX binding was not inhibited by NTII (short chain a-neurotoxin), a selective antagonist of muscle-type nAChRs. These results suggest that the adrenal gland expresses an a7 or a7-like nAChR with affinity for WTX in the low micromolar range. The results of radioligand analysis also clearly show that binding of WTX to nAChRs is orthosteric, in good agreement with recent modeling data [20]. Competition experiments with CTX indicated that a7ora7-like nAChRs represented only about 60% of the [ 125 I]WTX-binding sites in adrenal membranes. Binding to the remaining 40% was inhibited by the muscarinic antagonist atropine and by the nonselec- tive muscarinic agonist oxotremorine M, suggesting that WTX also interacts with mAChRs. To further characterize the WTX interaction with mAChRs, the muscarinic toxins MT1, MT2 and MT3 from D. angusticeps were used: MT3 had no effect on bind- ing, whereas MT1 and MT2 displaced 40% of bound [ 125 I]WTX with IC 50 values of 31 ± 8 nm and 52±14nm, respectively (Fig. 4). Both MT1 and MT2 are reported to bind efficiently to the M 1 recep- tor and less effectively to the M 3 [21] and M 4 recep- tors [22]. MT3 is a selective ligand of the M 4 receptor, with significantly lower affinity for the M 1 receptor [23]. Our results clearly demonstrate that WTX interacts with mAChRs in adrenal membranes, but do not allow the precise mAChR subtype to be determined. The literature regarding mAChR isotyp- ing in the adrenal gland is also controversial. Tobin et al. [24] reported that M 1 is the predominant sub- type in the canine adrenal gland, whereas Ballesta et al. [25] associated muscarinic ligand-binding sites in the bovine and feline adrenal glands with the M 2 sub- type. Functional tests have identified M 1 ,M 3 and M 4 receptors in rat adrenal glands [26,27]. It is notable that radioactive WTX failed to accumulate at high levels in either heart or trachea, tissues that are known to contain mAChRs of various types. This may be explained by low receptor expression levels and ⁄ or by the relatively low affinity of WTX for mAChRs ($ 0.1–1 lm). Table 2. Parameters of [ 35 S]GTP[cS] binding to the membranes from CHO cells expressing mAChRs. Half-efficiency concentrations (EC 50 ) and maximal effect (E max ) values were obtained by fitting data to Eqn (4) (see Experimental procedures). EC 50 (expressed as negative loga- rithm) and E max are means ± SEMs from four independent experiments performed in quadruplicate. WTX ACh ACh + 1 l M WTX pEC 50 E max pEC 50 E max pEC 50 E max M 1 6.62 ± 0.07 1.18 ± 0.05* 5.58 ± 0.04 2.06 ± 0.09 5.60 ± 0.04 1.86 ± 0.08** M 2 6.75 ± 0.03 1.39 ± 0.03* 5.54 ± 0.04 2.14 ± 0.15 5.47 ± 0.03 2.04 ± 0.10 M 3 6.43 ± 0.10 1.11 ± 0.05*** 5.56 ± 0.05 1.96 ± 0.08 5.58 ± 0.04 1.86 ± 0.07 M 4 6.08 ± 0.04 1.34 ± 0.03* 5.61 ± 0.04 2.19 ± 0.10 5.57 ± 0.04 1.50 ± 0.08** M 5 6.62 ± 0.03 1.41 ± 0.04* 5.62 ± 0.05 1.95 ± 0.08 5.15 ± 0.04* 1.52 ± 0.07** * P < 0.01, significantly different from basal values by one-tailed t-test; ** P < 0.05, significantly different from control without WTX (ACh alone) by ANOVA and Tukey–Kramer post hoc test; *** P < 0.05, significantly different from basal values by one-tailed t-test. Weak toxin binds two acetylcholine receptor types D. Yu. Mordvintsev et al. 5070 FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS To characterize the WTX interaction with mAChR subtypes in more detail, we investigated binding to human mAChR subtypes expressed in CHO cells. WTX at concentrations up to 3 lm was found either to have no effect on, or to increase, the binding of the orthosteric antagonist [ 3 H]NMS. WTX at concentra- tions up to 30 lm was not able to displace completely the natural orthosteric agonist [ 3 H]ACh. WTX there- fore exerts allosteric effects on the binding of both ligands. Analysis of the results of competition experi- ments (Fig. 5) using a ternary allosteric model demon- strated that WTX can bind to all subtypes of mAChRs with submicromolar affinities. Experiments with the competitor ligand NMS revealed partial WTX selectivity for M 2 receptors, whereas a different ligand, ACh, revealed