Correspondence to: Arthur Karlin, Center for Molecular Recognition, Columbia University, 630 West 168th Street, New York, NY 10032. Fax:212-305-5594 E-mail:ak12{at}columbia.edu.
Released online: 17 January 2000
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Abstract |
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A ring of aligned glutamate residues named the intermediate ring of charge surrounds the intracellular end of the acetylcholine receptor channel and dominates cation conduction ( subunit contained a Cys substituted for
Thr244, three residues away from the ring glutamate
Glu241. The rate constants for the reactions of
Thr244Cys with the neutral 2-hydroxyethyl-methanethiosulfonate, the positively charged 2-ammonioethyl-methanethiosulfonate, and the doubly positively charged 2-ammonioethyl-2'-ammonioethanethiosulfonate were determined from the rates of irreversible inhibition of the responses to acetylcholine. The reagents were added in the presence and absence of acetylcholine and at various transmembrane potentials, and the rate constants were extrapolated to zero transmembrane potential. The intrinsic electrostatic potential in the channel in the vicinity of the ring of charge was estimated from the ratios of the rate constants of differently charged reagents. In the acetylcholine-induced open state, this potential was -230 mV with four glutamates in the ring and increased linearly towards 0 mV by +57 mV for each negative charge removed from the ring. Thus, the intrinsic electrostatic potential in the narrow, intracellular end of the open channel is almost entirely due to the intermediate ring of charge and is strongly correlated with alkali-metal-ion conductance through the channel. The intrinsic electrostatic potential in the closed state of the channel was more positive than in the open state at all values of the ring charge. These electrostatic properties were simulated by theoretical calculations based on a simplified model of the channel.
Key Words: nicotinic, mutagenesis, reaction kinetics, conductance, selectivity
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INTRODUCTION |
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Lipid bilayers are almost impermeable to inorganic ions because the energy of a monovalent ion in lipid is 40 kcal/mol higher than in water (
Electrostatic interactions have been described in a number of channels. The gramicidin A channel, a most efficient conductor of alkali-metal ions, has no charged residues, but interactions with backbone carbonyl dipoles and with oriented waters stabilize monovalent cations in the channel (-helices, and occupation of two selectivity-determining sites is stabilized by backbone carbonyls (
The ACh receptor is a complex of five subunits, two and one each of ß,
(or
), and
, which surround the central channel (see Figure 1 A) (
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The intrinsic electrostatic potential in the channel due to fixed and induced charges in the receptor (at zero transmembrane potential) was determined at three positions along the M2 segment, near its cytoplasmic end at
T244, near its middle at
L251, and near its extracellular end at
L258 (
T244 to -25 mV at
L258. The determination was based on a comparison of the rate constants for the reactions of differently charged, but otherwise similar, organic reagents with Cys substituted by site-directed mutagenesis for residues facing the channel lumen. The reactions were monitored by their effects on receptor function. The reagents were derivatives of sulfhydryl-specific thiosulfonates (
The residue T244, and the aligned residues in the other subunits, are in a narrow region of the channel lumen that constitutes the selectivity filter (
G240 to
T244 (see Figure 1 C) and includes the activation gate (
E241, one ßE252, and one
E255 (see Figure 1 C). These Glu constitute what
7)5 ACh receptor reduced its cation conductance drastically and was a necessary but not sufficient alteration to switch the charge selectivity of this channel from cationic to anionic (
We now show that the intrinsic electrostatic potential in the vicinity of T244 in the open channel is almost entirely due to the intermediate ring of charge. The magnitude of the negative potential decreases linearly as the negative ring charge is decreased, extrapolating to zero potential at a total ring charge of zero. Thus, the negative intrinsic electrostatic potential in the vicinity of the selectivity filter correlates with the cation conductance.
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MATERIALS AND METHODS |
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Mutagenesis and Oocyte Expression
Site-directed mutations were generated in mouse muscle T244C, wild-type ß, and wild-type
by PCR with pfu DNA polymerase (Stratagene Inc.). The PCR product was ligated into the pSP64T vector using appropriate restriction sites, and the cassette was sequenced to confirm the mutation. Mutants were named as <subunit> <wild-type residue> <residue number> <mutant residue>, using single-letter codes for amino acid residues. Capped cRNA was produced by in-vitro transcription with SP6 polymerase. Defolliculated Xenopus laevis oocytes were prepared and injected with 50 nl cRNA (100500 ng/µl) at a ratio of 2
:1ß:1
:1
, as previously described (
Electrophysiology
Currents were recorded under two-electrode voltage-clamp from oocytes in a bath solution containing (mM): 115 NaCl, 2.5 KCl, 1.8 MgCl2, and 10 HEPES, pH 7.2. Oocytes were perfused with bath solution maintained at a temperature of 18°C, at a rate of 7 ml/min. Voltage-recording and current-passing glass electrodes (filled with 3 M KCl) varied in resistance between 0.5 and 1 M. The reference electrode was connected to the bath via an agar bridge. All reagents were applied via the bath. Peak ACh-induced current was measured during a 10-s application of ACh at a concentration 4x EC50 for each mutant. There was no evidence of slow desensitization during these 10-s applications. ACh was applied two or three times to each oocyte, and only if the peak currents varied by <5% was the oocyte used further.
