Abstract
We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-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 δD140N 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 α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.
Highlights
Congenital myasthenic syndromes are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms
The muscle acetylcholine receptor (AChR) belongs to the cystineloop (Cys-loop) superfamily of receptors and is a heteropentamer composed of homologous subunits with stoichiometry (␣1)21␦␥ in fetal and (␣1)21␦⑀ in adult muscle [2]
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]
Summary
Mutation Analysis—We directly sequenced muscle AChR ␣-, -, ␦-, and ⑀-subunit genes using the patient’s genomic DNA. The resulting global set of open and closed dwell times from wild-type and mutant AChRs was analyzed using the program MIL (QuB suite), which uses an interval-based maximum likelihood method that corrects for missed events [16] to yield fitted rate constants in a kinetic scheme for receptor activation. The rate constants for the wild-type and mutant AChRs derived from kinetic analysis of open and closed dwell times were applied to the model. These terms are related to the free energy of interresidue interaction, ⌬⌬Gint, as follows: ⌬⌬GXY ϭ ⌬⌬GX ϩ ⌬⌬GY ϩ ⌬⌬Gint [24, 25] Given this relationship and noting that ⌬⌬Gs for the individual mutant AChR is ⌬GX Ϫ ⌬GW, ⌬GY Ϫ ⌬GW, ⌬GXY Ϫ ⌬GW, ⌬⌬Gint can be calculated from ϪRTln((W ϫ XY)/(X ϫ Y)), where is the apparent channel gating equilibrium constant for wild-type (W) and mutant (X, Y, and XY) AChRs [4, 26]. The parents of the patient reported in this study provided informed consent to participate
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