Correspondence to: H. Peter Larsson, Neurological Sciences Institute, Oregon Health Sciences University, Portland, OR 97006. Fax:(503) 418-2501 E-mail:larssonp{at}ohsu.edu.
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Abstract |
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Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES-) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET+). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 Å) to a position close (8 Å) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling.
Key Words: electrostatics, cysteine reactivity, Shaker, voltage clamp, Xenopus oocytes
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INTRODUCTION |
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Voltage-gated ion channels respond to changes in the transmembrane electric field by opening or closing their ion conducting pore. The central part of the ion channel (from the fifth to the sixth transmembrane segments, S5S6) contains the pore and several of the gates, and is most likely similar to the structurally determined bacterial KcsA K channel (
Studies measuring charge movement per channel, in combination with S4 charge neutralizations, have suggested that the four most external S4 charges move across a large portion of the electric field (
The purpose of the present study was to estimate the distance between R362 in S4 and residues at the extracellular end of S5 (E418 and A419; Fig 1) in both the closed and open configurations. Information about the exact movement of S4 and its charges is crucial in the quest for an understanding of how the movement of S4 causes the channel pore to open and close. We estimated the distances between S4 and S5 by measuring the electrostatic impact that the charged residues of S4 have on residues in S5 at both resting and activated membrane potentials. We did this by mutating 418 or 419 (at the extracellular end of S5) to a cysteine and studying the rate of modification of the introduced cysteine by charged cysteine-specific reagents at two different potentials. The results show that the top charge of S4 (R362) moves from a distant location (>20 Å) to a very close location (8 Å) in relation to residue 418 during activation of the channels.
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MATERIALS AND METHODS |
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Molecular Biology
The experiments were performed on Shaker H4 channels (646 deletion (
Electrophysiology and Solutions
The currents were measured with the two-electrode voltage-clamp technique (CA-1 amplifier; Dagan Corp.). Microelectrodes were made from borosilicate glass and filled with a 3-M KCl solution. The resulting resistance varied between 0.5 and 2.0 M. The amplifier's capacitance and leak compensation were used, and the currents were low-pass filtered at 1 kHz. All experiments were performed at room temperature (2023°C). For the electrophysiological experiments, we used the 1-K solution (in mM: 88 NaCl, 15 HEPES, 1 KCl, 0.4 CaCl2, and 0.8 MgCl2; NaOH is added to adjust pH to 7.4, yielding a final Na concentration of
96 mM).
Cysteine Reagents: Application and Reactivity
The positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET+),* the negatively charged MTSES- (sodium (2-sulfonatoethyl) methanethiosulfonate), and the neutral MMTS0 (methyl methanethiolsulfonate) (Toronto Research Chemicals Inc.) were applied in the bath solution by a gravity-driven perfusion system. The modification was assayed functionally in two-electrode voltage-clamped oocytes, as described previously (
To be able to compare the state dependency of the modification rates between the different mutations, we have to assume that a majority of the S4 domains has moved from its fully deactivated position to its fully activated position at the two test potentials, -80 mV and 0 mV (or +40 mV for R362Q mutations). The major charge movement in wild-type (WT) Shaker channels occurs between -80 and -20 mV (
The modification rate k of a cysteine depends on the intrinsic reaction rate of the MTS reagent ki, the accessibility of the thiol group pAcc, the probability of finding the thiol group in its reactive unprotonated form pCys-, and the local concentration of the MTS reagent cMTS.
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(1) |
The accessibility of the thiol group pAcc(V) depends on the molecular configuration of the channel protein and, thus, for a voltage-gated ion channel, it can depend on the membrane potential V. The protonation of the thiol group pCys-(V) depends on the local (relative bulk) electrical potential Cys(V). For example, a negative value of
Cys attracts H+ to the thiol reagent group, giving a lower local pH.
Cys(V) could depend on the conformation of the channel. For example, the movement of the positive S4 charges close to the cysteine will make the local electric potential around the cysteine more positive and, hence, raise the local pH.
