[³H]-N-methylscopolamine ([³H]NMS) (76 Ci/mmol) was purchased from Amersham Biosciences (Buckinghamshire, UK). Fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR). Carbamoylcholine chloride (carbachol) and atropine hemisulfate were from Sigma-Aldrich (Helsinki, Finland). Recombinantly expressed MT7 was purified from insect cell medium as described previously [15] and the concentration was determined spectrophotometrically (A_M = 15900 M⁻¹cm⁻¹). Other chemicals used were of analytical grade quality.

The human M₁ and M₅ receptors have been previously described [19]. All site-directed mutagenesis constructs were generated using the PCR technique with specific oligonucleotide primers with desired mutations (TAG, Copenhagen, Denmark). Subcloning was performed using the pBluescript KS vector (Stratagene, La Jolla, CA) or the pFastBac1 vector (Invitrogen, Paisley, UK) and the correct sequences were verified by automated DNA sequencing (Turku Centre for Biotechnology, Turku, Finland). The chimeric receptor CH1 was a fusion of the M₁ and M₅-EYE receptor cDNAs at an ApaI site (nucleotide +474 in M₁ and nucleotide +489 in M₅). The chimeric receptor CH2 was a fusion of the M₁ and M₅-EYE receptor cDNAs between a StuI site in M₁ (nucleotide +811) and a PvuII site in M₅ (nucleotide +1099). All new receptor constructs were finally transferred to the pFastBac1 vector for the generation of recombinant baculoviruses with the Bac-to-Bac baculovirus expression vector system (Invitrogen).

Spodoptera frugiperda Sf9 cells were grown in suspension at 27 °C in Grace’s insect medium (Invitrogen, Paisley, UK) supplemented with 8% heat-inactivated fetal bovine serum (Invitrogen), 50 µg/mL streptomycin (Invitrogen), 50 U/mL penicillin (Invitrogen) and 0.02% Pluronic F68 (Sigma-Aldrich, Helsinki, Finland). For recombinant protein expression, Sf9 cells were plated on tissue culture dishes and allowed to attach. A high-titer virus stock was then added to give a multiplicity of infection of 3-5. For the functional assay, the infections were allowed to proceed for 26 h and cultures used for radioligand binding were harvested after 48 h.

Cells were detached from plates by gentle pipetting, centrifuged 150° g for 4 min, resuspended in a smaller volume growth medium and incubated for 20 min with 4 μM fura-2 acetoxymethyl ester at room temperature. Thereafter, cells were centrifuged 150° g for 4 min, washed once in assay buffer, resuspended in assay buffer and divided into aliquotes. The aliquotes were briefly spun down in a microcentrifuge, the medium was removed and cell pellets were stored on ice until use. The assay buffer was composed of 130 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 10 mM glucose, 1.2 mM MgCl₂, 4.2 mM NaHCO₃, 7.3 mM NaH₂PO₄, 63 mM sucrose and 20 mM MES, pH adjusted to 6.3. At the beginning of the experiment, the cells were resuspended in assay buffer with or without MT7 for 5 min. Thereafter the cells were placed in a cuvette in a thermostatted (27 °C) cuvette holder with magnetic stirring in a fluorescence spectrophotometer (Photon Technology International, Ford, UK) and fluorescence recording at alternating 340 nm and 380 nm (excitation) and 510 (emission) was started. Different concentrations of carbachol were added in a cumulative fashion and calibration of each experiment was performed with 0.04% triton X-100 to measure F_max and 11 mM EGTA to measure F_min. Intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) were calculated from fluorescence obtained at 340 nm according to:

[Ca²⁺]_i = (F − F_min)/(F_max − F) ° 255 nM (K_d for fura-2 − Ca²⁺ complex at 27 °C)     (1)