The response of mutant receptors to ACh was characterized by fitting the Hill equation to the currents evoked by at least five different ACh concentrations:
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(1) |
where IMAX is the asymptotic maximum current, EC50 is the concentration of ACh evoking half-maximal current, and n is the Hill coefficient.
Reagents
2-Ammonioethyl-methanethiosulfonate bromide [CH3SO2SCH2 CH2NH3+ Br-] (MTSEA) was purchased from Toronto Research Chemicals. 2-Hydroxyethyl-methanethiosulfonate [CH3SO2SCH2 CH2OH] (MTSEH) and 2-ammonioethyl-2'-ammonioethanethiosulfonate dichloride [H3N+CH2CH2SO2SCH2CH2NH3+ Cl-2] (AEAETS) were synthesized as previously described (
Reaction Rates
As previously described (
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(2) |
where It is the peak ACh-evoked current after t seconds of cumulative exposure to thiosulfonate reagent, I0 is the initial current, I is the peak current when the reaction is complete, and k* is the pseudo-first-order rate constant. The second-order rate constant,
, is given by Equation 3:
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(3) |
To determine the rate constant for the reaction at zero transmembrane potential, either we extrapolated the rate constants for the reaction at three non-zero membrane potentials to zero membrane potential, or we measured the reaction rate at 0 mV directly. Whatever the holding potential was during the application of the reagent, the holding potential was always -50 mV during the test responses to ACh.
Intrinsic Electrostatic Potential
We determined the rate constants, , for the reactions of MTSEA, MTSEH, and AEAETS with
T244C at zero transmembrane potential. We previously determined the rate constants, kME, for the reactions of these reagents with 2-mercaptoethanol [HSCH2CH2OH] in bulk solution (
0, of the rate constants for pairs of the thiosulfonate reagents (at zero transmembrane potential), as follows:
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(4) |
where 1 is the second-order rate constant for the reaction of either MTSEA or AEAETS with the Cys in
T244C and 2
is in each case the second-order rate constant for the reaction of MTSEH with
T244C. 1kME is the second-order rate constant for the reaction of either MTSEA or AEAETS with 2-mercaptoethanol in solution, and 2kME is the second-order rate constant for the reaction of MTSEH with 2-mercaptoethanol in solution. Under conditions of quasi-equilibrium and low saturation of the reaction site with the reagent:
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(5) |
where G 0 =
G 01 -
G 02, the difference in the standard free energies of association of reagent 1 and reagent 2 with the reaction site. The standard free energy of association of the first reagent,
G 01 = G 0S,1 - G 0EX,1 - G 0S, where G 0S,1 is the standard free energy of the complex of the site and reagent 1, G 0EX,1 is the standard free energy of the reagent 1 in the extracellular solution, and G 0S is the standard free energy of the unoccupied site. Similarly,
G 02 = G 0S,2 - G 0EX,2 - G 0S. The terms G 0S cancel in
G 0.