Cys(V) could also depend on the transmembrane voltage if the cysteine is located in the transmembrane electric field, for example, if the cysteine is located deep in the pore (
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(2) |
where pKa is the pH at which 50% of the cysteines are negatively charged (pH = 8.3), and pH is the bulk value. F, R, and T have their usual thermodynamic meanings. The local concentration of the MTS cMTS(V, MTS) will also depend on the local electric potential and can be described by:
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(3) |
where cMTS,bulk is the bulk concentration of the MTS reagent (in the solution), and z is the valency of the charge of the MTS reagent.
In the present paper, we will measure the time constant of the reactivity in millimolar seconds at different membrane potentials, to estimate the local electric potential
Cys(V) at the cysteine. The ratio of the reactivity of open channels (0 mV) to the reactivity of closed channels (-80 mV) is (combining Equation 1 and Equation 3):
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(4) |
If the difference in local potential at the cysteine between the open and the closed state is
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(5) |
then combining Equation 4 and Equation 5:
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(6) |
Assuming that the relative (sterical) accessibilities (0 vs. -80 mV) are the same for MTSET+ and MTSES-, then follows
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(7) |
Thus, it is possible to calculate the difference in local potential at the thiol between the open and the closed state without any knowledge about the intrinsic reactivity rates, the accessibility of the thiol, the absolute local potential, and the charge state of the thiol. Furthermore, if the extracellular surface potential (relative bulk solution) is assumed to be negative (
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(8) |
If we combine Equation 6 and Equation 8, then the relative accessibility can be calculated as:
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(9) |
Thus, if the reagent is positively charged, like MTSET+ (z = +1), then an increase in local concentration of the reagent (negative Cys attracts the positive MTSET+) factors out the decrease in reactive (unprotonated) cysteines and the Equation 9 is reduced to:
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(10) |
Thus, if there is no difference in reaction rate at -80 or 0 mV for MTSET+, then the sterical accessibility is the same at both potentials. For MTSES- (z = -1) the expression reads:
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(11) |
Electrostatic Calculations
To get a rough estimate of the distance between the reactive cysteine and a surface charge, we used the following equation (
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(12) |
where r is the potential at a distance r from an elementary charge e, assuming that the charge is located at the border between a low dielectric (membrane) and a high dielectric (water,
r = 80) medium.
is the inverse of the Debye length in the aqueous phase (9.8 Å in the 1-K solution; see
0 is the permittivity of free space.
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RESULTS |
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Activation of the Channel Affects the Modification Rates of 418C
To test the hypothesis that S4 is located close to S5 (
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Modification of E418C by MTSET+, MTSES-, or MMTS0 substantially reduced the current (Fig 3A and Fig B). The modification rate of 418C by MTSES- was faster by a factor of 18 when S4 was in the activated state (0 mV; = 2.5 mMs) than when in the resting state (-80 mV;
= 44.7 mMs; Fig 3 A and Table 1). However, the modification rate of 418C by MTSET+ was not significantly different in the activated state (
= 2.1 mMs) compared with that in the resting state of S4 (
= 2.2 mMs; Fig 3 B and Table 1). These results suggests that activation increases the local concentration of MTSES- 4.2-fold, decreases the local concentration of MTSET+ 4.2-fold, and increases the probability of finding 418C in the unprotonated state 4.2-fold. As expected, the modification rate of 418C by MMTS0 was faster by a factor of
4 when S4 was in the activated state (0 mV;
= 6.9 mMs) than when in the resting state (-80 mV;
= 25.3 mMs; Table 1). These results show that there is a positive change in the local electrostatic potential around 418C during activation of the channels. In the next section, we will show that a large part of this change in local potential is because the positive charges on S4 approach 418C.
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R362Q Decreases the State-dependent Effect of E418C Modification
During activation, three of the most NH2-terminal charges of S4 (R362, R365, and R368) are exposed on the surface of the ion channel (see INTRODUCTION). In a previous investigation, we suggested that, of the three charges, R362 is the one that comes closest to 418C ( = 11.6 mMs) was substantially slower than the modification rates of 418C channels (
= 2.5 mMs). The modification rate by MTSET+ of 362Q/418C channels was similar to that of 418C channels in both the activated state and the resting state of S4 (Fig 3 D), indicating that the (sterical) accessibility was not changed by the R362Q mutation (see Equation 10). Thus, the neutralization of R362 reduces the absolute value of the electrostatic potential at 418C in the activated state of S4, but it does not change the electrostatic potential around 418C in the resting state, nor the accessibility of 418C in either one of the states.
