Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates

Abstract

We identify two heteroallelic mutations in the acetylcholine receptor ␦-subunit from a patient with severe myasthenic symptoms since birth: a novel ␦D140N mutation in the signature Cysloop and a mutation in intron 7 of the ␦-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that ␦D140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the ␣-, ␤-, and ⑀-subunits also suppresses apparent channel gating, but the suppression is much greater in the ␣-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that ␣Asp-138 is energetically coupled to ␣Arg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved ␣Asp-138 and ␣Arg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.Congenital myasthenic syndromes are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Although no fewer than 20 congenital myasthenic syndrome disease genes have been identified, most congenital myasthenic syndromes arise from mutations in muscle acetylcholine receptor (AChR) 2 subunits (1). The muscle AChR belongs to the cystineloop (Cys-loop) superfamily of receptors and is a heteropentamer composed of homologous subunits with stoichiometry (␣1) 2 ␤1␦␥ in fetal and (␣1) 2 ␤1␦⑀ in adult muscle (2). Each subunit consists of an N-terminal extracellular domain composed of 10 ␤-strands that form inner and outer ␤-sheets, a transmembrane domain of four ␣-helices (M1-M4), a large cytoplasmic domain between M3 and M4, and an extracellular C-terminal tail.The AChR Cys-loop divides the inner from the outer ␤-sheets of the extracellular domain and is situated at the interface between the extracellular and pore domains. Its name derives from a disulfide bond between a pair of cysteine residues separated by 13 residues, four of which are highly conserved. One of these conserved residues, Asp-138 in the muscle AChR ␣-subunit, is present at equivalent positions in all subtypes and species of AChR subunits (Fig. 1A) and also across all subunits of all species of the Cys-loop receptor superfamily. However, little is known about the contributions of the Cysloop Asp of the different subunits to AChR expression and AChR activation.Recent crystal structures of eukaryotic Cys-loop receptors show that the residue equivalent to ␣Asp-138 localizes within the hydrophobic core of the subunit, where it establishes electrostatic contact with an invariant cationic residue equivalent to ␣Arg-209 in the pre-M1 domain of the muscle AChR (3). In addition, anionic residues from the ␤ 1-...

Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

“…In particular, the present experiments are performed in the absence of extracellular calcium, whereas calcium was present in the rapid application measurements. Evidence that extracellular calcium could affect the activation kinetics comes from determinations of the mean burst duration, which is 1.29 ms in the present work and 3.3 ms in previous work done in the presence of calcium ( Shen et al, 2012 , 2016 ). Referring to Eq.…”

“…ACh associates with the resting state of the wild-type AChR approximately 10-fold slower than the rate of diffusion, suggesting the binding site is not fully accessible to the surrounding solvent or dynamic conformational changes restrict agonist access. The rate constants are similar to those based on a scheme with a direct transition between closed and open states ( Hatton et al, 2003 ; Lee et al, 2009 ; Shen et al, 2016 ), although they are not expected to depend strongly on the presence of closed state intermediates or to require the ultimate in temporal resolution. The association rate constants do not contain a statistical factor, owing to structural nonequivalence of the binding sites, although such a factor would change the rate constants by a factor of two.…”

Mutations causing congenital myasthenia reveal principal coupling pathway in the acetylcholine receptor ε-subunit

“…from 3 extracellular loops surround an invariant Arg residue from the pre-M1 region ( Figure 1A) (8,(11)(12)(13)19). In the AChR, αR209 (aligned position equivalent to εR218) juxtaposes the conserved αE45 (aligned position equivalent to εE45) at the apex of the β1-2 loop (14), the conserved αE175 (aligned position equivalent to εE184) of the β8-9 loop (15), and the invariant αD138 (aligned position equivalent to εD138) of the Cys-loop (20) (Figure 1A). However, while these 3 negatively charged residues couple strongly to αR209 in promoting efficient and rapid channel opening, a key question is whether residues at equivalent aligned positions in the ε-subunit show similar functional coupling.…”

Structure-function analysis of interallelic complementation inROOTYtransheterozygotes

“…To our knowledge, this is the first time that these types of approaches have been used to structurally characterize transheterozygous mutations in plants. In other model organisms, the structural basis of interallelic complementation has been explored only in a handful of cases, such as for myosin light chain protein Cdc4p in Schizosaccharomyces pombe and acetylcholine receptor AChR in humans (Slupsky et al, 2001; Shen et al, 2016). We argue that combining genetics with structural modeling yields unique insights into the function of proteins harboring amino acid substitutions, as demonstrated by the characterization of mutant proteins encoded by the missense alleles of RTY and their interallelic combinations (see below).…”