For two reagents that are similar in all respects except charge, we assume that all contributions to G 0 cancel, except the difference in electrostatic free energies of association for the two reagents that depend on reagent charge. We equate this difference in electrostatic free energies and, hence,
G 0 to (z1 - z2)F
S, where z1 is the charge of MTSEA (+1) or AEAETS (+2), z2 is the charge of MTSEH (0), and
S is the intrinsic electrostatic potential in the channel close to the charge on the reagent when it is reacting with the Cys substituted for
T244. Therefore, as derived previously (
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(6) |
Theoretical Calculation of the Standard Free Energy of Association
In the absence of a high-resolution structure for the receptor, we modeled the channel lumen as a water-filled cylinder embedded in a uniform, low-dielectric slab, representing both the membrane-spanning domain of the receptor and the phospholipid bilayer (see Figure 8). The electrostatic standard free energy, G 0, of transfer (or association) of a reagent molecule or a Na+ from bulk solution to a given location within the model channel was defined as
G 01 above and calculated as the electrostatic energy of the complex of the reagent or inorganic ion at a given position in the channel, minus the self-energy of the reagent or ion in bulk solution, and minus the electrostatic energy of the unoccupied channel with its charges (
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We built molecular models of the reagents MTSEA, MTSEH, and AEAETS with the Biopolymer module of the program InsightII (Molecular Simulations Inc.). The models were optimized using MOPAC in the MOPAC/AMPAC module of InsightII. Subsequently, the structures were energy-minimized with the program CHARMM (T244C. We used these models to include the effects of size and charge distribution on the electrostatic interactions of the reagents with the channel. In the case of Na+, the Born radius of 1.68 Å was used (
Because anions cannot penetrate the cation-selective ACh channel to the vicinity of T244 (
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RESULTS |
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Expression of Glutamate-ring Mutants
The aligned residues E241, ßE252, and
E255 (Figure 1 C) were mutated to alter the ring charge. We designate the subunits with a superscript to indicate the charge of the residue at the ring position. The wild-type subunits are designated
-, ß-,
0, and
-. The mutant
E241Q is designated
0, ßE252Q is designated ß0, ßE252K is designated ß+, and
E255Q is designated
0. In all cases,
contained the mutation T244C, so that
- contains the mutation T244C, and
0 contains both mutations, T244C and E241Q. The complexes tested were:
-2ß-
0
- (i.e., the pseudo-wild type receptor with
T244C),
-2ß0
0
-,
-2ß-
0
0,
02ß-
0
-,
-2ß+
0
-,
02ß0
0
-, and
02ß0
0
0. These combinations had -4 to 0 net charges in the glutamate ring.
The responses of each of these complexes to a range of ACh concentrations were fitted by the Hill equation (Equation 1), which yielded IMAX, EC50, and n. Despite the expected lower single-channel conductance in the mutants with decreased ring charge (02ß0
0
0, which never gave more than -50 nA in response to ACh and was not tested further. For the oocytes and mutants used, the mean IMAX varied from approximately -500 to -2,000 nA (Figure 2 A). These whole-cell currents, of course, depend on the extent of expression and the gating and desensitization kinetics as well as on the single-channel conductance.
-2ß+
0
- gave unstable currents that often underwent spontaneous runup or rundown of repeated responses, and we could not use it for measuring reaction rates.
With decreasing ring charge, the EC50 for ACh increased modestly from 13 µM for the pseudo-wild type to ~50 µM for 02ß0
0
- or 1.6-fold increase in EC50 per unit decrease in negative charge (Figure 2 B). Under the conditions of our experiments, wild-type receptor has an EC50 for ACh of ~3 µM (
Thiosulfonate Reaction Rates
For each of the mutants, we recorded ACh-induced currents between repeated exposures to thiosulfonate reagents. Typical results are shown for 02ß0
0
- exposed to MTSEA in the absence (Figure 3 A) and in the presence (Figure 3 C) of ACh. The peak currents of the ACh-induced test responses declined as a first-order process (Figure 3B and Figure D). The exponential fits to these data yielded pseudo-first order rate constants for the reactions of MTSEH, MTSEA, and AEAETS with each of the Glu-ring mutants.
For most mutants and reagents, rate constants were measured at three holding potentials. A typical set of rate constants are shown in Figure 4 for the reactions of MTSEH, MTSEA, and AEAETS, in the absence and presence of ACh, with 02ß0
0
-. Only for AEAETS in the presence of ACh did the rate constant depend significantly on the holding potential, as previously found with
-2ß-
0
- (
-2ß-
0
0 were carried out at a holding potential of 0 mV, and thus the rate constants at 0 mV were determined directly.
The rate constants at zero transmembrane potential for each reagent varied with the total charge in the Glu ring (Figure 5). The log (rate constant) was a linear function of ring charge. The rate constants for MTSEA decreased by a multiplicative factor of 0.6 in the absence and 0.4 in the presence of ACh, per unit decrease of negative charge. The rate constants for AEAETS increased by a factor of 2.0 in the absence of ACh, and decreased by a factor of 0.3 in the presence of ACh, per unit decrease of negative charge. The rate constants for MTSEH increased by a factor of 3.1 in the absence and 5.6 in the presence of ACh, per unit decrease of negative charge.
The increase in the rate constant for MTSEH, as the magnitude of the negative ring charge decreased, was ascribed to an increase in the local pH and a concomitant increase in the probability of the target T244C sulfhydryl being in the reactive -S- deprotonated state (see DISCUSSION). The changes in the rate constants of the positively charged MTSEA and AEAETS with decrease in the negative charge of the Glu ring were then a combination of (a) a rate increase due to increased deprotonation of the sulfhydryl, and (b) a rate decrease due to decreased interaction of the charged reagents with the Glu ring charge. The rate increase due to deprotonation of the SH should be equal for the charged and uncharged reagents, and this factor should cancel in the ratio of the rate constant of a charged reagent to the rate constant of the uncharged reagent. What remains is due to the interaction of the charged reagent with the intrinsic electrostatic potential.