Activation of the Channel Affects the Electric Potential at E418C by 35 mV, of which 19 mV Is Contributed by R362
To estimate the magnitude of the change in the local electrostatic potential at 418C induced by the activation of the channel, we calculated the ratio of the relative change in the modification rate for MTSET+ compared with the relative change for MTSES- during activation (Equation 7). This procedure factors out the different intrinsic reaction rates of the two MTS reagents and the possible changes in the accessibility or reactivity of the cysteine at the different membrane voltages (see MATERIAL AND METHODS).
The ratio of the change in the modification rate for MTSES- and MTSET+ at the two different voltages is 17.9, which corresponds to an (activation induced) increase of the electrostatic potential at 418C by 35 mV (Equation 7). For R362Q/E418C channels, the electrostatic potential at position 418 is increased by only 16 mV during activation (Equation 7). This indicates that R362 adds 19 mV to the electrostatic potential at position 418 during activation. Because the neutralization of R362 has no significant effect on the modification rates in the resting state but a large effect in the activated state, this suggests that R362 is located far away from 418C in the resting state and is coming very close to 418C in the activated state of S4. Assuming that both R362 and 418C are at the interface between the membrane phase and the aqueous phase, we calculated that the 19-mV contribution by R362 corresponds to a distance of 8.1 Å between the charge at 362 and the sulfur atom of 418C in the activated state (Equation 11). In the resting state, we estimate that R362 is located >20 Å away from 418C. These distances are in agreement with the position of S4 that we suggested in our previous study (
418C Is Located on the Surface of the Protein
In the calculation above, we have presumed that 418C is located on the protein surface and that 418C does not sense the transmembrane voltage directly. If 418C is located, for example, in a narrow crevice, then 418C will sense a part of the transmembrane voltage and the modification rate by MTSES- will increase when the membrane voltage is made more depolarized. We tested if the increase in modification rate is partly due to the change in transmembrane voltage or if the increase is due to the conformational changes during activation, for example, the movement of S4 charges closer to 418C. Since the voltage dependence of activation (and the movement of S4) saturates at 0 mV, while any direct effect of the transmembrane voltage will not saturate, we measured the modification rate for MTSES- also at +60 mV. The modification rate at +60 mV was not significantly different from that at 0 mV (
(0 mV) = 60 ± 5 s, n = 2;
(60 mV) = 68 ± 6 s, n = 3). This shows that the transmembrane voltage is not directly affecting the modification rate, and suggests that the modification rate of 418C is affected primarily by conformational changes during activation.
The Neighboring A419C Is Also Affected by S4 but to a Smaller Degree (19 mV)
To show that the change in electrostatic potential is not unique for 418C, we similarly investigated the neighboring residue A419. Modification of 419C by MTSET+ shifts the voltage dependence of the activation of the channels to more positive voltages, whereas MTSES- shifts it to more negative voltages ( = 0.6 mMs) was not significantly different from that in the activated state of S4 (0 mV;
= 0.8 mMs; Table 1). This suggests, as for 418C, that the (sterical) accessibility for 419C does not change by activation of the channel. However, the modification rate for MTSES- increased by a factor of 3.5, from
= 27.8 mMs in the resting state to
= 8.0 mMs in the activated state of S4 (Table 1). Using a similar analysis as for 418C, we calculated that the electrostatic potential at 419 increases by 19 mV during activation (Table 1). Thus, both 418C and 419C are electrostatically affected by the activation (S4 movement) of the channel. Furthermore, 418C is affected more than 419C by the activation of the channel, which is compatible with the earlier suggestion that 418 is located closer to S4 than 419 (
S4 Charges Approach Residues in the Pore Region during Activation but not during Slow Inactivation
Our results show that the reactivity of the cysteines in the external end of S5 is affected by the activation of the channel and the movement of S4. However, because of the long depolarizing pulses used during the perfusion of MTS reagents (45 s) our results were obtained with a mixed population of channels, a majority in a slow-inactivated state (>80%) and a minority in the open state. The position of S4 relative to the pore could change when the channel goes from the open state to the inactivated state ( = 75 ms) and slow recovery from inactivation (
= 26 s;
10% undergo slow inactivation. During the hyperpolarizing pulse, 419C closes quickly and most of the inactivated channels recover from inactivation. Thus, during the alternating pulse protocol, the channel should spend
4550% in a closed state and
4550% in the open state (S4 in the activated position). The slow-inactivated state is occupied <10%.
