INTRODUCTION

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Ion channels open, conduct ions selectively, and close. The mechanisms for these functions must reside largely in the residues that line the open channel or obstruct the closed channel. To uncover these mechanisms, we try to determine how the external signals and forces that alter the functional states of an ion channel protein affect both the residues that line the channel and the ions in the channel. Small, charged reagents can serve as surrogates for permeant ions to probe the environment within a channel (Akabas et al., 1992; Stauffer and Karlin, 1994). The rates of reactions of such reagents with cysteines substituted in membrane-spanning segments can be used to identify channel-lining residues, to determine the accessibility of these residues both in the conducting and nonconducting states of the channel, to locate selectivity filters and gates, and to estimate the electrostatic potential in the vicinity of these residues (Akabas et al., 1994a, 1994b; Akabas and Karlin, 1995; Kurz et al., 1995; Lu and Miller, 1995; Pascual et al., 1995; Kuner et al., 1996; Sun et al., 1996; Xu and Akabas, 1996; Cheung and Akabas, 1997; Liu et al., 1997; Yang et al., 1997; Zhang and Karlin, 1997, 1998). In this paper, we explore the accessibility of channel-lining residues and the electrostatic potential in their vicinity in different functional states of the acetylcholine receptor channel.

The five subunits, ₂, of the muscle-type acetylcholine (ACh)¹ receptor surround the central channel quasi-symmetrically (Unwin, 1993; Galzi and Changeux, 1995; Karlin and Akabas, 1995). The NH₂-terminal half of each subunit is extracellular, and the COOH-terminal half forms three membrane-spanning segments (M1, M2, and M3), a large cytoplasmic loop, a fourth membrane-spanning segment (M4), and a short, extracellular tail. The two ACh binding sites are formed in the extracellular domain in the interfaces between the NH₂-terminal halves of two pairs of subunits, and the channel through the membrane is formed by the membrane-spanning segments of all of the subunits. Residues lining the ion-conducting pathway have been identified on the basis of the functional effects of mutagenesis and by affinity labeling in the M1 segment (DiPaola et al., 1990) and the M2 segment (Hucho et al., 1986; Imoto et al., 1988; Charnet et al., 1990; Revah et al., 1990; Pedersen et al., 1992) of the different subunits. From the effects of the mutations of charged residues bracketing M2 on rectification and on the sidedness of channel block by Mg²⁺, the NH₂-terminal end of M2 was shown to be intracellular and its COOH-terminal end, extracellular (Imoto et al., 1988).

The systematic identification of all of the channel-lining residues in the M1 and M2 segments of the  and  subunits was approached by the substituted-cysteine- accessibility method (SCAM) (Akabas et al., 1992, 1994a; Akabas and Karlin, 1995; Zhang and Karlin, 1997, 1998). In this method, each residue in the membrane-embedded segments of a channel protein is mutated one at a time to Cys, the mutants are expressed in heterologous cells, and the susceptibility of these substituted Cys to reaction with small, charged, sulfhydryl-specific reagents is determined. If the application of reagent results in an irreversible alteration in the function of the channel, it is inferred that the substituted Cys reacted and, therefore, was exposed in the water-filled lumen of the channel. This inference is based on certain assumptions: in membrane-embedded channel proteins, the sulfhydryl (SH) group of a native or engineered cysteine residue (Cys) is in one of three environments: in the water-accessible surface, in the lipid-accessible surface, or in the protein interior. We assume that the channel lining is part of the water-accessible surface (Dani, 1989) and further that, in the membrane-spanning domain of the protein, the channel lining is the only water-accessible surface. We assume that hydrophilic, charged reagents will react much faster with sulfhydryls in the water-accessible surface than in the lipid-accessible surface or in the interior of the protein. We synthesized a set of polar sulfhydryl-specific reagents, methanethiosulfonate derivatives, that reacted by the same mechanism and were similar in size, but that differed in their charge (Stauffer and Karlin, 1994). These reagents are directed at water-accessible SH both because they are polar and because they react at least 5 × 10⁹ faster with dissociated S than with undissociated SH (Roberts et al., 1986). In the lipid- accessible surface and in the protein interior, ionization of SH is suppressed because of the low dielectric constant of the environment.

In both M1 and M2 of the ACh receptor, we observed markedly different reactivities in the presence and absence of ACh of several substituted Cys residues to reagents added extracellularly (Akabas et al., 1992, 1994a; Akabas and Karlin, 1995; Zhang and Karlin, 1997, 1998). We ascribed these differences in reactivities to conformational changes concomitant with gating. Because the changes in reactivity were scattered over the length of the channel, with residues affected by ACh near residues not affected by ACh, it was more likely that the reactivities were affected by local conformational changes rather than by a general increase in accessibility due to the opening of a gate closer to the extracellular side than these residues.

In the current work, we determined the rate constants for the reactions of thiosulfonate reagents during brief applications of ACh and in the absence of ACh, with nine susceptible Cys-substituted residues in the M2 segment of the  subunit. We found that the rate constants for the reactions at several, but not all, of the residues were very different in the presence and absence of ACh. In addition, the rates of reaction with positively charged reagents were dependent on the transmembrane holding potential, and this dependence was characteristic of the open state. Even at zero holding potential, the reaction rates of different reagents depended on their charge, indicating that in addition to the extrinsic holding potential there is an intrinsic electrostatic potential in the channel. Furthermore, the profile of intrinsic potential is different in the open and closed states of the channel. The dependence of methanethiosulfonate reaction rates on transmembrane potential was demonstrated in cystic fibrosis transmembrane conductance regulator (Cheung and Akabas, 1997). The intrinsic electrostatic potentials have been estimated from the relative rates of reaction of differently charged methanethiosulfonate reagents in the ACh receptor binding site (Stauffer and Karlin, 1994) and in a vestibule of the Na channel containing residues of the voltage-sensing S4 segment (Yang et al., 1997). A preliminary report of the work in this paper has appeared previously (Pascual and Karlin, 1997b).

