Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

Abstract

The nicotinic acetylcholine (ACh) receptors cycle among classes of nonconducting resting states, conducting open states, and nonconducting desensitized states. We previously probed the structure of the mouse-muscle ACh receptor channel in the resting state obtained in the absence of agonist and in the open states obtained after brief exposure to ACh. We now have probed the structure in the stable desensitized state obtained after many minutes of exposure to ACh. Muscle-type receptor has the subunit composition alpha(2)betagammadelta. Each subunit has four membrane-spanning segments, M1-M4. The channel lumen in the membrane domain is lined largely by M2 and to a lesser extent by M1 from each of the subunits. We determined the rates of reaction of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl methanethiosulfonate with cysteines substituted for residues in alphaM2 and the alphaM1-M2 loop in the desensitized state and compared these rates to rates previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states of the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-aminoethyl methanethiosulfonate, we previously located the closed gate in the resting state between alphaG240 and alphaT244, in the predicted M1-M2 loop at the intracellular end of M2. Now, we have located the closed gate in the stable desensitized state between alphaG240 and alphaL251. The gate in the desensitized state includes the resting state gate and an extension further into M2.

Figure 1

The arrangement of the ACh receptor subunits (A) and their common topology (B). The extracellular (EX) and intracellular (IN) sides of the membrane are indicated. The region of Cys substitution is enclosed in a dashed rectangle.

Figure 2

Desensitization due to prolonged application of 1 mM ACh on receptor. (A) A typical recording from an oocyte expressing αL251C and wild-type β, γ, and δ, with I_PEAK, I_PLAT, and I_POST indicated. (B) The fraction of current remaining at the end of the desensitizing application of 1 mM ACh, I_PLAT/I_PEAK, for each mutant and wild type. (C) The degree of recovery of current after a 45-s washout of 1 mM ACh, I_POST/I_PEAK, for each mutant and wild type. Means ± SEM and numbers of experiments are shown.

Figure 3

Measurement of the rate of reaction of MTSEA applied extracellularly to the L251C mutant in the desensitized state. (A) Control, no MTSEA. (B) Repeated application of MTSEA for 5 s (●), 10 s (■), and 15 s (⧫). (C) The peak current was normalized and plotted against cumulative time of exposure either to buffer (○, plotted at 15-s intervals) or 200 μM MTSEA (●). The solid line is the least-squares exponential fit to the data (see Methods).

Figure 4

The second-order rate constants for the reaction of extracellularly applied MTSEA with mutant ACh receptors in different states. (A) Second-order rate constants for receptors in the desensitized state (shaded squares; n = 3–7; this work) and in the resting (●) and open (○) states (from ref. 61). (B) The log of the quotient of the rate constant, k_D, in the desensitized state divided by the rate constant, k_R, in the resting state, for each mutant. (C) The log of the quotient of the rate constant, k_D, in the desensitized state divided by the rate constant, k_O, in the open state, for each mutant.

Figure 5

The effect of the intracellular application of MTSEA to αE241C in the desensitized state. The current evoked by ACh for 10 s was recorded, and the application of ACh continued, with the addition of 5 μM proadifen, for 25 min. The cell was washed for 5 min, and ACh was applied again for 10 s. The voltage clamp was turned off 1 min after the initial application of ACh and turned on again 1 min before the second application of ACh. Traces are shown for wild type (A) and αE241C (B).

Figure 6

The time courses of the reactions of intracellular MTSEA. MTSEA was applied as in Fig. 5, for the times indicated along the abscissa. The current evoked by ACh after application of MTSEA was normalized by the initial pre-MTSEA current and plotted against the time elapsed between the two ACh pulses. Effects are shown for wild type (A), αT244C (B), αE241C (C), and αG240C (D). The means, standard errors of the mean, and the number of individual time points (and cells) are shown. In C and D, the data were fit by an exponential decay function with k = 0.0017/s for αG240C and k = 0.00073/s for αE241C (solid lines). Based on the rate constants obtained previously under otherwise similar conditions (62), we plot the time courses of the reactions of intracellular MTSEA with the mutants in the resting state (long dashes) and open state (dash-dots).

Figure 7

The reactivities of Cys substituted for the residues in αM2 and the αM1–M2 loop in resting, open, and desensitized states. Two αM2 segments are represented schematically, facing the channel lumen. Where the lumen is black, the channel is relatively impermeable to MTSEA and presumably to inorganic cations. The second-order rate constants are divided into order-of-magnitude bins by rounding log(k) to the nearest integer. The rate constants are color-coded as indicated so that the warmer the color, the greater the rate constant. MTSEA was applied extracellularly by continuous superfusion of the cells, and its concentration was constant and known; hence we could calculate the second-order rate constant by dividing the pseudo first-order rate constants by this concentration. MTSEA was applied intracellularly, however, by diffusion out of the patch pipette, and its concentration at the intracellular surface of the membrane was neither constant nor known, both because of the hydrolysis of MTSEA and the unknown rate of diffusion from the pipette to the membrane. For the purpose of representation, we converted the time constants for the reactions of intracellular MTSEA to second-order rate constants, on the same scale as the second-order rate constants for the reactions with extracellular MTSEA, by multiplying the inverse of the time constants by 28100. For each of G240C, E241C, and K242C, we divided the second-order rate constant for the reaction of extracellular MTSEA by the inverse of the time constant for the reaction of intracellular MTSEA, both reactions in the open state. Log(28100) equals the average of the logs of these quotients. In the case of the desensitized state, all rate constants for the reactions with extracellular MTSEA were obtained in oocytes. Substituted Cys that reacted with MTSEA but for which the rate constants have not been determined are indicated by gray filled circles.