some WTX selectivity for the M 2 ,M 4 and M 5 receptors (Table 1). The observed competition between WTX and atropine for binding to adrenal membranes most probably also occurs through an allo- steric mechanism. WTX displayed neutral or positive cooperativity with NMS and neutral or negative cooperativity with ACh. A general feature of allosteric modulation is that both the direction and magnitude of cooperatively are remarkably specific for a given combination of alloste- ric ligand, orthosteric ligand (in our case WTX and NMS or ACh, respectively), and receptor subtype [28,29]. The allosteric action of WTX argues that the toxin binds to a domain distinct from the orthosteric binding site on the mAChR. A diverse range of musca- rinic allosteric ligands have been reported to bind to the extracellular portion of the receptor [30,31]. It is notable that mamba muscarinic toxins, which are simi- lar to WTX with respect to polypeptide chain length and general folding patterns, also interact allosterically with mAChRs. For example, muscarinic toxin 7 (MT7) was found to demonstrate clear negative cooperativity with NMS [32]. It is, however, possible that the binding sites for WTX and muscarinic toxins only partially overlap. WTX alone is able to activate mAChRs, as demon- strated by WTX-mediated stimulation of [ 35 S]GTP[cS] binding. The level of G-protein activation by WTX was lower than that produced by the full agonist ACh. However, at the M 2 ,M 4 and M 5 receptors, the level of G-protein activation reached a significant fraction (34%, 29%, and 43%, respectively) of E max of ACh. Because WTX targets an allosteric binding site, WTX can be considered to be an allosteric partial agonist. Activation of mAChRs by allosteric ligands has been reported previously [33]. WTX did not change the potency of ACh stimulation of GTPcS binding at M 1 – M 4 receptors, in agreement with neutral or weak nega- tive cooperativity between WTX and ACh at these receptors. At M 5 receptors, WTX decreased ACh potency three-fold, in agreement with an approxi- mately three-fold decrease in ACh binding (Table 1). It markedly decreased ACh potency at M 1 ,M 4 and M 5 receptors, demonstrating not only that the toxin changes the receptor affinity for ACh, but also that the activity of the ternary WTX–ACh–receptor com- plex is different from that of the binary WTX–receptor or ACh–receptor complexes, supporting the theory that the receptor has multiple active conformations [34]. It was previously reported that c-bungarotoxin, a nonconventional toxin from another snake genus (Bungarus multicinctus), can interact with both nAChRs and mAChRs [8]. The toxin inhibited [ 125 I]Bgt binding to Torpedo nAChR with an IC 50 of $ 100 nm, and inhibited the binding of [ 3 H]QNB to rat ventricle membranes (M 2 receptor) with an IC 50 also of $ 100 nm [8]. No competition was observed between c-bungarotoxin and [ 3 H]QNB for binding to the M 1 receptor. As compared with WTX, c-bungaro- toxin displayed a higher affinity for Torpedo nAChRs and slightly lower affinity for the M 2 receptor. We have shown here that WTX interacts with all mAChR subtypes and, moreover, that WTX is itself able to activate mAChRs by binding to allosteric site(s). Whereas the allosteric influence of WTX on receptor affinity for ACh was generally marginal, the allosteric modulator thiochrome was observed to alter ACh affinity for the M 4 receptor by an order of magnitude, as reported here for WTX at the M 5 receptor, and thiochrome was shown to have a significant effect on ACh release in rat striatal slices [35]. In addition, drugs acting as ectopic agonists induced only about one-third of the maximal increase of GTPcS binding evoked by the full agonist acting via M 1 receptors in intact CHO cells [36] or via M 2 receptors in rat cortical slices [37]. However, the role of perturbed muscarinic transmis- sion in the pathophysiology of WTX poisoning will require detailed physiological studies. It is noteworthy that nonconventional toxins form a phylogenetically heterogeneous group, and c-bungaro- toxin and WTX belong to