	METHODS

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Mutagenesis and Expression

All mutations were introduced in the M2 segment of the mouse muscle  subunit and capped, runoff cRNA transcripts were obtained for the -subunit mutants and for wild-type , , , and  subunits after linearization of the plasmid cDNA as previously described (Akabas et al., 1994a). cRNAs at a concentration of 1 mg/ml in water were stored at 80°C. They were diluted and mixed for injection at a ratio of 2:1:1:1. Stage V and VI Xenopus laevis oocytes were collected and defolliculated in collagenase following standard procedures (Akabas et al., 1992). Oocytes were injected with 60 nl of cRNA diluted to 1-100 ng/µl, depending on desired current expression levels. Cells were kept in culture for 1-10 d before recording.

ACh-induced Current

Currents were recorded under two-electrode voltage-clamp. The oocyte bath solution contained (mM) 115 NaCl, 2.5 KCl, 1.8 MgCl₂, 10 HEPES, pH 7.2, except where indicated otherwise. Solutions flowed at 7 ml/min first through a stainless steel coil immersed in a thermostat at 18.0°C, and then past the oocyte, which was held in a rectangular chamber with a cross-section normal to the direction of solution flow of 4 mm². An agar bridge connected a Ag:AgCl reference electrode to the bath and was placed as close as possible to the oocyte. The bath was clamped at ground potential. We used beveled agarose-cushion (Schreibmayer et al., 1994) glass micropipettes filled with 3 M KCl, resistance ~0.5 M, for both current-passing and voltage-recording electrodes. A few uninjected oocytes from each batch were tested for the presence of endogenous ACh-induced currents, which were never found. The function of wild-type and mutant receptors was assayed as the ACh-induced current elicited by the application of brief (10-20-s) pulses of ACh, at a concentration 10× the EC₅₀, as determined for each mutant, and at a holding potential of 50 mV, except where indicated otherwise. ACh-induced currents ranged from 1 to 25 µA.

Synthesis and Use of Thiosulfonate Derivatives

The positively charged 2-aminoethyl-methanethiosulfonate, CH₃ SO₂SCH₂CH₂NH₃⁺ (MTSEA), (Bruice and Kenyon, 1982), and 2-tri-methylammonioethyl-MTS, CH₃SO₂SCH₂CH₂N(CH₃)₃⁺ (MTSET), (Stauffer and Karlin, 1994), and the neutral 2-hydroxyethyl-MTS, CH₃SO₂SCH₂CH₂OH (MTSEH), are a set of rapidly reacting, sulfhydryl-specific reagents that differ only in their head groups and whose rates of reaction with a Cys-substitution mutant are readily compared. MTSEA and MTSET were synthesized as previously described and were also purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

MTSEH was newly synthesized by dissolving 40 g of sodium methanethiosulfonate and 41 g of 2-bromoethanol (95%) in acetonitrile. The stirred mixture under argon was refluxed overnight. The mixture was cooled and filtered; the filtrate was concentrated, mixed with methylene chloride, filtered, and concentrated again to yield a yellow oil. 1 ml yellow oil was mixed with 9 ml chloroform, and the small amount of precipitate that formed was removed by centrifugation in a clinical centrifuge. The supernatant was layered on a silica gel (grade 9385, 230-400 mesh; Merck, Darmstadt, Germany) column (32 cm length, 1.9 cm diameter), pre-equilibrated with chloroform (stabilized with 0.75% ethanol). The column was eluted under mild pressure at 3-4 ml/ min with 90 ml chloroform and with 260 ml 98% chloroform/2% methanol; the next 100 ml contained the pure product, as determined initially by thin-layer chromatography on silica developed in 98% chloroform/2% methanol. The components were visualized under UV light (254 nm) and by spraying with a mixture containing 1 mM DTNB (5,5'-dithio-bis-2-nitrobenzoate), 0.5 mM dithiothreitol, and trimethylamine in methanol, in which the product gave a white spot against a yellow background. The 100 ml containing the product was reduced to ~10 ml on a rotary evaporator; 20 ml of methylene chloride was added, and the volume was reduced again, first by rotary evaporation, and then on a high vacuum line with liquid nitrogen traps. Approximately 0.6 g of liquid was recovered. By assay with TNB (2-nitro-5-thiobenzoate) (Stauffer and Karlin, 1994), the average purity of three preparations was 98%. The nuclear magnetic resonance spectrum was consistent with the structure of MTSEH. Mass spectrometry, +FAB ionization, gave MH⁺ 157.

A doubly positively charged thiosulfonate, 2-aminoethyl-2-aminoethanethiosulfonate, NH₃⁺CH₂CH₂SO₂SCH₂CH₂NH₃⁺ (AEAETS), adds the 2-aminoethylthio group to the Cys SH, just like MTSEA. It was synthesized as previously described (Field et al., 1961, 1964). It was recrystallized by dissolving in methanol, adding about one-third volume of diethyl ether, and storing at 4°C for 2 d. By thin-layer chromatography on cellulose, developed in 60% ethanol/30% 0.1 N HCl/10% t-butanol, the product in methanol gave one ninhydrin-positive spot with an R_f = 0.26. TNB assay gave 95% purity based on a mol wt of 256.9. The melting point was 168-170°C. Mass spectrometry by direct probe electron impact with no solvent gave peaks of 256 and 258, corresponding to the compound with two ³⁵Cl and to the compound with one ³⁵Cl and one ³⁷Cl. The nuclear magnetic resonance spectrum was consistent with the structure of AEAETS.