Intrinsic Electrostatic Potential
As previously noted (S and as the corresponding
G 0 (Equation 5).
The ratio of ratios, 0, for the pair MTSEA and MTSEH, decreased as the magnitude of the negative charge in the Glu ring decreased, in both the presence and absence of ACh (Figure 6 A). The corresponding intrinsic electrostatic potential in the vicinity of the site of reaction, z
s, and the corresponding
G 0, increased linearly (Figure 6 B). (z
s for
-2ß-
0
0 in the closed state falls off the line, reflecting the deviation of the rate constant for the reaction of MTSEA with this mutant, shown in Figure 5 B.) The linear least-squares fits yield slopes of 59 mV/ring charge (presence of ACh) and 54 mV/ring charge (absence of ACh). The extrapolated values at zero ring charge are -4 mV (presence of ACh) and 72 mV (absence of ACh). The two lines are nearly parallel, but displaced by 75100 mV. Remarkably, in the open state of the channel, the intrinsic electrostatic potential in the vicinity of
T244 is almost entirely due to the Glu ring charge.
The ratio of ratios, 0, for the pair AEAETS and MTSEH, also decreased as the magnitude of the negative charge in the Glu ring decreased, markedly in the presence but only slightly in the absence of ACh (Figure 7 A). The corresponding z
s, increased linearly (Figure 7 B). (z
s for
-2ß-
0
0 is again somewhat anomalous, but in this case in the open state, reflecting the deviation of the rate constant, shown in Figure 5 C.) The linear least-squares fits yield slopes of 62 mV/charge (presence of ACh) and 14 mV/charge (absence of ACh). The extrapolated values at zero ring charge are 80 mV (presence of ACh) and 92 mV (absence of ACh). In this case, the two lines are not parallel, but rather nearly converge at zero ring charge.
For the pseudo wild-type receptor (-2ß-
0
-, ring charge -4), the values of z
S and
G 0 determined with AEAETS and MTSEH (-215 and -5.0 kcal/mol) and those determined with MTSEA and MTSEH (-230 and -5.3 kcal/mol) are nearly the same. At first glance, this is surprising given that the charge of AEAETS is +2 and the charge of MSTEA is +1. A theoretical calculation discussed below indicates that the second positively charged ammonium of AEAETS, located ~10 Å away from the first positively charged ammonium and from the ring of charged glutamates, may have little net effect on
G 0 for the association of AEAETS with the site of reaction in the channel and hence little effect on
G 0 and z
s.
Lysine Ring Charge
Adjacent to the ring of four Glu and a Gln is a ring of five Lys (Figure 1 C). Mutations in these Lys have been made previously, and the mutants that were functional did not show marked changes in conductance or other properties (T244C, wild-type
, and wild-type
. The total charge in the lysine ring was thereby changed from +5 to +3. The charge in the Glu ring remained -4.
S was estimated with the pair MTSEA and MTSEH in the presence and absence of ACh. z
S for ßK253E in the presence of ACh was 31 mV less negative, and in the absence of ACh, 15 mV less negative than
S for the pseudo wild type,
-2ß-
0
-. z
S was also estimated with the pair AEAETS and MTSEH. z
S for ßK253E was 56 mV less negative in the presence of ACh, and 16 mV more positive in the absence of ACh, than
S for
-2ß-
0
-. Thus, in all cases, making the charge in the ring of Lys less positive changed z
S modestly in the positive direction. This direction is the opposite of what we would expect if the charges in the ring of Lys interacted directly with cations in the channel.
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DISCUSSION |
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Using an approach (
Estimating the Intrinsic Electrostatic Potential
We estimate S using Equation 6, which we derived initially from the simple two-barrierone-well kinetic model of
, for the reaction of reagent added, say, to the extracellular side of the membrane depends on four reaction rate constants; these characterize the transfer from the extracellular side to the site (i.e., association with the site), the transfer from the site back to the extracellular side, the transfer from the site to the intracellular side, and the covalent reaction between reagents associated with the site and the Cys in the site. The rate constant for the transfer from the intracellular side to the site is eliminated because the reagent concentration on the intracellular side is close to zero.