The modification rate by MTSES- of 419C using this protocol was 1/(-80 mV | 0 mV) = 1/(12.7 ± 2.0 mMs) (mean ± SEM, n = 6). The reaction rate for MTSES- in the closed state was earlier shown to be 1/
(-80 mV) = 1/(27.8 mMs). The reaction rate for the open state could easily be calculated as Equation 13:
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(13) |
The calculated reaction rate in the open state 1/(0 mV) = (1/8.3) is not significantly different from the rate in the (mainly) inactivated state 1/(8.0 ± 1.1 mMs). This suggests that the change in modification rate occurs during activation (outward movement of S4), and that there is no further increase during slow inactivation. Thus, S4 moves close to 419C during the opening of the channel with no additional approach during inactivation.
Neutralization of R362 or R362/R365 Has no Effect on Slow Inactivation Rate
In a previous study (
Fig 4 shows that neutralization of the outermost arginine in S4 (R362Q) does neither slow down the macroscopic inactivation rate nor the recovery from inactivation. The time constant for a single exponential fitted to the inactivation time course at +60 mV was 5.9 ± 0.4 s for WT (mean ± SD, n = 5) and 6.1 ± 0.3 s for 362Q (n = 3). For the recovery at -80 mV, there was a slight difference: 1.6 ± 0.8 s for WT (n = 5) and 0.8 ± 0.2 s for R362Q (n = 3). However, because the recovery process, in contrast to the inactivation, is voltage dependent (the recovery from inactivation is faster at more hyperpolarized potentials) this difference arises most likely because R362Q channels activate at more depolarized potentials ( = 5.2 ± 1.0 s, n = 4). This suggests that neither R362 nor R365 play an important role in the conformational changes during slow inactivation.
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DISCUSSION |
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Our results show that the extracellular end of S4 comes very close to the extracellular end of S5 during activation of the channels. We estimate that the distance between R362 and E418 is 8 Å in the open state and >20 Å in the closed state. This confirms earlier suggestions of the location of S4 relative to the pore (
Quantitative Evaluation of the Electrostatic Effects
Our results indicate that R362 contributes with 19 mV of the 35-mV change in the electrostatic potential at position 418 that occurs during activation. The remaining 16 mV could be caused by R365 and R368, which also become exposed during activation (
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Functional Effects of the Large Changes in Electrostatic Potential during Activation
The close approach of R362 to E418 and the electrostatic interaction between the two, could have significant effects on the gating of the channel. We have suggested earlier that E418 function as a surface charge, altering the electric field that S4 is experiencing (
Several studies have recently suggested that S4 charges (in particular R362) and E418 interact (and move relative to each other) during slow inactivation (
A Hypothesis for Slow Inactivation Coupling
We now favor our alternative hypothesis, that S4 movement causes the hydrogen bond between 418 and the P-S6 loop to break by an indirect, allosteric mechanism (
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Footnotes |
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* Abbreviations used in this paper: MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide; WT, wild-type.
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Acknowledgements |
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We are grateful to Mattias Elmér and Daniel Erichsen for some of the recordings. We also thank Drs. Russell Hill, and Bo Rydqvist for manuscript comments.
This work was supported by grants from the Swedish Medical Research Council (No. 13043 to F. Elinder and No 12554 to P. Larsson), Åke Wibergs Stiftelse, Magnus Bergvalls Stiftelse, and The Swedish Society of Medicine and Jeanssons Stiftelser. F. Elinder and P. Larsson have junior research positions at the Swedish Medical Research Council. R. Männikkö has a Ph.D. position supported by the National Network in Neuroscience.
Submitted: 22 February 2001
Revised: 10 May 2001
Accepted: 15 May 2001
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