Mutation causing congenital myasthenia reveals acetylcholine receptor beta/delta subunit interaction essential for assembly

Abstract

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the beta subunit gene. One mutation causes skipping of exon 8, truncating the beta subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (beta426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing beta426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that beta426delEQE impairs AChR assembly by disrupting a specific interaction between beta and delta subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the beta subunit is crucial for interaction with the delta subunit. The findings imply that the mutated residues are positioned at the interface between beta and delta subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.

Figure 1

EP fine structure and localization of AChR at patient (a) and control subject (b) EP regions with peroxidase-labeled α-bgt. Note the small nerve terminal, small postsynaptic region, no openings from the primary synaptic cleft into secondary clefts, and patchy and attenuated reaction for AChR in patient EP.

Figure 2

(a) Schematic presentation of the positions of the identified mutations in the AChR β subunit gene. (b) Multiple alignment of the long cytoplasmic loop of AChR subunits around the β1276del9 mutation. β1276del9 predicts an in-frame deletion of EQE codons at 426–428 (underlined). (c) Size fractionation of PstI-digested PCR products amplified from genomic DNA from blood of family members. The β1276del9 mutation results in a 61-bp fragment, whereas the wild-type allele gives rise to a 70-bp fragment. The father and 3 affected siblings are heterozygous for β1276del9. Arrow indicates patient; filled symbols indicate affected individuals. (d) Size fractionation of RT-PCR products of the AChR β subunit gene amplified with primers in exon 7 and 9. The normal transcript yields a 350-bp fragment; skipping of exon 8 results in a 126-bp fragment. Mother and patient carry both transcripts. Skipping of exon 8 was confirmed by direct sequencing.

Figure 3

Haplotype analysis of family members using β1276del9, 5 single nucleotide polymorphisms, and 3 microsatellite markers. The 3 affected siblings carry the same haplotypes for the β subunit gene. The allele with β1276del9 (asterisk) is shown by a solid box; the allele expected to cause skipping of β exon 8 is shown by a dotted box. Distances from the β subunit gene locus are shown in centimorgans (cM). pterm, p-terminal direction; qterm, q-terminal direction.

Figure 4

Expression of pentameric AChRs containing wild-type or mutant β subunits. (a) α-bgt binding to surface receptors on intact HEK cells transfected with the indicated AChR subunits. (b) Total α-bgt binding to saponin-permeabilized cells transfected with the indicated AChR subunits. Amounts of bound [¹²⁵I] α-bgt are normalized to that measured for wild-type AChR (α₂βεδ).

Figure 5

Expression of pentameric AChRs containing mutant β subunits. Local sequences of the mutant β subunits are shown at the top, with the asterisk indicating the patient mutation. (a) α-bgt binding to cell-surface receptors from HEK cells transfected with the indicated β subunits along with complimentary α, ε, and δ subunits. (b) Same determinations as a, but with saponin-permeabilized cells. Amounts of bound [¹²⁵I]α-bgt are normalized as in Figure 4.

Figure 6

Expression of subunit-omitted α₂βδ₂ and α₂βε₂ pentamers containing mutant β subunits. Local sequences of the mutant β subunits are shown at the top, with the asterisk indicating the patient mutation. (a) α-bgt binding to surface receptors on intact HEK cells transfected with either α, β, and δ subunits (to form α₂βδ₂) or α, β, and ε subunits (to form α₂βε₂). For each combination of subunits, the type of β subunit is indicated on the x-axis. (b) Same determinations as a, but with saponin-permeabilized cells. Amounts of bound [¹²⁵I]α-bgt are normalized as in Figure 4.

Figure 7

Sucrose gradient centrifugation of AChRs from saponin-permeabilized cells transfected with the indicated subunits. Following saponin treatment, cells were incubated with [¹²⁵I]α-bgt, washed free of unbound α-bgt, solubilized, and centrifuged on sucrose gradients as described in Methods. Radioactivity in each fraction is normalized to the peak value in each gradient. Arrows and S values indicate α-bgt (1.3 S) and α₂βδε or α₂βε₂ pentamer (9 S) peaks.

Figure 8

Single-channel currents elicited by 10 μM ACh from AChRs containing wild-type or mutant β subunits and complimentary α, δ, and ε subunits. Currents are shown filtered at 8 kHz with openings as upward deflections (a). Corresponding open- and closed-duration histograms (b and c) are shown fitted by the sum of exponentials with the following parameters. Wild-type: open durations, τ_o = 0.65 ms, number of events = 7681; closed durations, τ₁ = 0.45 ms, a₁ = 0.47, τ₂ = 2.5 ms, a₂ = 0.53. Mutant βdelEQE: open durations, τ_o = 0.99 ms, number of events = 2422; closed durations, τ₁ = 0.44 ms, a₁ = 0.39, τ₂ = 3.9 ms, a₂ = 0.61.