Correspondence to Sergei Sukharev: sukharev{at}umd.edu
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ABSTRACT |
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Key Words: mechanosensitive channel voltage tension inactivation osmoregulation
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INTRODUCTION |
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Martinac and coworkers (Martinac et al., 1987), who were the first to apply patch-clamp techniques to giant Escherichia coli spheroplasts, reported a pressure-gated bacterial channel of 1 nS conductance. MscS, a mechanosensitive channel with 1-nS conductance was identified later as a product of the orphan yggB gene, sharing similarity with the NH2-terminal half of the potassium efflux protein MscK (formerly KefA) (Levina et al., 1999
). MscK was also shown to be a mechanosensitive channel with conducting characteristics similar to that of MscS (Levina et al., 1999
). However, while MscS exhibited time-dependent inactivation (Koprowski and Kubalski, 1998
; Levina et al., 1999
), MscK was characterized by more sustained activities under constant stimulation. MscK gating was also found to be critically dependent on the presence of potassium ions in the external medium (Li et al., 2002
).
Crystallographic work by the Rees group recently produced the crystal structure of E. coli MscS (Bass et al., 2002), revealing a homo-heptameric complex with three transmembrane helices per subunit and a large cytoplasmic COOH-terminal domain. Two transmembrane helices M1 and M2 were bundled together, resembling the "paddle" of the KvAP voltage sensor (Jiang et al., 2003
). Due to the presence of several arginines, these helices were proposed to serve a similar function as the MscS voltage sensor. The third transmembrane helix (M3) lined a relatively wide but very hydrophobic pore, deemed to represent the open conformation (Bass et al., 2002
). Our computational studies suggested that the MscS pore constriction is dehydrated and likely nonconductive (Anishkin and Sukharev, 2004
), thus raising the question of what functional state the crystal structure actually represents. Independent computational studies also confirmed the dehydrated and low-conducting nature of the MscS pore (Sotomayor and Schulten, 2004
). Biochemical cross-linking experiments suggested that the native closed conformation of the channel is more compact as compared with the crystal conformation in both the transmembrane and cytoplasmic domains (Miller et al., 2003
).
The available molecular information on MscS facilitates tremendously many aspects of functional studies. While cloning and tagging of MscS permitted purification and functional reconstitution of the protein in liposomes (Okada et al., 2002; Sukharev, 2002
), generation of the MJF465 triple mutant (mscL, mscS, mscK) E. coli strain prepared a "clean" genetic background on which the channel could be studied it its native setting (Levina et al., 1999
). The first brief report of MscS behavior at different voltages measured in a clean system (Vasquez and Perozo, 2004
) gave a different picture of gating compared with the previous phenomenology (Martinac et al., 1987
; Koprowski and Kubalski, 1998
) collected on mixed channel populations.
The present work characterizes the behavior of MscS in the MJF465 triple mutant E. coli strain (Levina et al., 1999). Data collection was aided by a high-speed pressure clamp apparatus to deliver reproducible ramps of pressure to patches. Software developed in-lab allowed us to generate doseresponse curves directly from the resulting two-channel recordings of current and pressure. The results indicate that MscS activation is essentially voltage independent, however the net channel activity is strongly influenced by the process of reversible inactivation. MscS inactivation was found to be both tension and voltage dependent. The interplay of these two processes, activation and inactivation, makes MscS most responsive to abruptly applied mechanical stimuli. We propose a kinetic scheme and gating mechanism in an attempt to reconcile the observed phenomenology with the crystal structure.
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MATERIALS AND METHODS |
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Electrophysiology
Patch-clamp recordings of MscS were taken in an excised patch, voltage clamp, configuration on an Axon 200B amplifier attached to an Axon DigiData 1320A A/D converter (Axon Instruments). Electrodes were borosilicate capillaries pulled to a bubble number of 4.5 (resistance 2.8 ± 0.2 M, in a buffer of 39 mS/cm conductivity) on a micropipette puller (Sutter Instruments Company). Recording was performed in symmetrical potassium (200 mM KCl, 90 mM MgCl2, 10 mM CaCl2, 5 mM HEPES titrated to pH 7.4 with KOH) or sodium (replaces above KCl with 200 mM NaCl) buffers in the pipette. The bath solution differed only in the addition of 300 mM sucrose. Pressure ramps were applied using an HSPC-1 high-speed pressure clamp apparatus (Besch et al., 2002
) controlled via analogue output from the DigiData1320A. ALA Scientific Instruments' P-V Pump unit was used as the pressure and vacuum source. Vacuum and pressure were calibrated at both the pumps and the headstage using a PM015D pressure monitor (World Precision Instruments). Pressure traces were then recorded directly from the HSPC-1 head stage. Output commands to the HSPC-1 were controlled by Axon pClamp8 software in episodic stimulation mode (Axon Instruments).
