Molecular conversion of muscarinic acetylcholine receptor M(5) to muscarinic toxin 7 (MT7)-binding protein

Abstract

Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M(1) muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M(1) receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M(5) subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M(1) receptor. An M(5) receptor construct (M(5)-E(175)Y(184)E(474)), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent K(i) = 24 nM; apparent K(i) for M(1) = 0.5 nM). After screening for different domains and residues, we found a specific residue (P(179) to L in M(5)) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M(5)-EL(179)YE, apparent K(i) = 0.5 nM). Mutation of P(179) to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors.

Keywords: G protein-coupled receptor; acetylcholine receptor; ligand binding; muscarinic toxin.

Figure 1

The M₅ receptor and the M₅-EYE mutant in Sf9 cells were assayed in the absence and presence of muscarinic toxin 7 (MT7). (a and b) Carbachol (CCh) concentration-response curves for [Ca²⁺]_i increases. Data points are means ± SD from two experiments; (c) Amino acid sequences of extracellular loops ECL2 and ECL3. TM4-7 denotes the helical transmembrane domains of the receptors. Introduced mutations in M₅ are in boldface; (d) Schematic presentation of the chimeric receptors used to localize regions of toxin binding.

Figure 2

Sf9 cells expressing mutated M₅ receptor constructs were assayed for carbachol-induced [Ca²⁺]_i increases and inhibition of [³H]NMS binding. (a and c) Functional analyses of the M₅-ELYE and M₅-EAYE receptor constructs in the absence and presence of MT7. Data points are means ± SD from two experiments. The apparent K_i values for MT7 were 0.52 ± 0.14 nM and 4.41 ± 1.09 nM (means ± SD, n = 4) for M₅-ELYE and M₅-EAYE, respectively; (b and d) Cell homogenates of each receptor construct were preincubated with different concentrations of MT7 before addition of 0.5 nM [³H]NMS. Bound radioactivity was converted to receptor concentration and presented as percent of control values in the absence of toxin. Data points are means ± SEM from three experiments.

Figure 3

Sf9 cells expressing the M₅-ELY construct were assayed for carbachol-induced [Ca²⁺]_i increases and inhibition of [³H]NMS binding. (a) Representative concentration-dependent increases in [Ca²⁺]_i for control and in the presence of 50 nM MT7. The apparent K_i was determined to 2.06 ± 0.86 nM (mean ± SD, n = 3); (b) Titration of MT7 against [³H]NMS with M₅-ELY. Data points are means ± SEM from three experiments.

Figure 4

Deceleration of [³H]NMS dissociation by MT7. The M₅-ELY and M₅-ELYE constructs were preincubated with [³H]NMS and dissociation initiated by addition of atropine with or without different concentrations of MT7. Samples were collected at different time points and [³H]NMS binding (B_t) determined. Data presented are from one experiment performed with triplicate samples. Two additional experiments with 10 and 100 nM MT7 gave similar results. Only the trendlines are shown for clarity. The control rate for M₅-ELY was 0.113 min⁻¹ and 0.003 min⁻¹ with 100 nM MT7. The control rate for M₅-ELYE was 0.147 min⁻¹ and 0.004 min⁻¹ with 100 nM MT7.

Figure 5

Reversibility test for MT7. Receptor constructs were preincubated with MT7 (30 nM with M₁ and M₅-ELYE, 100 nM with M₅-ELY) for 45 min and then subjected to [³H]NMS binding (control (C) and zero washes) or centrifuged and washed one to three times before [³H]NMS binding. Data are means ± SEM from three experiments.