different groups: II and V, respectively [38,39]. WTX and other cobra weak toxins occupy a phylogenetically intermediate position between snake muscarinic toxins and long a-neurotox- ins, whereas c-bungarotoxin is more similar to long a-neurotoxins. This similarity may explain the higher c-bungarotoxin affinity for nAChRs. Because WTX targets two different receptor types, it may be regarded as a multifunctional or multitarget three-finger toxin. Multitarget three-finger toxins have D. Yu. Mordvintsev et al. Weak toxin binds two acetylcholine receptor types FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS 5071 been described previously; for instance, MT1 and MT2 also affect target a-adrenergic receptors [22]. Multi- functionality is not unique to toxins. For example, the intracellular calcium sensor calmodulin controls many important biological functions, ranging from gene transcription and neurotransmitter release to muscle contraction and cell survival [40–42], by binding to hundreds of target proteins. It regulates the activity of diverse cellular proteins (enzymes, ion channels, tran- scription factors, and cytoskeletal proteins) but partici- pates in G-protein-coupled receptor signaling via a different mechanism. However, highly active proteins, including toxins generally, have a single major function. MT7 is a good example of a three-finger toxin with high selectivity. It selectively and potently targets M 1 mAChR. To achieve this high selectivity and affinity, evolution has ‘polished’ the toxin molecule to fit only one specific binding site. In contrast to MT7, WTX binds to different binding sites with comparatively low affinity. However, targets are available in several organs of the prey or in different prey species. Therefore, the significance of multitargeted toxins for prey capture may be greater than that of highly specific toxins. In summary, we report for the first time the use of an 125 I-labeled derivative of a weak (nonconventional) toxin to study the in vivo distribution of binding sites in mice; this demonstrated high levels of accumulation in the adrenal glands. Binding of radioactive WTX to rat adre- nal membranes was analyzed in detail. We have shown for the first time that WTX from cobra venom interacts with both mAChRs and nAChRs. However, the mecha- nism of the interaction is different at the two receptor types: the WTX interaction with nAChRs is orthosteric, whereas the interaction with mAChRs is allosteric. Experimental procedures WTX and CTX were purified from the N. kaouthia venom as described previously [6]. Chloramine T was from Serva (Hei- delberg, Germany), acetonitrile from Kriochrom (St Peters- burg, Russia), trifluoroacetic acid from Merck (Darmstadt, Germany), dithiothreitol from BioRad (Hercules, CA, USA), oxotremorine M from Sigma, [ 3 H]atropine and [ 3 H]QNB from Amersham, [ 3 H]ACh from ARC, and [ 3 H]NMS and [ 35 S]GTP[cS] from NEN. Other reagents were of the highest commercially available quality. nAChR-enriched membranes from the electric organs of Torpedo californica were kindly provided by F. Hucho (Free University of Berlin, Germany). Radioactive toxin derivatives Toxin iodination was performed using the chloramine T method under the modified conditions of Wang and Schmidt [43]. In brief, 20 lL of aqueous potassium iodide (0.7 mgÆmL )1 ) solution, 40 lL of 0.15 m sodium phosphate buffer (pH 7.4) containing 20 MBq Na 125 I (Leipunovski Physical Energetic Institute, Obninsk, Russia) and 36 lLof aqueous chloramine T (0.5 mgÆmL )1 ) were added to 200 lL of toxin (1 mgÆmL )1 ) in 0.2 m sodium phosphate buffer (pH 7.4). After 7 min of incubation, the mixture was applied to a Reprosil Pur C18-AQ column (4.6 · 250 mm), and iodinated toxin was isolated by elution with a gradient of acetonitrile in water (from 20% to 50% over 60 min) in the presence of 0.1% trifluoroacetic acid. Acetonitrile was removed by evaporation on a SpeedVac concentrator (Savant Instruments, Holbrook, NY, USA), and the iodin- ated toxin was dissolved in 0.2 m sodium phosphate buffer (pH 7.4). The CTX and WTX derivatives had specific