When the rate constant for transfer of reagent from the site back to the extracellular side is much greater than both the rate constant for transfer from the site to the intracellular side and the rate constant for reaction at the site, the reagent at the site is close to equilibrium with reagent in the extracellular solution. Also, as we know from the absence of reversible channel-blocking by the reagents at the concentrations used here, the degree of occupation of the reaction site is low; i.e., the rate constant for transfer of reagent from the site back to the extracellular side is much greater than the rate constant for transfer of reagent from the extracellular side to the site times the extracellular concentration. Under these conditions, the concentration of sites occupied by reagent is approximately equal to the total concentration of not-yet-reacted sites times the equilibrium affinity constant times the extracellular reagent concentration, and the second-order rate constant for the reaction of the site, , is the intrinsic rate constant for the reaction of the occupied site times the equilibrium affinity constant (see
G 0/RT), where
G 0, the standard free energy of association (or transfer) was defined in MATERIALS AND METHODS; i.e.,
kS exp(-
G 0/RT).
For two reagents, 1 and 2, that are similar except for their charges, we form the ratio, 1/2
(1kS/2kS) exp(-
G 0/RT). We assume that the ratio of the rate constants for the reactions of the reagents with the Cys in the site, 1kS/2kS, is the same as the ratio of the rate constants for their reactions with a simple thiol, 2-mercaptoethanol, in bulk solution; i.e., 1kS/2kS = 1kME/2kME. With
0 defined by Equation 4, Equation 5 follows. We also assume that the only contributions to
G 0 that do not cancel are those due to the difference in the charges of the two reagents; i.e.,
G 0 is just the difference in the electrostatic free energies of association of the two reagents with the site of reaction. The more similar the two reagents, the smaller the errors in these two assumptions. The reaction mechanisms of the three reagents used here, MTSEA, AEAETS, and MTSEH, are the same. MTSEA is also very similar in size and shape to MTSEH; however, the doubly charged AEAETS is significantly longer than MTSEH (13 compared with 10 Å). For the more reliable pair, MTSEA and MTSEH,
G 0 is the difference in the free energies of association with the site of the positively charged ammonium head group of MTSEA and of the neutral hydroxyl head group of MTSEH.
Neglecting any differences in the nonelectrostatic contributions to the binding of an ammonium group and a hydroxyl group, we equate G 0 to (z1 - z2) F
S; i.e., to F
S for z1 = 1 and z2 = 0. The electrostatic free energy of transfer of a charged reagent from bulk solution to a site in the channel would have the form zF
S, however, if
S were due to fixed charges only and independent of the reagent charge z. Because there are dielectric boundaries in the system, the free energy of transfer also contains a term equal to 0.5 zF
Dielectric, where
Dielectric is the potential due to the reaction field generated by the reagent charge.3 The electrostatic potential at the position of the reagent charge due to all other fixed charges and to dielectric boundaries is
S' =
Fixed +
Dielectric; however,
G 0 = (z1 - z2)F(
Fixed + 0.5
Dielectric), and we do not have separate measurements of
Fixed and
Dielectric. Thus,
S, calculated as
G 0/[(z1 - z2)F], equals
Fixed + 0.5
Dielectric and differs from
S' by 0.5
Dielectric. In the theoretical calculations discussed below,
Dielectric is much smaller than
Fixed, so that
S
S'.
In equating the electrostatic free energy of transfer to zFS, we assume that the charge of the reagent is at a unique location and electrostatic potential. This is valid for MTSEA (z1 = +1), but not for AEAETS, which has two ammonium groups separated by as much as 10 Å. When the ether sulfur of AEAETS is in position to react with the Cys of
T244C, one positively charged ammonium group is close to
E241 and the other glutamates in the intermediate ring while the other positively charged ammonium is at the level of
S248. From previous measurements of
S at different positions in the channel (
Because the two charges on AEAETS are separated, G 0 for the pair AEAETS and MTSEH, with a charge difference of +2, is not twice the magnitude of
G 0 for the pair MTSEA and MTSEH, with a charge difference of +1. The experimentally derived values of
G 0 for the two pairs of reagents in the open channel were almost identical (Figure 6 B and 7 B).
The unequal contributions of the two charges of AEAETS are rationalized by a theoretical analysis of a simplified model of the channel (Figure 8). In the open channel with a ring charge of -4, we calculate that G 0 for the pair AEAETS and MTSEH is 1.4x
G 0 for the pair MTSEA and MTSEH (Figure 9 A), not 2x. This result can be explained as follows: the ammonium group of MTSEA and one of the ammonium groups of AEAETS sit in the plane of the negative charges (representing the ring of Glu) when the ether sulfur is in position to react with the Cys. The electrostatic free energies for the transfer of these ammonium groups from bulk solution to their position in the channel are the same for the two reagents. The electrostatic free energy for the transfer of the second ammonium of AEAETS, however, is less favorable than the first because the second ammonium is farther from the ring of negative charges and is further from bulk water at the end of the channel. In general, the electrostatic free energies of transfer of spatially separated charges are unlikely to be equal. To avoid the uncertainty in the appropriate value of z to use for AEAETS in Equation 6, we calculate instead z
S (=
G 0/F) for both pairs of reagents (Figure 6 B and 7 B).