The thiosulfonate reagents are relatively unstable at neutral and alkaline pHs. They hydrolyze to a sulfenic acid (RSOH) and a sulfinate (R'SO₂). In a second step, the sulfenic acid disproportionates to a thiol (RSH) and a sulfinate (RSO₂). At pH 7 and 20°C, the half-times for the hydrolysis are 12 min for MTSEA, 11 min for MTSET, and 6 min for AEAETS (Karlin and Akabas, 1998; Stauffer and Karlin, 1994). These reagents are stable for hours, however, in unbuffered water at 4°C. Thus, stocks of the reagents were made daily by dissolving reagent to a concentration of 1-100 mM in water and kept on ice. They were diluted in bath solution just before use. The diluted reagent was placed in a syringe barrel and kept cool by ice-water in a surrounding jacket. The solution was warmed to 18°C as it passed through a thermostated coil just before reaching the oocyte (see above).

The rate constants for the reactions of the thiosulfonates with 2-mercaptoethanol were determined by stopped-flow, rapid-mixing spectrophotometry as previously described (Stauffer and Karlin, 1994). All determinations were at 20°C in 58 mM NaPO₄, 0.1 mM EDTA, pH 7.0, ionic strength 0.130.

Determination of Reaction Rates in Different Receptor States

The time-course of the reaction of a reagent with a substituted-Cys mutant in the absence of ACh was determined by recording the initial response to ACh and subsequent responses to ACh during several repeats of the following sequence: a short application of reagent, a wash with bath solution, an application of ACh, and another wash. Positively charged ammonium reagents could have either agonist activity or channel blocking activities (Sanchez et al., 1986), and these activities could vary with the mutant. Therefore, the reagents were monitored for such reversible actions on each mutant.

In the presence of ACh, the receptor first opens and then desensitizes in two steps, one fast and one slow (Katz and Thesleff, 1957; Sakmann et al., 1980; Neubig et al., 1982; Heidmann et al., 1983; Hess, 1993). To determine the rate of reaction in the open state, we applied reagent plus ACh for short times (10-20 s), during which the extent of slow desensitization was slight. Slow desensitization of all mutants used in this paper took place at a rate 0.005 s¹ (not shown), which was not a significant correction to the decrease in current due to the reaction of the MTS reagents. Therefore, during the first 20 s of application of reagent plus ACh, the reaction was with receptor mainly in the open state and the fast desensitized state.

The reactions of MTSEA and AEAETS with T244C in the presence of ACh were relatively fast and the end-points were complete inhibition of the ACh-induced current. In these cases, the reactions over intervals as brief as 3 s were detectable. Over this short interval, the amplitude of the current decreased approximately linearly, and the second-order-rate constant, , was estimated by

(1)

where I is current, t is time, x is the concentration of reagent, and subscripts 1 and 2 refer to the beginning and end, respectively, of the measurement interval. In this case, it was possible to change the holding potential in successive reaction intervals, and thus obtain the rate constant as a function of holding potential in a single experiment (first protocol). Each such experiment was repeated on at least three different oocytes.

For more slowly reacting mutants and reagents, a second protocol was used to determine the rate constant for the reaction in the presence of ACh. The following sequence of solutions was applied several times: ACh at 10× EC₅₀ for 10-20 s to test the response, bath solution for 2-4 min, reagent plus ACh for 2-20 s, and bath solution for 3-4 min. The holding potential was fixed during the experiment. We determined the rate constant by fitting the peaks of the test currents to

(2)

where t is the cumulative time of reagent application, the subscripts refer to the cumulative time at which the current was recorded, x is the concentration of reagent, and  is the second- order-rate constant. These experiments were repeated on oocytes at different holding potentials.

In the presence of ACh, MTSET increased the rate of desensitization so that the decrease in the amplitude of the current in the presence of MTSET had both an irreversible component due to the reaction and a reversible component due to desensitization. We estimated the rate constant for the reaction of MTSET with the open state using the second protocol. We fit the following equation (see Appendix b for derivation) to the test currents:

(3)

where  is the second-order rate constant, x is the concentration of reagent, I_PRE is the peak current induced by ACh at 10× EC₅₀ obtained before the reaction, Q_DUR is the total charge flow (current integrated over time) during the reaction with MTSET in the presence of ACh, Q_POST is the total charge flow during the test response to ACh at 10× EC₅₀ after the reaction, and Q_PRE is the total charge flow during the test response before the reaction. In practice, MTSET and ACh were applied several times to each oocyte, each preceded and followed by test responses so that the test response after one application of MTSET was the test response preceding the next application. The rate constant for the reaction occurring during each MTSET application was calculated by Eq. 3. Several rate constants were thus obtained from each experiment, and these were averaged using each one to recalculate the degree of reaction in each time period, exp(xt), and from these we calculated the cumulative extent of reaction as a function of the cumulative time of exposure to MTSET. These points were then fit by Eq. 2. This averaging procedure gave the most weight to the first rate constant, involving the greatest change in response, and the least weight to the last, involving the smallest change in response.

A third protocol was used to determine the rate constants for reactions in the absence of ACh, which was identical to the second protocol except that reagent was added in the absence of ACh. The data were fit by Eq. 2.

All oocytes were tested for stability of responses to ACh before any reagents were applied by three to five applications of ACh over a period of 5-15 min. The criterion for acceptable stability was that the peak currents varied <3% from each other. Thus, run-down or run-up of the responses was <3%/5 min or 0.0001/s.