The Infected cells from monolayer culture were harvested in phosphate-buffered saline solution, centrifuged 700× g and stored as pellets at −70 °C until use. For binding experiments the cells were resuspended in binding buffer (100 mM NaCl, 20 mM HEPES, 10 mM MgCl₂, 1 mM EDTA, pH adjusted to 7.4) and homogenized with an Ultra-Turrax homogenizer. The homogenates (50–100 μg of protein) were incubated with different concentrations (0.01–10 nM) of [³H]NMS in a volume of 150 μL for 45 min at room temperature to determine K_d values for the radioligand. Atropine (10 μM) was used to determine nonspecific binding. The inhibition curves for MT7 were obtained by preincubation of homogenates with different concentrations (0.001–300 nM) of MT7 diluted in binding buffer for 45 min at room temperature. Thereafter 0.5 nM [³H]NMS was added and incubation continued for another 45 min. The concentrations of MT7 reported were obtained in the final reaction volume. When measuring the radioligand dissociation rate, receptors were first preincubated with 1.5 nM [³H]NMS for 30 min. Following this, dissociation was started by addition of 10 μM atropine with or without MT7. Aliquots were removed at regular intervals from 0 to 100 min. In testing the reversibility of MT7 binding, cell homogenates were first incubated with toxin in test tubes for 45 min, thereafter centrifuged 12,000 × g for 30 min and then resuspended in fresh buffer. After 15 min the homogenates were added to the binding reactions with 0.5 nM [³H]NMS or subjected to another round of centrifugation and resuspension. All reactions were terminated by rapid filtration through prewashed GF/B filters followed by five washes with cold buffer containing 180 mM NaCl, 25 mM MgCl₂, and 20 mM HEPES, pH 7.4, in a microplate harvester. Radioactivity on filters was determined in a TopCount microplate scintillation counter (PerkinElmer, Waltham, MA).

Non-linear curve-fitting of the concentration-response data was performed using GraphPad Prism (GraphPad Software, La Jolla, CA). The pIC₅₀ values were obtained through non-linear regression to fit log IC₅₀, and the The pK_x values and the cooperativity factor α were obtained through curve-fitting to the allosteric ternary complex model.

The apparent K_i values in the functional assay were obtained from data fitted to the equation:

Δ[Ca²⁺] = [A]Δ[Ca²⁺]_max/([A](1 + [I]/K_i') + EC₅₀(1 + [I]/K_i))     (2)

[A] and [I] are the concentrations of agonist and inhibitor, respectively, EC₅₀ is the [A] producing half-maximal stimulation and K_i and K_i' are the surmountable (competitive) and the insurmountable (noncompetitive) inhibition constants, respectively [20]. The K_i value gives a measure of the right-shift of the concentration-response curve and the K_i' value gives a measure of the suppression of the maximum response. Only the apparent K_i values are given because the apparent K_i' values did not obey inhibitor concentration dependency, as previously also found when using MT7 as inhibitor [18].

Functional inhibition of receptors by MT7 was assessed by measuring the [Ca²⁺]_i responses in Sf9 cells expressing intact or modified receptor proteins. Activation of the M₅ receptor with carbachol resulted in concentration-dependent increases in [Ca²⁺]_i as determined from fura-2 fluorescence (Figure 1a). MT7 did not affect the activation of the intact M₅ subtype. To try to gain binding of MT7, we introduced two mutations in ECL2 and one in ECL3 of M₅ (M₅-EYE, Figure 1c). The glutamate residue in the proximal part of ECL2 of M₁ (E¹⁷⁰ in M₁, K¹⁷⁵ in M₅) and a tyrosine residue adjacent to the conserved cysteine (Y¹⁷⁹ in M₁, Q¹⁸⁴ in M₅) were both previously recognized as important for MT7 binding [18]. These residues together with the V⁴⁷⁴ to E mutation in ECL3 rendered the M₅ subtype sensitive to MT7 (Figure 1b). The apparent K_i (>20 nM, Table 1) was, however, significantly higher than what we obtained with the M₁ subtype (apparent K_i = 0.45 ± 0.18 nM, mean ± SD, n = 3). This prompted us to look for additional interaction sites between the toxin and the receptor. First we used chimeric M₁:M₅-EYE receptors to locate the region involved (Figure 1d). These chimeras (CH1 and CH2) indicated that we should refocus on the ECL2, since CH2 incorporating the ECL2 of M₁ increased the affinity for MT7 about tenfold as compared to M₅-EYE (Table 1). After screening for individual residues in ECL2 of M₅, we found that the additional P¹⁷⁹ to L (M₅-ELYE) mutation increased significantly the affinity for MT7 (Figure 2a). This result was further confirmed in radioligand binding experiments with a calculated IC₅₀ of 0.6 nM for MT7 (Figure 2b, Table 2). As for the M₁ subtype (IC₅₀ ≈ 0.5 nM) [18,21], MT7 does not compete directly with the orthosteric antagonist and a fraction of the radioligand binding remains even at saturating MT7 concentrations. Thus, the potencies for inhibition are presented as pIC₅₀ values and not converted to K_i values as for competitive ligands (Table 2).