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zS and the Glutamate Ring Charge
Receptor complexes with Glu ring charges of -4, -3, -2, and -1 were probed with MTSEA, MTSEH, and AEAETS. Based on the rate constants for the reactions of MTSEA and MTSEH with T244C in the presence of a near-saturating concentration of ACh (the open state4), z
S increased linearly in a positive direction from -230 mV at a ring charge of -4 to the extrapolated value of -4 mV at a ring charge of 0 (Figure 6 B). The linear increase with ring charge indicates that z
S and
G 0 are electrostatic in origin and that the conditions on Equation 5 and Equation 6 are not seriously violated. The extrapolation of z
S at 0 ring charge to close to 0 mV indicates that in the open state, at least, the Glu ring is the only net source of z
S. The contributions of all other charges, fixed or induced, balance.
Theoretical calculation of zS based on MTSEA and MTSEH in the open state gave values that corresponded closely to the experimental values (Figure 9 A). In the model, there are no fixed charges other than the ring of charges representing the Glu. When these charges, and thereby
Fixed, are eliminated, the only component of the electrostatic potential is
Dielectric. With the charge of MTSEA close to the end of the channel, as in the model,
Dielectric (calculated with the ring charge equal to zero) is close to zero. We do not know the actual magnitude of
Dielectric when MTSEA is in the lumen of the receptor. If
Dielectric is not close to 0, then
Fixed due to fixed charges other than the intermediate ring Glu must be half the magnitude of
Dielectric and opposite in sign to yield the overall
S close to zero.
In the closed state4 of the receptor, zS determined with MTSEA and MTSEH also increased linearly as the negative ring charge was decreased, and the fitted line was parallel to the line fitted to the open state data, displaced by 74 mV in the positive direction at a ring charge of 0 (Figure 6 B). This difference between the closed and open states is consistent with a gate closing between the Glu ring and the target Cys substituted for
T244 (
S and
G 0 for the pair MTSEA and MTSEH were similar to the experimental values at ring charge -4; however, the theoretical values rose more quickly than the experimental values as the magnitude of the ring charge approached zero (Figure 9 A). This discrepancy is likely a consequence of the over-simplified gate in the model, which, although appropriately located between the ring of charge and
T244, occludes the channel more than would be necessary to block the passage of alkali metal ions.5
Determined with the pair AEAETS and MTSEH, zS in the open state once again increased linearly with the decrease in the magnitude of the ring charge (Figure 7 B), generally supporting the electrostatic basis of z
S. In this case, z
S did not extrapolate to ~0 mV at 0 ring charge. Also, in the closed state, z
S was positive at all values of the ring charge. Both phenomena are seen in the theoretical model (Figure 9 A). In the closed state, however, the slope of the line fitted to the experimental z
S was not significantly different from 0. AEAETS is larger than MTSEH and, because of its greater size, the narrow channel could retard its reaction much more than that of MTSEH. If channel closing involves a narrowing of the lumen from
G240 to
T244 (
T244C in the closed state could be severely hindered. In this case, nonelectrostatic contributions might not be eliminated in
0 and might indeed dominate over the electrostatic contributions.
One question is whether the changes in 0 with changes in ring charge could have been due to changes in gating kinetics. The mutations resulted in as much as a fourfold increase in the EC50 for ACh (Figure 2 B). The changes were likely the results of changes in gating kinetics, which might have affected the rates of reaction of the reagents in the presence of ACh. There was no correlation, however, between the EC50 of the different mutants and the rate constants for the reactions of MTSEA or of AEAETS (plot not shown). The rate constants for MTSEH increased, albeit nonlinearly, as the EC50 increased; however, this could not be due to changes in gating because in most mutants the rate constants for MTSEH were nearly the same in the open and closed states (Figure 5 A). Thus, the changes in
0 and z
S with changes in the Glu ring charge were not due to changes in gating kinetics.
We conclude that in the open state of the channel the intrinsic electrostatic potential in the vicinity of T244 is largely due to the negatively charged Glu in the intermediate ring of charge. All other electrostatic interactions with the reagents and, presumably, with permeant inorganic cations must more or less balance.