radioactivities of 404 and 220 MBqÆlmol )1 , respectively. MALDI MS measurements showed that the product was a di-iodinated WTX derivative. Animals Adult virgin female mice (28–31 g body weight) and adult male mice (specific pathogen-free; 30–35 g body weight) were employed for radioactive toxin distribution studies. Virgin female Wistar rats (270–300 g body weight) were used for adrenal membrane preparations. Animals were maintained on a 12 h : 12 h light:dark cycle with food and water ad libitum. Membrane preparations Adrenal membranes were obtained as described previously [44]. The procedure was performed in a cold room, and all labware was washed with 1 mm EDTA (pH 7.0) before use. Animals were killed by decapitation, and dissected organs were homogenized in two volumes of cold buffer (10 mm sodium phosphate buffer, pH 7.4, 1 mm magnesium chlo- ride, 30 mm sodium chloride, 1 mm dithiothreitol, 0.005 mm phenylmethanesulfonyl fluoride, and DNase I) using a Potter S homogenizer (B. Braun Melsungen AG, Germany). The suspension was layered onto aqueous sucrose solution (41% w ⁄ v) and centrifuged for 1 h in a Beckman SW27 rotor at 95 000 g. The material retained at the interface was collected, resuspended in 10 volumes of buffer, and centrifuged for 20 min under the same condi- tions. Membranes were aliquoted and stored at )72 °C. Protein concentrations were determined as described by Peterson [45]. Body distribution of [ 125 I]WTX in mice Radioactive WTX ($ 20 kBq) was injected intraperitoneally at a dose of 20 mgÆkg )1 in a volume of 130 l L. This dose is not lethal for mice [13]. Animals were killed by cerebral Weak toxin binds two acetylcholine receptor types D. Yu. Mordvintsev et al. 5072 FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS dislocation at 5, 10, 15, 20 or 30 min postinjection. Three animals were used for each time point. Brain, spinal cord, heart, lungs, diaphragm, liver, kidney, adrenal and thyroid glands, adipose tissue and hip muscles were dissected. Blood was taken from the liver artery. Urine was also col- lected. Organs were washed three times in water and counted on a Compugamma c-counter (LKB, Sweden). Radioligand analysis Binding to adrenal membranes Suspensions of adrenal membranes (1.1 mg proteinÆmL )1 ) were incubated with different concentrations of [ 125 I]WTX for 1 h at room temperature in 50 lLof50mm Tris ⁄ HCl (pH 8.0) containing 0.02% BSA. The mixture was filtered through GF ⁄ F filters (Whatman, UK) presoaked for 2 h in 0.5% aqueous polyethyleneimine. Filters were washed three times with 1 mL of 50 mm Tris ⁄ HCl containing 0.1% Tween-20, and bound radioactivity was determined by c-counting. For competition experiments, different concen- trations of competitor compound were incubated for 1 h at room temperature with the adrenal membrane suspension (1.1 mg proteinÆmL )1 )in50mm Tris ⁄ HCl (pH 8.0) and 0.02% BSA. [ 125 I]WTX (16 pmol) was added to give a final volume of 50 lL; the mixture was incubated for 1 h at room temperature before being filtered through GF ⁄ F fil- ters. Filters were washed as above, and bound radioactivity was determined. Nonspecific binding was measured in the presence of 100 lm WTX. The equations used to calculate binding parameters were Eqn (1) for one binding site and Eqn (2) for two binding sites (see below). Binding to Torpedo membranes Varying concentrations of WTX or CTX were incubated for 1 h at room temperature with a suspension of Torpedo membranes (5 pmol of toxin-binding sites) in 50 mm Tris ⁄ HCl (pH 8.0) containing 0.02% BSA. [ 125 I]WTX (16 pmol) was added to a final volume of 50 lL, and this was followed by 1 h of incubation and filtration through GF ⁄ F filters (Whatman, UK) as before. Nonspecific bind- ing was determined in the presence of 100 lm WTX. [ 3 H]Atropine and [ 3 H]QNB binding Competition experiments using 3 H-labeled low molecular weight muscarinic ligands and muscarinic toxins from Dendroaspis venom were performed essentially as described above for [ 125 I]WTX competition binding, with the difference that [ 3 H]atropine and [ 3 H]QNB were incu- bated