Congenital myasthenia-related AChR delta subunit mutation interferes with intersubunit communication essential for channel gating

Abstract

Congenital myasthenias (CMs) arise from defects in neuromuscular junction-associated proteins. Deciphering the molecular bases of the CMs is required for therapy and illuminates structure-function relationships in these proteins. Here, we analyze the effects of a mutation in 1 of 4 homologous subunits in the AChR from a CM patient, a Leu to Pro mutation at position 42 of the delta subunit. The mutation is located in a region of contact between subunits required for rapid opening of the AChR channel and impedes the rate of channel opening. Substitutions of Gly, Lys, or Asp for deltaL42, or substitutions of Pro along the local protein chain, also slowed channel opening. Substitution of Pro for Leu in the epsilon subunit slowed opening, whereas this substitution had no effect in the beta subunit and actually sped opening in the alpha subunit. Analyses of energetic coupling between residues at the subunit interface showed that deltaL42 is functionally linked to alphaT127, a key residue in the adjacent alpha subunit required for rapid channel opening. Thus, deltaL42 is part of an intersubunit network that enables ACh binding to rapidly open the AChR channel, which may be compromised in patients with CM.

Figure 1. Structural model of extracellular and transmembrane domains of the AChR α and δ subunits (Protein Data Bank code 2BG9).

αW149 is at the center of the ligand binding site. In the transition zone, the indicated N41 and L42 residues are in the β₁ strand of the δ subunit. δN41 is near Y127 in the α subunit.

Figure 2. Acetylcholinesterase-reactive EP regions.

Note dispersion of EP regions over an extended length of the muscle fiber. Scale bar: 20 μm.

Figure 3. EM localization of AChR with peroxidase-labeled α-bgt at patient (A) and control (B) EPs.

Scale bars: 1 μm.

Figure 4. Mutation analysis.

(A) Multiple sequence alignments of the β₁, β₂, and β₄ strands of AChR subunits. L42 is conserved in all human AChR subunits and in δ subunits of all species. Note the δL42P, δI58K, and δV93L in the β₁, β₂, and β₄ strands, respectively. (B) Family analysis. The parents and 2 sisters (all heterozygous for δL42P; half-shaded symbols) of the proposita (closed circle; arrow) were asymptomatic.

Figure 5. α-bgt binding studies.

(A) [¹²⁵I]α-bgt binding to surface receptors on intact HEK cells transfected with the indicated AChR subunits. The results were normalized to results from α-bgt binding to wild-type AChR (α₂βδε) and represent mean ± SD of 3–6 experiments. (B) Total α-bgt binding to saponin-permeabilized cells transfected with the indicated subunit cDNAs. Amounts of bound [¹²⁵I]α-bgt were normalized to that measured for the wild-type dimer (αδ).

Figure 6. Single-channel currents elicited by 50 nM ACh from HEK cells expressing wild-type and mutant AChRs.

Left: Representative channel openings, shown as upward deflections. Right: Logarithmically binned burst-duration histograms fitted to the sum of exponentials. Arrows indicate mean durations of burst components.

Figure 7. Activation kinetics of wild-type and δL42P AChR and open channel probabilities.

(A and B) Left: Representative single-channel currents at the indicated ACh concentrations recorded from HEK cells expressing the indicated AChRs. Currents are shown as upward deflections; bandwidth, 10 kHz. Center and right: Histograms of closed and open durations corresponding to each ACh concentration are shown with the 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 4. On the y axes, each histogram entry is the probability of occurrence on a square root (Sqrt.) scale. (C) P_open as function of ACh concentration. Symbols and vertical lines indicate means ± SD. Smooth curves indicate the P_open predicted by the fitted rate constants shown in Table 4.

Figure 9. Structural model of the AChR α and δ subunits and mutant cycle analyses.

(A) An enlarged view of the coupled intersubunit residues αY127 and δN41 in the structural model of the Torpedo AChR (Protein Data Bank code 2BG9). (B) A mutant cycle for the mutations αY127T, δL42P, and εL40P. Single-channel currents correspond to each AChR elicited by 100 μM ACh, as in Figure 6. Changes in gating free energy along each limb of the cycle are shown, and the overall coupling free energy (ΔΔG_int) in units of kcal/mol computed from –RTln[(θ_wwθ_mm) / (θ_wmθ_mw)], where θ (β/α) is a gating equilibrium constant for diliganded receptors for wild-type, single-mutant, or double-mutant AChRs (Table 4). Horizontal bar indicates 20 ms for wild-type and 100 ms for mutant AChRs. Vertical bar indicates 5 pA. (C–E) Mutant cycles for the indicated mutations are shown, as described in B.

Figure 8. Kinetic scheme for AChR activation.

AChR activation involves the reversible binding of 2 molecules of agonist (A) to the AChR in the resting, closed state (R), followed by reversible formation of the open state (R*). High concentrations of ACh block the open channel, producing a nonconducting blocked state (R_B). Rate constants are indicated next to each arrow.