Reaction Rate Constants of MTSEH and the Glu Ring Charge
Methanethiosulfonates react at least 5 x 109 faster with dissociated thiolates (RS-) than with undissociated thiols (RSH) (T244C depends on, among other factors, the ionization of the thiol. The increase in the rate constant for neutral MTSEH as the magnitude of the negative ring charge decreased (Figure 5A), we ascribe to an increase in the local pH and a concomitant increase in the probability of the target
T244C sulfhydryl being in the reactive -S- deprotonated state. This can be modeled by assuming that the proton concentration, hS, in the vicinity of the Cys is at equilibrium with the extramembranous proton concentration, hE, according to a Boltzmann distribution, hS/hE = exp(-
G 0S*H/RT),
G 0S*H is the standard free energy of transfer of a proton from the extramembranous solution to the vicinity of the target Cys when the reagent is also in the channel in position to react; i.e.,
G 0S*H = G 0S*H - G 0S* - G 0H, where G 0S*H is the standard free energy of the proton in the site also occupied by reagent, G 0S* is the standard free energy of the site occupied by reagent, but not a proton, and G 0H is the standard free energy of the proton in bulk solution. We can define an electrostatic potential,
S* =
G 0S*H/F. Note that
G 0S*H is different than
G 0 in Equation 6, and
S* is different than
S.
For MTSEH, for which z = 0, the observed rate constant for the modification of the Cys, , is given by Eq. 7 in
= kSk', where kS is the rate constant for the reaction of MTSEH at the site with the Cys, and k' is an expression containing the transfer rate constants, which do not vary with electrostatic potential because MTSEH is neutral. The rate constant kS applies to the rate of reaction of the target Cys in terms of the total concentration of the Cys. Only a small fraction of the Cys is in the reactive thiolate form. If k- is the rate constant for the reaction of the thiolate form with MTSEH at the site, then kS = (k-KA/hE)exp(F
S*/RT), where KA is the acid dissociation constant of the Cys sulfhydryl. The observed overall rate constant,
, is then given by
= (k'k-KA/hE)exp(F
S*/RT) and ln(
) = ln(k'k-KA/hE) + F
S*/RT.
For uncharged MTSEH, only S* is a function of q, the ring charge. Furthermore, the experimental ln(
) versus ring charge is well fit by a straight line (Figure 5 A). Thus,
S* is a linear function of q; i.e.,
S* = mq + b. The slopes of the least-squares lines in Figure 5 A imply that m is 43 mV/ring charge in the open state and 27 mV/ring charge in the closed state. The comparable slopes of
S, determined with the pair MTSEA and MTSEH, as a function of q, were 59 mV/ring charge in the open state and 54 mV/ring charge in the closed state (Figure 6 B). The linear relationships between ln(
) and q for the reaction of MTSEH are comparable to the linear relationships between
S and q, determined with MTSEA and MTSEH. This correspondence supports the notion that the ring of Glu exerts an electrostatic effect in the channel lumen at the level of
T244.
The actual pH in the channel lumen is not known. If it were low enough around the Glu, then it would seem unlikely that all four Glu carboxyls would be deprotonated simultaneously. Nevertheless, each step of Glu mutation to Gln has an equivalent effect on S (Figure 6 B), consistent with all four Glu being ionized, at least while reagent is in the site.
Lysines
Adjacent to the ring of Glu is a ring of five Lys, one residue closer in the sequence to the target Cys in T244C. Mutation of one of these Lys to Glu, in ßK252E, had a paradoxical effect on
S. Even though the charge in this ring was made less positive by 2 charges,
S in the open state in this mutant was more positive by ~30 mV than in the pseudo wild type. The direction of change is opposite to that which would result from a direct interaction of the Lys residues with the charge of the reagents. The magnitude of this change is also small compared with the change in
S caused by a change of 2 charges in the Glu ring (Figure 6 B).
Despite this indication that the Lys do not interact directly with the reagents, when K242 was mutated to a Cys and expressed with wild-type ß,
, and
subunits in HEK 293 cells, the Cys reacted in the open state (but not in the closed state) of the channel with MTSEA added either extra- or intracellularly (
K242 and the aligned Lys in the other subunits do not ordinarily face the channel in wild-type receptor and play primarily a structural role. It is possible that the side chains are so oriented that even though the lysines are one residue closer in the sequence than the glutamates to the target Cys in
T244C, the
-NH3+ of the Lys side chains are considerably farther from the target Cys than the
-COO- of the glutamates.
Supporting this interpretation is the previous finding that the mutation of the homologous Lys to Glu in Torpedo ACh receptor ß, , or
subunits had no effect on the conductance of this receptor (
7)5, the combination of neutralization of the Glu ring and two additional mutations was sufficient to change the charge selectivity of the channel from cationic to anionic, albeit severely reducing the ACh-induced current. Further mutation to neutralize the Lys ring, however, did not alter the anionic selectivity or the current (
Two other channel-flanking rings of net negative charge, the outer ring of residues aligned with E262 and the inner ring aligned with
D238, contribute to the conductance of the muscle-type ACh receptor channel, but considerably less than does the intermediate ring of charge (
7)5 channel, neutralization of the inner ring of charge had no detectable effects on ACh-evoked currents (
Implications for Conductance of Alkali Metal Ions
Cation conductance through the ACh receptor is linearly dependent on the number of charged residues in the intermediate ring of charge (G240 to
T244 (
G 0 and the equivalent z
S, measured with the pair MTSEA and MTSEH, are -5.3 kcal/mol and -230 mV.
