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 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.

Keywords: Cys-loop receptor superfamily; congenital myasthenic syndrome; invariant Cys-loop aspartate; invariant pre-M1 arginine; ion channel; mutant cycle analysis; nicotinic acetylcholine receptors (nAChR); patch clamp; receptor structure-function; structure-function.

FIGURE 1.

A, structural model of extracellular domains of human AChR viewed from the intracellular side indicating positions of invariant Cys-loop Asp and pre-M1 Arg residues (based on the crystal structure of the ACh binding protein (Protein Data Bank entry 1I9B) and mutational analyses of the human AChR binding domain (36)). B, side view of the α subunit showing that the pre-M1 Arg serves as a cationic hub linking the Cys-loop, β_1-2 loop, and β_8-9 loop in the AChR α subunit (Protein Data Bank entry 2BG9).

FIGURE 2.

Position of the δD140N mutation in the δ subunit of AChR (Protein Data Bank entry 2BG9). Left, extracellular and transmembrane domains of the δ subunit. Right, structure of the interface between extracellular and transmembrane domain demarcated by a rectangle in the left panel.

FIGURE 3.

Specific α-bgt binding to intact (A and B) and saponin-permeabilized (C) HEK cells transfected with the indicated AChR subunit cDNAs (see “Experimental Procedures”). The results are normalized for α-bgt binding to wild-type AChR (A and B) or wild-type αδ dimers (C) and represent mean ± S.D. (error bars) of at least three experiments.

FIGURE 4.

Single channel currents elicited by ACh from HEK cells expressing wild-type, δD140N, and αD138N AChRs. Left, channel openings are shown as upward deflections. Right, logarithmically binned burst duration histograms fitted to the sum of exponentials. Arrows, peaks of burst components. The values for major components are indicated for each AChR.

FIGURE 5.

Activation kinetics of wild-type (A), δD140N (B), and αD138N AChRs (C). The left column shows representative single channel currents at the indicated AChR concentrations recorded from HEK cells. Bandwidth was 10 kHz. The center and right columns show corresponding histograms of closed and open duration dwell times with superimposed global fits for Scheme 1. Fitted rate constants are shown in Table 2.

FIGURE 6.

A, simulated MEPC at the control EP for wild-type AChR and at the patient EP for δD140N AChR. The rate constants for wild-type and mutant AChRs shown in Table 2 were used in the simulations. Other parameters for the simulation are indicated under “Experimental Procedures.” MEPC decay times for wild-type and δD140N AChR are obtained by exponential fitting of the curves. B, nonlinear relations between the peak MEPC amplitude and the AChR density at the control and the patient's EPs.

FIGURE 7.

A, stereo view of the interface between extracellular domains and transmembrane domains shows spatial disposition of interacting charged residues in Cys-loop, pre-M1, β_1-2 loop, and β_8-9 loop (based on the crystal structure of 5-HT_3A (Protein Data Bank entry 4PIR)). B, a mutant cycle of energetic interaction among αAsp-138 and αArg-209. Single channel currents correspond to each species of AChR elicited by 300 μm ACh. Numbers over the arrows indicate the difference in the apparent gating free energy between the two different AChRs in kcal/mol. S.E. values were computed as described under “Experimental Procedures.” The 95% confidence limit, or twice the S.E., indicates a coupling energy significantly different from zero. The indicated diagonal arrow shows the coupling free energy (ΔΔG_int) for αD138E/αR209K.