	RESULTS

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Protocols for Determining Rates

We used three different protocols (see METHODS) to determine the rate constants for the reactions of the thiosulfonate reagents with the Cys-substituted mutants in the presence and absence of ACh and as a function of holding potential. We applied the first protocol to reactions with large rate constants and large effects, in which case short applications of low concentrations of reagent had readily measurable functional effects. In this protocol, we applied the thiosulfonate and ACh continuously for a few seconds, during which time we stepped the membrane potential to four different values. As a control for this protocol, we tested for voltage-gated channels in the oocyte that might respond to the jumps in holding potential and found none (Fig. 1 A). We also found that during the brief applications of ACh, slow desensitization was negligible, and the current was constant (Fig. 1 B). During brief applications of ACh and reagent, the current magnitude declined linearly, as illustrated by the effect of the reaction of T244C with 5 µM AEAETS in the presence of 60 µM ACh (Fig. 1 C). In other experiments, we found that the change in current was irreversible (data not shown). The rate constant at each holding potential was estimated using Eq. 1.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Determination of the rate constants for the reactions of thiosulfonates with substituted cysteines. (A-C) Reaction of AEAETS with T244C in the presence of ACh. (A) Current recorded in the absence of ACh while clamping an oocyte expressing T244C receptor to 25 mV for 1 s, 25 mV for 0.8 s, 75 mV for 0.4 s, and 50 mV for 0.25 s. (B) Currents induced by 60 µM ACh under the same voltage protocol as in A. (C) Currents in the presence of 5 µM AEAETS plus 60 µM ACh under the same voltage protocol as in A. Reagent and ACh were added before the start of the first voltage step. The time course of inhibition was approximately linear, and the pseudo-first order reaction rate constant at each voltage was estimated by the slope divided by the current amplitude at the beginning of each voltage interval. Linear fits and slope values are shown for the reactions at 25 and 100 mV. The records in A-C were obtained from the same cell. (D-E) Reaction of AEAETS with T244C in the absence of ACh. (D) ACh-induced currents before and after applications of 1 mM AEAETS in the absence of ACh. The following sequence of applications was repeated five times: 60 µM ACh for 10 s, wash 4 min, AEAETS for 2-4 min (beginning at arrows), and wash 3 min; ACh was applied for 10 s to obtain the final response. The cumulative duration of exposure to AEAETS before each ACh-induced response is indicated next to the peak of current. The clamp potential was 50 mV. (E) Peak ACh-induced currents in D (), normalized to the initial ACh- induced current, plotted against the cumulative time of exposure to AEAETS. The solid line is a single exponential fit (see METHODS). (F-G) Reaction of 10 mM AEAETS with S248C in the presence of ACh. (F) An oocyte was alternatively exposed to 25 µM ACh in the absence and presence of AEAETS and allowed to recover for 4 min after each application. The following sequence of applications was repeated seven times: ACh for 10 s, wash 4 min, ACh plus AEAETS for 2-20 s (beginning at arrows), and wash 4 min. ACh for 10 s was added again at the end. The clamp potential was 50 mV. The cumulative duration of exposure to AEAETS before each ACh-induced response is indicated next to the peak of current. (G) As in E, normalized peak currents were plotted against the cumulative duration of exposure to AEAETS ().

In the second protocol, which we applied to slower reactions, the membrane potential in each experiment was fixed, and we repeatedly applied the sequence: ACh, wash, reagent plus ACh, and wash. This protocol is illustrated for the reaction of S248C with 10 mM AEAETS plus 100 µM ACh (Fig. 1 F). In this case also, slow desensitization was negligible during each reaction period (2-20 s). The rate constant was determined by an exponential fit (Eq. 2) to the test ACh responses as a function of the preceding cumulative duration of exposure to reagent (Fig. 1 G).

The third protocol was used for reactions in the absence of ACh. In each experiment, the reagent was applied several times (at a fixed holding potential), interspersed as in the second protocol, with washes and test responses. This is illustrated for the reaction of 1 mM AEAETS with T244C (Fig. 1 D). The rate constant was determined by fitting Eq. 2 to the test responses as a function of the preceding cumulative duration of the reaction (Fig. 1 E).

Dependence of Rate Constants on Functional State

We determined the rate constants for the reactions of MTSEA with nine substituted Cys in and bracketing M2, previously found to be accessible (Akabas et al., 1994a). The mutants were E241C at the intracellular end of the channel, T244C, L245C, S248C, L251C, S252C, V255C, L258C, and E262C at the extracellular end of the channel. The rate constants were determined for the reactions in the absence and presence of ACh (Fig. 2, Table I).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Rate constants for the reaction of MTSEA with substituted Cys mutants in the absence () and presence () of ACh. When reagent was added with ACh, the ACh concentration was 10× the EC50 for the mutant. The protocols are given in METHODS. MTSEA concentrations ranged from 2.5 µM to 5 mM. Horizontal lines connect the mean rate constants in the two conditions. Each symbol is the mean of three to seven independent determinations. Thick lines represent the SEM where it extends beyond the symbol. The holding potential was 50 mV.

For the reactions in the absence of ACh, the rate constants ranged from 0.21 M¹ s¹, for the reaction with L258C, to 480 M¹ s¹, for the reaction with T244C, a range of 2,300-fold. For the reactions in the presence of ACh, the rate constants ranged from 2.2 M¹ s¹, for L245C, to 16,800 M¹ s¹, for T244C, a range of 7,600-fold. Both in the absence and presence of ACh, extracellularly applied MTSEA reacted fastest with T244C, close to the intracellular end of the channel.

MTSET was tested only on T244C and S248C. Both in the presence and absence of ACh, it reacted more slowly than MTSEA with T244C, and its rate of reaction with S248C was too slow to be measured (Fig. 3, Table I).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Comparison of the rate constants for the reactions of AEAETS (diamonds), MTSEA (circles), MTSET (squares), and MTSEH (triangles) with substituted Cys mutants in the absence (filled symbols) and presence (open symbols) of ACh. Thick lines indicate the SEMs of the rate constants where larger than the symbol. The reactions of MTSET and MTSEH with S248C, both in the presence and absence of ACh, were undetectable. For L258C, the rate constant for the reaction with AEAETS in the absence of ACh is an upper limit because AEAETS induced some current. The holding potential was 50 mV. Each symbol is the average of two to seven independent measurements.