We also calculated G 0 and the equivalent z
S in a cylindrical model of the channel (Figure 8), which is highly simplified compared with a moderate-resolution structure of the open ACh receptor channel (
G 0 and z
S very close to the experimental ones (Figure 9 A).
In addition, we calculated the electrostatic contribution to G 0, and z
S for the transfer of a Na+ from the extracellular solution to various positions along the axis of the model channel (Figure 9 B). The electrostatic contribution to
G 0 and z
S reach minima of -6.3 kcal/mol and -273 mV at a distance from the channel midpoint of -13 Å (i.e., 2 Å from the intracellular end of the model channel). By comparison, the electrostatic contribution to the free energy of transfer of a K+ from bulk water to the central cavity of the KcsA potassium channel was calculated to be -8.5 kcal/mol (
In the ACh receptor channel, the large negative electrostatic potential could be the basis for a high affinity cation-binding site in the vicinity of the intermediate ring. We cannot calculate an equilibrium binding constant, however, because neither the experimentally derived G 0 for the pair MTSEA and MTSEH nor the theoretically calculated electrostatic contribution to
G 0 for the transfer of a Na+ is equivalent to the total free energy of transfer of a cation from bulk solution to the channel site.6 If, however, the free energy of cation transfer were in the range of -5 to -6 kcal/mol, the equivalent equilibrium dissociation constant would be in the range of 40200 µM, far lower than the half-saturation concentration for Na+ conductance of ~100 mM (
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Footnotes |
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2 Although the accessibilities of these residues from the two sides of the membrane in the open and closed states of the channel are known (
1 Abbreviations used in this paper: ACh, acetylcholine; AEAETS, 2-ammonioethyl-2'-ammonioethanethiosulfonate dichloride; MTSEA, 2-ammonioethyl-methanethiosulfonate bromide; MTSEH, 2-hydroxyethyl-methanethiosulfonate.
3 Because Dielectric depends on z, 0.5 zF
Dielectric is proportional to 0.5 z2, and only if
Fixed >>
Dielectric is
G 0 proportional to the first power of the reagent charge, z.
4 The receptor exists in at least four different functional states, closed, open, and fast- and slow-desensitized. Only in the open state is the channel conducting. As discussed previously (20 s) application of reagent together with ACh the reaction is with a mixture of receptors in the open state and with receptors in the fast-desensitized state. In the case of reactions with
T244C, the rate constant for the reaction of MTSEA with receptor that has been driven into the slow-desensitized state is two to three orders of magnitude smaller than with receptor in the mixed open and fast-desensitized states (our unpublished results). Furthermore, the dependence on transmembrane potential of the rate of reaction of
T244C with AEAETS in the presence of ACh suggests that the reaction is predominantly with receptor in the conducting open state, and not with receptor in the closed state or in either of the nonconducting desensitized states. For convenience, we call the mixed state within the first 20 s of ACh application the open state.
5 In the future, we will explore the theoretical implications of more subtle gate structures, of a more realistic lumen geometry, of moving the intermediate ring, of including the inner and outer rings of charges and the ring of lysines, and of varying the orientation of the reagents in the channel.
6 Even if the ammonium group of MTSEA were representative of a permeant cation and the hydroxyl group of MTSEH were representative of a water molecule displaced by the cation, G 0 does not include the entropy of transfer of the cation from bulk solution to the site, the value of which is unknown. The calculated electrostatic contribution to
G0 for the transfer of Na+ from bulk solution to the site is the electrostatic contribution to the enthalpy of transfer. It does not include van der Waals contributions to the enthalpy of transfer and does not include the entropy of transfer.
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Acknowledgements |
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We thank Eberhard von Kitzing, John Dani, and Christopher Miller for helpful discussions and Barry Honig for advice and use of his computer facilities.
This work was supported by research grants NS07065 (A. Karlin) from the National Institute of Neurological Disorders and Stroke and MCB-980892 (B. Honig) from the National Science Foundation. Diana Murray was supported by a Postdoctoral Fellowship from the Helen Hay Whitney Foundation.
Submitted: 20 October 1999
Revised: 8 December 1999
Accepted: 9 December 1999
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References |
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