Naturally occurring mutations at the acetylcholine receptor binding site independently alter ACh binding and channel gating

Abstract

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, epsilon N182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant alphadelta site. Studies of the analogous mutation in the delta subunit, deltaN187Y, disclose rate constants for ACh occupancy of the nonmutant alpha epsilon site. The second CMS mutation, epsilon D175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. epsilon D175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, epsilon N182Y localizes to the interface with the alpha subunit, and epsilon D175 to the entrance of the ACh binding cavity. Both epsilon N182Y and epsilon D175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring epsilon N182 and epsilon D175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

Figure 1.

AChR channel currents recorded from control and patient EPs. Left column shows AChR channel currents elicited by 1 μM ACh from control EP and by 5 μM ACh elicited from patient EP, with openings as upward deflections. Channel opening events are briefer at patient than at control EP. Right column shows the corresponding burst duration histograms with dotted lines indicating burst components. Time constants, τ_n, and fractional areas, a_n, for each component of bursts: Control: τ₁ = 0.04 ms, a₁ = 0.09, τ₂ = 2.38 ms; a₂ = 0.91, total events, 1,315. Patient: τ₁ = 0.185, a₁ = 0.67, τ₂ = 0.66 ms, a₂ = 0.33; total events, 4,025. Bandwidth = 12 kHz for control, 8 kHz for patient; membrane potential = −80 mV, T = 22°C ± 0.5.

Figure 2.

(A) Allele-specific PCR (ASP) for ɛD175N and RsaI restriction analysis for ɛN182Y of genomic DNA from patient (arrow) and family members. The patient carries ɛD175N and ɛN182Y; patient, mother, and brother harbor ɛN182Y. Open and closed arrowheads indicate wild-type and mutant fragments, respectively. (B) The ɛD175N and ɛN182Y mutations are heteroallelic. Allele-specific PCR selectively amplifies the ɛD175N allele (left), and the ɛD175N allele (right). PCR product amplified from each allele is digested with RsaI that only cuts the ɛN182Y allele. That only allele “b” is cut by RsaI indicates that ɛN182Y is on allele “b”. (C) Aspartate at ɛ codon 175 (D175) is conserved in human α, δ, and γ subunits, and in ɛ subunits of all species. Asparagine at ɛ codon 182 (N182) is conserved in human β, δ, and γ subunits, and in ɛ subunits of all species.

Figure 3.

Kinetics of activation of wild-type and mutant receptors. Left column displays individual clusters of single channel currents recorded at the indicated ACh concentrations from cells expressing adult human AChRs containing the indicated mutant ɛ subunits. Currents are displayed at a bandwidth of 10 kHz, with channel openings shown as upward deflections. Center and right columns display the corresponding closed and open duration histograms with the fits for Scheme I superimposed. Fitted rate constants are given in Table III.

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Figure 4.

Kinetics of activation of receptors with site-directed mutations. Left column displays individual clusters of single channel currents recorded at the indicated ACh concentrations from cells expressing adult human AChRs containing the indicated mutant subunits, as in Fig. 3.

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Figure 5.

Dependence of channel open probability (P_open) on ACh concentration for receptors containing the indicated mutant ɛ subunits plus complementary α, β, and δ subunits. The mean fraction of time the channel was open during a cluster (P_open) was determined at the indicated concentrations of ACh. The theoretical P_open was calculated from Scheme I using the fitted rate constants in Table III, and superimposed on the P_open measurements. Error bars indicate standard deviations of P_open for the population of clusters at the specified ACh concentration.

Figure 6.

ACh binding to receptors in the presence of the desensitizing agent proadifen. (A) Comparison of ACh binding to wild-type and ɛN182Y receptors. (B) Comparison of ACh binding to wild-type and ɛD175N receptors. Binding of ACh was determined by competition against the initial rate of ¹²⁵I-α-bgt binding. Intact HEK cells expressing the indicated receptor type were incubated in the presence of ACh, with or without 100 μM proadifen (pro), 30 min before measuring the initial rate of α-bgt binding. Smooth curves are fits to the Hill equation (measurements in the absence of proadifen) or to an equation describing the sum of two binding sites (measurements in the presence of proadifen). Fitted parameters (A): wild-type, K_ov = 7.9 × 10⁻⁷, n = 1.4; wild-type plus proadifen, K_A = 3.3 × 10⁻⁸, K_B = 2.1 × 10⁻⁹; ɛN182Y, K_ov = 1.4 × 10⁻⁷, n = 0.9; ɛN182Y plus proadifen, K_A = 2.3 × 10⁻⁸, K_B = 1.2 × 10⁻⁹. Fitted parameters (B): wild-type, K_ov = 1.5 × 10⁻⁶, n = 1.1; ɛD175N, K_ov = 1.2 × 10⁻⁵, n = 1.1; wild-type plus proadifen, K_A = 2.9 × 10⁻⁸, K_B = 8.9 × 10⁻⁹; ɛD175N plus proadifen, K_A = 2.7 × 10⁻⁸, K_B = 1.7 × 10⁻⁶.

Figure 7.

Location of residues ɛD175 and ɛN182 in a structural model of the binding site interface formed by α and ɛ subunits of the human AChR (Sine et al., 2002). Top panel shows the major extracellular domain of the α subunit in magenta, that of the ɛ subunit in yellow and the other subunits in gray. View is from the periphery of the receptor normal to the central vestibule. Bottom panel shows a stereo view of the ligand binding site. Residue side chains are shown in stick representation, with residues at the center of the binding site (αW149 and ɛW55) and other residues mutated in CMS (ɛP121, αG153; see text) shown for reference.