The rate constants for the reactions of AEAETS with T244C, S248C, L251C, and L258C were all smaller than those for MTSEA in the absence of ACh and were all larger than those for MTSEA in the presence of ACh (Fig. 3). (The reaction of AEAETS in the absence of ACh with S248C was too slow to be measured.)

The rate constants for the reactions of the uncharged MTSEH was fast enough to be determined only with T244C, L251C, and L258C. MTSEH reacted more slowly than the other reagents both in the presence and absence of ACh, except with L258C in the absence of ACh, where all reagents reacted very slowly (Fig. 3).

As a rule, the charged reagents reacted faster than the uncharged MTSEH. Among the charged reagents, size and charge both influenced the rates of reaction. MTSEA (+1) is smaller than MTSET (+1), which is smaller than AEAETS (+2). In the absence of ACh, size appears to be more important than charge; in the presence of ACh, both charge and size were important (Fig. 3).

In most cases, the rate constants for the reactions of the thiosulfonates with substituted Cys in the channel were orders of magnitude slower than the rate constants for their reactions with 2-mercaptoethanol in homogeneous solution (Table I). Even the fastest reaction of MTSEH in the channel, with L251C in the presence of ACh, was 1,200× slower than the reaction of MTSEH with 2-mercaptoethanol in solution. The fastest reactions of MTSEA and MTSET, with T244C in the presence of ACh, were only four times slower than the reactions with 2-mercaptoethanol, but the reactions with T244C in the absence of ACh were five to six orders of magnitude slower than the reactions with 2-mercaptoethanol. As discussed later, the reactions in the channel can be slowed by low accessibility to the Cys, steric hindrance around the Cys, and suppressed ionization of the Cys SH. These retarding influences can be partly compensated by the electrostatic potential in the channel.

The reactions of MTSEA with six of the nine substituted Cys were much faster in the presence of ACh than in its absence (Fig. 2). The rate constants were larger by factors ranging from 35 for T244C to 1,970 for V255C (Table I). For MTSET and AEAETS, also, the rate constants for the reaction with T244C in the presence of ACh were much larger than in the absence of ACh (Fig. 3). For AEAETS, the rate constant was larger by a factor of 51,000 (Table I). Also, AEAETS reacted much faster with S248C, L251C, and L258C in the presence of ACh than in its absence.

MTSET evoked a small current in T244C in the absence of ACh. Similarly, AEAETS evoked a small current in L258C in the absence of ACh. Even in these two cases, where the reagents themselves evoked a detectable current, the reactions were still far faster in the presence of ACh, when the current was large, than in the absence of ACh, when the current was small (Fig. 3).

Not all reactions were accelerated by the addition of ACh. For the reactions of L245C and S252C with MTSEA, the rate constants in the presence and absence of ACh were barely distinguishable, and, for the reaction of MTSEA with S248C, the rate constants differed only by a factor of 3.6 (Fig. 2). Also, the rate constant for the reaction of the uncharged MTSEH with T244C was unchanged by the addition of ACh. There is no obvious correlation between the distance of a substituted Cys from the extracellular end of the channel and the effects of ACh on the rate constant. If there were a gate in the middle of M2, then the opening of this gate should have increased the rates of reaction of all substituted Cys distal to it. No such simple pattern of effects of ACh is apparent.

Reaction Rates in the Desensitized State

In the presence of ACh, the receptor opens and also undergoes transitions in two steps, fast and slow, to desensitized states (Katz and Thesleff, 1957; Sakmann et al., 1980; Neubig et al., 1982; Heidmann et al., 1983; Hess, 1993). To estimate the rate constant for the reaction of MTSEA with T244C in the slow desensitized state, we applied 60 µM ACh for several minutes until the current had decreased to ~20% of its peak value. At this point, at least 80% of the receptors were in the desensitized state and some fraction of the remainder were in the open state. After a wash of 15 s, brief compared with the half-time of ~1 min for recovery from desensitization, we added 85 µM MTSEA for 30 s (Fig. 4). Based on the estimates of the rate constants above, this application of MTSEA would have modified over 99% of receptors if ACh had been added simultaneously with MTSEA and 25% of receptors in the absence of ACh. After the MTSEA application, the responses to brief applications of ACh recovered to 75% of the initial amplitude; i.e., there was 25% irreversible inhibition, as would be expected in the absence of ACh. A subsequent application of MTSEA in the absence of ACh caused a similar irreversible inhibition of the response. In five similar experiments using different MT-SEA concentrations, we estimated the rate constants for the reaction with the desensitized state from the extent of irreversible inhibition due to a 30-s application of 2.5 or 85 µM MTSEA to mostly desensitized receptors. The rate constant for the reaction with the desensitized state was 110 ± 26 M¹ s¹, compared with 480 ± 190 M¹ s¹ in the absence of ACh, and 16,800 ± 3,600 M¹ s¹ during brief applications of ACh. The difference between the first two rate constants is not statistically significant.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Reaction of T244C in the desensitized state. (A) Experimental protocol for determining the extent of MTSEA reaction with T244C in the desensitized state. Applications of 60 µM ACh are indicated by downward arrows, of bath solution by upward arrows, and 85 µM MTSEA by horizontal bars. The time scale is given as the abscissa in B. Two initial responses elicited by 10-s applications of ACh were followed by a 4-min application of ACh, during which the current declined to 20% of its peak value by desensitization. Bath solution was applied for 15 s, and then MTSEA was applied for 30 s. After a 15-s wash, ACh was reapplied several times and the responses increased as the receptors recovered from desensitization. MTSEA was applied again for 30 s to the receptors in the resting state, and the effect of this application was assayed by a final application of ACh. The holding potential was 50 mV. (B) The inhibition, 1  It/I0, due to both desensitization and the reaction of MTSEA is plotted as a function of the recording time, corresponding to the experiment in A. The recovery from desensitization was fit by a single exponential function with a time constant of 106 ± 27 s (n = 5), characteristic of the mutant T244C.

