Highly fatal fast-channel syndrome caused by AChR ε subunit mutation at the agonist binding site

Abstract

Objective: To characterize the molecular basis of a novel fast-channel congenital myasthenic syndrome.

Methods: We used the candidate gene approach to identify the pathogenic mutation in the acetylcholine receptor (AChR) ε subunit, genetically engineered the mutant AChR into HEK cells, and evaluated the level of expression and kinetic properties of the mutant receptor.

Results: An 8-year-old boy born to consanguineous parents had severe myasthenic symptoms since birth. He is wheelchair bound and pyridostigmine therapy enables him to take only a few steps. Three similarly affected siblings died in infancy. He carries a homozygous p.W55R mutation at the α/ε subunit interface of the AChR agonist binding site. The mutant protein expresses well in HEK cells. Patch-clamp analysis of the mutant receptor expressed in HEK cells reveals 30-fold reduced apparent agonist affinity, 75-fold reduced apparent gating efficiency, and strikingly attenuated channel opening probability (P(open)) over a range agonist concentrations.

Conclusion: Introduction of a cationic Arg into the anionic environment of α/ε AChR binding site hinders stabilization of cationic ACh by aromatic residues and accounts for the markedly perturbed kinetic properties of the receptor. The very low P(open) explains the poor response to pyridostigmine and the high fatality of the disease.

Figure 1. Agonist binding sites of acetylcholine receptor (AChR)

(A) Structural model of extracellular domains of human AChR viewed from the synaptic space indicating positions of Trp residues at the α/δ and α/ɛ binding sites. (B) Side view of the α and ɛ subunits showing position of loops E, D, G, and F in the ɛ subunit, and loops A, B, and C in the α subunit. (C) Stereo view of the binding site showing positions of aromatic residues shrouding the binding pocket. In each panel the mutated ɛTrp55 at the α/ɛ binding site is highlighted in red. (Based on the crystal structure of the ACh binding protein [PDB 1I9B] and lysine scanning mutagenesis delineating the structure of the human AChR binding domain.)

Figure 2. Single-channel currents elicited from human embryonic kidney fibroblast cells transfected with wild-type acetylcholine receptor (AChR) and ɛW55R-AChR

Left: Representative channel openings elicited by 50 nM ACh. Right: Logarithmically binned burst duration histograms fitted to the sum of exponentials. Arrows indicate mean duration of burst components.

Figure 3. Activation kinetics of wild-type and ɛW55R-acetylcholine receptor (AChR) and channel open probabilities

(A, B) Left column shows individual clusters of single-channel currents recorded at indicated ACh concentrations from human embryonic kidney fibroblast (HEK) cells. Right columns show histograms of closed and open durations at each ACh concentration with superimposed probability density functions (smooth curves) generated from a global fit of the scheme to dwell times obtained for the entire range of ACh concentrations. Fitted rate constants are shown in table 2. (C) Channel open probability (P_open) as function of ACh concentration. Symbols and vertical lines indicate means and standard deviations. Smooth curves are P_open predicted by the fitted rate constants in table 2.