The P¹⁷⁹t to L mutation in the middle of ECL2 of the M₅ receptor appeared to have an important role in the binding of MT7. The corresponding residue of the M₁ receptor we did not specifically investigate in our previous study, and the M₃ double mutant with the corresponding P²¹⁷ to L and the additional P²¹⁸ to A mutations did not show an inhibitory effect of MT7 [18]. To further investigate whether the aliphatic side chain of the leucine is important for the binding or if the proline only postures a steric hindrance for other interactions to take place, we mutated the P179 to A and analyzed the receptor construct in the functional assay (Figure 2c) and with radioligand binding (Figure 2d, Table 2). MT7 was found to possess lower potency for M₅-EAYE in both assays when compared to M₅-ELYE, indicating a contribution to the overall binding for the leucine side chain.

In our previous study using chimeric M₁:M₃ receptors, we found that a lysine residue (K⁵²³) in M₃ ECL3 sequence resulted in a tenfold loss in the apparent K_i value for MT7 [18]. It was not clear, however, whether this was due to a loss of binding contact with the corresponding negatively charged glutamate (E³⁹⁷) of M₁ or if the long acyl chain and positively charged head group of the M₃ lysine somehow precluded other types of interactions between the receptor and the toxin. In the M₅ receptor, the corresponding residue is a valine (V⁴⁷⁴), which should at least not exhibit any charge repulsion. We thus exchanged the mutated ECL3 of M₅-ELYE for the intact M₅ sequence and measured the binding characteristics of MT7 (Figure 3). In the functional assay, the M₅-ELY construct lost an approximately fourfold blocking potency (apparent K_i = 2.06 ± 0.86 nM, mean ± SD, n = 3) as compared to M₅-ELYE, and about tenfold loss in binding affinity (Table 2). In addition to the loss in binding affinity, we noticed that the inhibitory capacity of MT7 in terms of displacement of radioligand was significantly lower than for the other receptor constructs, i.e., the remaining bound radioligand in the presence of saturating concentrations of MT7 mounted to about 30% for M₅-ELY (Figure 3b), whereas for M₅-ELYE (Figure 2b) and for M₁ the corresponding level was about 15%. We do not currently have an explanation for these different behaviors.

In addition to the high affinity binding of MT7 to the M₁ receptor, the characteristics of this toxin−receptor complex also include an allosteric slowing of antagonist dissociation [22,23], and a pseudo-irreversibility of binding, i.e., the toxin does not dissociate from the receptor in spite of extensive washing [7,24]. To find out if our M₅ constructs exhibited similar properties, we first tested the ability to slow down the dissociation of prebound [³H]NMS. With both M₅-ELY and M₅-ELYE there was a concentration-dependent deceleration of antagonist dissociation (Figure 4). The pIC_50,diss values (affinity for [³H]NMS-occupied receptor) were 7.73 ± 0.06 and 8.47 ± 0.06 for M₅-ELY and M₅-ELYE, respectively. We also tested the irreversibility of MT7 binding at M₁ and the mutated M₅ constructs. We have previously used a protocol with repeated centrifugations to demonstrate the reversibility of MTα binding to the α_2B adrenergic receptor [25]. Using the same protocol for MT7, we could not recover control binding sites with either M₁ or the mutated M₅ constructs (Figure 5). These data indicate that the M₅ constructs have been converted to M₁ phenotypes in the known pharmacological aspects of MT7 binding.