Dependence of the Reaction Rates on the Transmembrane Electrostatic Potential

The electrostatic potential at each point in the channel is a sum of an intrinsic electrostatic potential, _S, due to charges in the surrounding protein and in the channel, and of a fraction, , of the extrinsic, transmembrane potential, _M. We now consider the effect of _M on the rate constants of the reactions of the thiosulfonates with the various substituted Cys. As a first approximation (see DISCUSSION), we characterize these effects in terms of the equation,  = ₀exp(z_M), where ₀ is the effective rate constant at zero holding potential, z is the algebraic charge on the reagent,  is the electrical distance from the extracellular medium to the probed residue, and  is F/(RT).

In the absence of ACh, the rate constants for the reactions of T244C with AEAETS, MTSEA, and MTSEH were not significantly dependent on _M in the range of 100 to 0 mV (Fig. 5). The least-squares fit of the above equation yields z equal to 0.04 ± 0.02, 0.05 ± 0.05, and 0.008 ± 0.04, respectively. The addition of these reagents caused no detectable increase in leak current, and hence the channel remained predominantly closed. The reaction with MTSET, however, was significantly dependent on holding potential, and the least-squares fit of  versus _M yielded z equal to 0.38 ± 0.02. In this case, however, MTSET (7.5 µM) induced a small current, ~5% as large as that induced by 60 µM ACh, and it is likely that the voltage dependence of the rate constant was characteristic of the open state in which the reaction was predominantly occurring.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Rate constants for the reactions of thiosulfonate derivatives with T244C in the absence of ACh as a function of membrane potential. Second-order rate constants for reactions with AEAETS (filled diamonds), MTSEA (filled circles), MTSEA at pH 6.5 (unfilled hexagons with dot), MTSET (filled squares), and MTSEH (filled triangles) are shown as a function of membrane potential. Nonlinear-least-squares fit of the data by Eq. 7 yielded the following parameters (parameters without errors were assumed): for MTSEA, C = 390 ± 55, D = 0, z = 0.05 ± 0.05; for AEAETS, C = 1.10 ± 0.06, D = 0, z = 0.040 ± 0.021; for MTSET, C = 1.28 ± 0.08, D = 0.01, z = 0.40 ± 0.02. These parameters were used to generate the curves. For MTSEH, for which z = 0, a line of zero slope was drawn at the mean .

In the presence of ACh, reactions of AEAETS and MTSET with T244C were dependent on the holding potential (Fig. 6). The least-squares fit of  versus _M yielded z equal to 0.33 ± 0.05 for AEAETS and equal to 0.42 ± 0.06 for MTSET. (These values are based on the simple equation above; the values of z given in the legend to Fig. 6 are based on the more complicated Eq. 7 in the DISCUSSION.) The reaction rates of MTSEA and MTSEH, however, were not significantly dependent on _M, with z equal to 0.008 ± 0.04 and 0.09 ± 0.12, respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Rate constants for the reactions of Cys-substituted mutants in the presence of ACh with AEAETS (), MTSEA (), MTSET (), and MTSEH () as a function of membrane potential. Means and SEM for three to seven determinations are shown. Nonlinear least squares fit of the data by Eq. 7 yielded the following parameters (parameters without errors were assumed), which were used to generate the curves: for T244C and MTSEA, C = 46,900 ± 4,190, D = 2, z = 0.38 ± 0.04; for T244C and AEAETS, C = 21,200 ± 410, D = 0.007 ± 0.0004, z = 0.54 ± 0.009; for T244C and MTSET, C = 307 ± 60, D = 0.01, z = 0.44 ± 0.06; for S248C and MTSEA, C = 6.6 ± 1.8, D = 1, z = 0.28 ± 0.12; for S248C and AEAETS, C = 11 ± 0.5, D = 0.007, z = 0.31 ± 0.02; for L251C and MTSEA, C = 1,680 ± 16, D = 0.09 ± 0.003, z = 0.15; for L251C and AEAETS, C = 6,060 ± 270, D = 0.007, z = 0.26 ± 0.01; for L258C and AEAETS, C = 131 ± 130, D = 0.007, z = 0.12 ± 0.04. For T244C and MTSEH, z = 0, and the overall mean of the mean  at the three M, 0.21 ± 0.05, is plotted with zero slope.

That the rate of reaction of MTSEH was independent of holding potential is consistent with its neutrality. The absence of voltage dependence of the reaction of MTSEA, however, was unexpected. One possibility is that MTSEA could enter the channel and react as a deprotonated, uncharged amine. MTSEA is partly deprotonated at pH 7.2, the pH of the bath solution. Because MTSEA hydrolyzes rapidly at alkaline pH, it is difficult to obtain a titration curve, but from the initial part of such a curve, we could estimate that the pK_a is no lower than 8.5. MTSEA would be 5% deprotonated if the pK_a of the amine were 8.5. Lowering the pH of the bath solution to 6.5, however, which decreased the fraction of deprotonated MTSEA fivefold, did not alter the rate of reaction of MTSEA with T244C in the absence of ACh, at either 100 or 50 mV (Fig. 5, unfilled hexagons with dot). Thus, it was predominantly the charged form of MTSEA that reacted with T244C, and the apparent absence of voltage dependence of the reaction rate was not due to the fraction of uncharged MTSEA. We will argue below that the lack of voltage dependence of MTSEA is likely a result of its permeability through the open channel.