with the membranes for 1 h prior to the addition of different concentrations of muscarinic toxins. Nonspe- cific binding was determined in the presence of 100 lm atropine. WTX interaction with heterologously expressed mAChRs CHO cell membranes expressing individual subtypes of human mAChRs were prepared, and all binding measure- ments were performed essentially as described by Jakubik et al. [46]. Briefly, radioligand-binding experiments were carried out in quadruplicate in 96-well-plates (round-bot- tomed; Packard) at 30 °C in incubation medium consisting of 100 mm NaCl, 10 mm MgCl 2 , and 20 mm Na-Hepes (pH 7.4), supplemented with fresh 1 mm dithiothreitol, for [ 35 S]GTP[cS] binding. The final incubation volume was 200 lL. The affinities of individual mAChR subtypes for [ 3 H]NMS and [ 3 H]ACh were determined in saturation bind- ing experiments using Eqn (1) (below). Allosteric interac- tions of WTX with individual mAChR subtypes were determined in competition experiments using a fixed con- centration (well below K d )of[ 3 H]NMS (125 pm)or [ 3 H]ACh (5 nm). Nonspecific binding was determined in the presence of 10 lm cold NMS. Incubations for lasted 60 min. Data were analyzed using Eqn (3) (below). For determination of [ 35 S]GTP[cS] binding to G-proteins, final [ 35 S]GTP[cS] concentrations were 200 pm (for M 1 ,M 3 and M 5 receptors) or 500 pm (for M 2 and M 4 receptors), in the presence of 5 lm (M 1 ,M 3 and M 5 receptors) or 50 lm (M 2 and M 4 receptors) of GDP. Nonspecific binding of [ 35 S]GTP[cS] was determined in the presence of 1 lm non- radioactive GTPcS. Incubations were for 20 min at 30 °C. Data were analyzed using Eqn (4) (below). In control experiments using membranes from nontransfected CHO cells, WTX had no effect on [ 35 S]GTP[cS] binding: retained radioactivities were 6505 ± 130, 6615 ± 98 and 6416 ± 233 d.p.m. in control membranes and in the presence of 1 or 10 lm WTX, respectively [means ± standard errors of the mean (SEMs), n = 6]. Incubations were terminated by filtration through glass fiber Whatman GF ⁄ F filters using a Tomtech Mach III cell harvester. Filters were dried under vacuum for 1 h, and heated to 80 °C, and the solid scintillator Meltilex A was then melted onto the filters (105 ° C, 90 s) using a hotplate. Filters were cooled and counted using a Wallac Microbeta scintillation counter. The following equations were used to evaluate data: For one-site or two-site saturable radioligand binding: y ¼ B max x=ðK d þ xÞð1Þ y ¼ B max x=ðK d þ xÞþB 0 max x=ðK 0 d þ xÞð2Þ where y is the binding of radioactive ligand at concentra- tion x, K d is the equilibrium dissociation constant of radio- ligand, and B max is maximal binding. For the allosteric interaction between WTX and [ 3 H]NMS or [ 3 H]ACh (high-affinity binding): D. Yu. Mordvintsev et al. Weak toxin binds two acetylcholine receptor types FEBS Journal 276 (2009) 5065–5075 ª 2009 The Authors Journal compilation ª 2009 FEBS 5073 y ¼ 100 ½C ðLÞ þ K dðLÞ =½C ðLÞ þ K dðLÞ ðK a þ xÞ=ðK a þ x=aÞ ð3Þ where y is the binding of radioligand L ([ 3 H]NMS or [ 3 H]ACh) in the presence of WTX at concentration x nor- malized to the absence of WTX, K d is the equilibrium dis- sociation constant of the radioligand, K a is the equilibrium dissociation constant of WTX, and a is the cooperativity factor between radioligand and WTX. For the concentration response of [ 35 S]GTP[cS] binding induced by ligands: y ¼ 1 þðE max À 1Þ=½ 1 þðEC 50 À xÞ nH  ð4Þ where y is [ 35 S]GTP[cS] binding in the presence of ACh at concentration x, expressed as fold increase of binding in the absence of ACh, E max is the maximal effect, EC 50 is the concentration of ACh producing 50% of maximal effect, and nH is the Hill coefficient. Acknowledgements We are grateful to F. Hucho (Berlin) for Torpedo membranes. 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Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors Dmitry Yu. Mordvintsev 1 ,. CTX, a-cobratoxin; mAChR, muscarinic acetylcholine receptor; MT1, muscarinic toxin 1; MT2, muscarinic toxin 2; MT3, muscarinic toxin 3; MT7, muscarinic toxin