The reactions of AEAETS in the presence of ACh with substituted Cys closer to the extracellular end of the channel than T244C were also voltage dependent (Fig. 6). The value of z decreased as the distance from the extracellular end of the channel decreased. The fit of  versus _M yielded z equal to 0.21 ± 0.04 for S248C, 0.17 ± 0.04 for L251C, and 0.007 ± 0.01 for L258C. Thus, the reactions of AEAETS with T244C, S248C, and L251C were significantly dependent on _M. For MTSEA, z was equal to 0.08 ± 0.09 at S248C and 0.04 ± 0.008 at L251C. There was no significant dependence of the reactions of MTSEA on membrane potential.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Controlling the States of the Receptor and the Channel

We have determined the rates of reaction of substituted Cys in the channel in the presence and absence of ACh, and we would like to associate these rates with the open and closed states of the channel. Neither in the presence nor absence of ACh, however, is the receptor and its channel in a single state. Four different principal functional states of the receptor have been characterized, resting, active, fast desensitized, and slow desensitized (Katz and Thesleff, 1957; Sakmann et al., 1980; Neubig et al., 1982; Heidmann et al., 1983; Hess, 1993). The channel is open only in the active state and is closed in the other three states. Immediately upon binding ACh, the receptor undergoes a sub-millisecond transition from the resting state to the active state. In the continued presence of ACh for hundreds of milliseconds, the receptor enters the fast desensitized state and then, in tens of seconds, the slow desensitized state. The occupied receptor reaches an equilibrium distribution among the different states, which favors the desensitized state.

Could the reaction rates measured in the absence of ACh be due to reaction occurring during spontaneous openings? In the absence of ACh, wild-type receptor opens spontaneously, but with a probability <10⁵ (  Jackson, 1989). One of the mutants used here, L251C, however, has a higher spontaneous open probability than wild type, although the open probability of unliganded receptor is still orders of magnitude lower than the open probability of doubly liganded receptor (Auerbach et al., 1996). We did not detect any difference between leak currents in uninjected oocytes and leak currents in oocytes expressing either wild-type receptor or any Cys-substituted mutant. The leak current was often ~0.5% of the ACh-induced current. We also did not detect any change in leak current with any mutant when we added the open-channel blocker QX-314. Therefore, the fraction of receptors that was open in the absence of ACh was very small compared with the fraction that was open immediately after adding ACh.

The reagents themselves could activate the receptor, as do high concentrations of some other amines (Sanchez et al., 1986). The quaternary ammonium MTSET does act as a low affinity agonist of the receptor, and AEAETS is a weak agonist of the mutant L258C. In these cases, reagent-induced current is readily detected. In no other cases, however, did we detect a reagent-induced increase in current. Also, the second-order rate constant for the reaction of MTSEA with T244C in the absence of ACh was independent of MTSEA concentration, which would not be the case if MTSEA were both activating the receptor and reacting with it. Furthermore, all of the accessible Cys mutants from E241C to V255C are protected against reaction with MTSEA by the open-channel blocker QX-314 in the presence of ACh, but not in the absence of ACh (Pascual and Karlin, 1997a). This is evidence that the reaction in the absence of ACh is predominantly with the closed state of the channel and not with a spontaneously open state or with a reagent-induced open state. We will also argue below that the dependence of reaction rates on the transmembrane potential is a characteristic of the open state and not of the closed states, and we observed voltage dependence of the rates only when we also detected receptor-mediated currents.

After brief exposure to ACh, the receptors are distributed among the resting, active, and desensitized states. This distribution, which is dependent on the kinetics of the transitions between states, could vary somewhat among the mutants, although, from the maximum currents obtained, none of the mutants appeared to have a low open probability. Nevertheless, some of the variation among the mutants in the effects of ACh on reaction rates could be partly due to differences in gating kinetics.

For one mutant, T244C, MTSEA reacted 35× faster during a brief exposure to ACh than in the absence of ACh. To get an estimate of the rate in the desensitized state, we carried out the same reaction after the mutant receptor was ~80% (or more) desensitized (Fig. 4). The rate constant in this largely desensitized state was no faster than that in the resting state and much slower than that in the open state. We do not know the rate constant for the reaction in the fast desensitized state. It is likely, however, that those reactions in the presence of ACh that were dependent on holding potential or that were retarded by open channel blockers were predominantly with the open state and not with any of the closed states, including the fast desensitized state.

Factors Determining the Reaction Rates

The rate constants for the reactions of the thiosulfonates with the substituted Cys depend on properties of the channel pathway to the Cys, properties of the Cys, and properties of the reagent. Obviously, changes in the structure of the channel underlying changes in its functional state could affect both access to a Cys residue and its local environment.

The overall rate of reaction cannot be faster than the rate of passage of the reagent from the bath to the vicinity of the SH. We assume that the pathway is the water-filled channel. For a number of substituted Cys, the voltage sensitivity of the reaction rate and protection against the reaction by open-channel blockers support this assumption. The rate of passage through the channel from the extracellular medium to the Cys could be very different in the open and closed states of the channel because of differences in the structure of the lining or of water in the channel (Green and Lu, 1995). In addition, the movement of charged reagents to the target Cys could be affected by the electrostatic field along the pathway (see below).

The reactivity of a target Cys is largely determined by its local environment. A major factor in the reactivity is the pK_a of the SH, because thiosulfonates react 9-10 orders of magnitude faster with a deprotonated S than with a protonated SH (Roberts et al., 1986). A Cys facing the water-filled channel should react faster than one facing other residues or lipid, both because the ionization of the SH is more likely in the environment with the higher dielectric constant and because there is more room to form an activated complex. The positions and configurations of the residues surrounding the channel are likely to be fluctuating and, therefore, so are the extent of exposure and the degree of steric hindrance to the formation of an activated complex. The reactions with Cys SH exposed in rare or short-lived fluctuations should be much slower than reactions with residues exposed most or all of the time. The wide range of rate constants for the reactions with the set of substituted Cys that we consider exposed could be due in part to differences in their pK_a and in part to their degrees of exposure and steric hindrance. These factors also could account for the Cys in the channel reacting much slower, in most cases, than 2-mercaptoethanol in solution (Table I).

Although the thiosulfonate reagents used here have a common reaction mechanism, there are differences in size and charge. We observed in some mutants that the addition of ACh had different effects on the reactions of the different reagents (Fig. 3). For example, at T244C, the rate constants for MTSEA, MTSET, and AEAETS were all much larger in the presence of ACh than in its absence, whereas for MTSEH the rate constants in the presence and absence of ACh were the same. Size differences cannot explain these results because MTSEH is the same size as MTSEA. Also, the rate constant for MTSEH is much smaller than the rate constant for MTSEA both in the presence and absence of ACh. That MTSEH is uncharged must be a major factor in its smaller rate constant and in the lack of effect on the rate constant of channel opening. At S248C, by contrast, the size of the reagents and local steric hindrance around the SH must play an important role in the reactions, because MTSET, with a relatively bulky trimethylammonium head-group, did not react with S248C, even though it can pass this position to react with T244C. MTSEA and AEAETS, with unsubstituted ammonium head groups, did react with S248C. At L251C, the rate constants for the reactions of MTSEH, MTSEA, and AEAETS are all larger in the presence of ACh than in its absence (Fig. 3), and the qualitative similarity of the effects of ACh on the rates is consistent with an increase in the exposure of this residue to all three reagents or an increase in the pK_a of the Cys. That the effect is 10,000-fold for AEAETS, 200-fold for MTSEA, and 40-fold for MTSEH indicates that factors in addition to changes in exposure or pK_a affect these rates.

Electrostatics and the Kinetics of Reactions in the Channel

Uncharged MTSEH reacted much more slowly than positively charged MTSEA, MTSET, and AEAETS with T244C, especially in the open state (Fig. 3). This result and others discussed below suggest that charge and, therefore, electrostatic potential play important roles in the reactions of these reagents in the channel. The electrostatic potential sensed by a charged reagent in the channel is the electrical distance times the extrinsic transmembrane potential, _M, plus the intrinsic electrostatic potential, _S. The intrinsic potential in the channel arises from permanent charges in the surrounding protein, from the difference in the dielectric constants of the channel and the surrounding protein, and from other ions and water in the channel (Dani and Eisenman, 1987; Green and Andersen, 1991; Konno et al., 1991; Green and Lu, 1995; Eisenberg, 1996). We present a simple model for the kinetics of a reaction in a channel and for the dependence of the reaction rate on electrostatic potential. This model places the target Cys in a site in the channel, with a barrier on either side, and the rate constants for crossing these barriers are treated according to absolute-reaction-rate theory (Woodhull, 1973; Dani and Eisenman, 1987; Hille, 1992). The advantages and limitations of this simple approach to ion permeation have been discussed elsewhere (Dani and Levitt, 1990; Hille, 1992). We assume that the jumps to and from the site, but not the reaction itself, depend on the electrostatic potential. The electrostatic contribution to the heights of the barriers involves both _M and _S, and the kinetic equations based on the model allow us to estimate the electrical distance, , to the site of reaction, and the intrinsic electrostatic potential, _S, at the site. The kinetic steps are indicated in Scheme I.

View larger version (6K): [in this window] [in a new window]   Scheme I.

X_EX is the reagent in the extracellular medium, X_IN is the reagent in the intracellular medium, S is the unoccupied site with an unreacted Cys, S' is the site reversibly occupied by the reagent, and S* is the site with the Cys covalently modified by the reagent. The rate constants for the jumps (associations and dissociations) of X are k₁ from the extracellular medium to S, k₁ from S to the extracellular medium, k₂ from S to the intracellular medium, and k₂ from the intracellular medium to S. k_S is the pseudo-first-order rate constant for the covalent reaction of X and the Cys in the complex S'.

The concentration of unreacted sites (and Cys) is s₀  s*, where s₀ is the initial and total concentration of sites and s* is the concentration of modified sites. Experimentally, the concentration of unreacted Cys is estimated from the current, I, elicited by ACh before the addition of X and after the reaction with X (and removal of unreacted X) as

(4)

where I is the current after reaction of all channels and I₀ is the current before any reaction (see Eq. 2). The solution of the differential equations corresponding to Scheme I is

(5)

where  is a combination of the rate constants and concentrations of reactants (Appendix a). When the reactant is applied just from the extracellular side, and x_IN = 0, then  = x_EX, where  is the effective second- order rate constant for the reaction and

(6)

Applying absolute-reaction-rate theory to the movement of X to and from the site, we obtain expressions for the rate constants that depend on free energy differences between ground and transition states at the peaks of the barriers. For X with charge z, these free energies contain electrostatic terms that depend on z. The electrical distances to the barriers are taken to be midway between the site and the medium on either side; i.e., the electrical distances from the extracellular medium to the barriers are /2 for barrier 1 and (1 + )/2 for barrier 2 (Woodhull, 1973). We assume that k_S is independent of _M and _S; i.e., that any separation of charge that might occur in the formation of the activated complex between X and the Cys is over too short a distance to be influenced by the gradients in _M and