Data Collection and Treatment
Axon pClamp 8 software was used to record the integral current through patches with a bandwidth of 15 kHz, digitizing at 1,0002,5000 samples/s depending on the duration of the recording. Two-channel (current and pressure vs. time) electrophysiological recordings were converted into Axon Text Format and subsequently analyzed using HISTAN, software custom-written in Matlab. A doseresponse curve (Po/Pc vs. pressure) calculation was implemented using the following protocol. The whole pressure range was divided into bins of 1 mm Hg width and all-point current histograms were created for each pressure value. Following baseline correction, an average integral current value I(p) was calculated for every pressure bin. An opening probability for the MscS population was estimated by Po(p) = I(p)/Imax, where Imax represents the maximal integral current observed in the recording. Under typical conditions (applied pipette voltage range of 30 to 80 mV), where the relative occupancy of subconducting states was very low, this approach gave an adequate estimation of the open probability of MscS. However, out of this range, subconducting states are dominant and this approach provides only an approximation of Po since it relies on an assumption that the contribution of the substates to integral current, relative to the fully open state, does not change significantly with tension. Proper treatment of MscS traces recorded at high positive and negative voltages will be a focus in future research.
Pressure to Tension Conversion
The midpoint pressure (p1/220) at which half of the MscS population was activated under +20 mV during the fastest (1 s) pressure ramp was used as a tension calibration point. p1/220 was calculated for each patch individually as an average of all traces recorded under a single applied voltage (usually 310 recordings). This average pressure corresponds to 5.5 dyne/cm, earlier shown to be the tension midpoint for MscS activation in spheroplasts (Cui and Adler, 1996) and liposomes (Sukharev, 2002
). p1/220 was then used in tension calculations for the remaining traces, recorded from the same patch, under different applied voltage. Pressures were converted to tensions using the law of Laplace for spherical surfaces:
= (p / p1/220) x 5.5 dyne/cm. This conversion implies that in the range of pressure needed to activate MscS patch, geometry does not substantially change. Based on video observations of liposome patches (Sukharev et al., 1999
), the membrane was found to be essentially flat at a zero pressure, becoming hemispherical under a pressure gradient. The curvature of liposome patches saturated with increasing pressures and remained stable in a range of tensions from 45 dyne/cm. These findings are likely true for spheroplast patches as well.
Fitting Procedures
Doseresponse curves for MscS were fitted with a two-state Boltzmann-type model where
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Po and Pc are the probabilities for the open and closed states, E is the difference in energy between the states in an unstressed membrane,
A is the in-plane protein area change during the gating transition,
is the membrane tension, and kT has the conventional meaning (Sachs, 1992
; Sukharev et al., 1999
). Fitting was performed in Microsoft Excel 2000 using a linear least squares method in semilogarithmic coordinates (log(Po/Pc) vs. tension). Only the first linear part of activation curve was fitted (see RESULTS), representing activation of the main population of MscS channels.
To characterize the time course of the MscS inactivation at constant pressure, the falling phase of the integral current was fitted with a single standard exponent of the first order (I(t) = Aet/ + C, where I is an integral current, t time, A amplitude, C additive constant, and
time constant). Fitting was performed in Axon Clampfit 9 using a Levenberg-Marquardt search method and sum of squared errors minimization criterion.
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RESULTS |
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Values of E and
A for the MscS gating transition, extracted from sequential ramps applied to the patch shown in Fig. 1 A, averaged 27 ± 2 kT and 20 ± 1.6 nm2 respectively (n = 11). The combined values from five different patches taken from independent spheroplast preparations produced
E = 24 ± 4 kT and
A = 18 ± 3 nm2 (n = 27). A plot of
E versus
A from the same patch (Fig. 1 D) illustrated strong correlation in the variation of these parameters. A previous study of population responses in MscL (Chiang et al., 2004
) showed that slightly nonhomogeneous populations of channels display strong linear correlations of
E and
A. This appeared to be the case for MscS, where variation of the slope of the doseresponse curves was substantially larger than variation in the midpoint position. Note that at the tension midpoint (
1/2), Po = Pc, and therefore from Eq. 1
1/2 =
E/
A. Linearly correlated pairs of
E and
A arise from doseresponse curves that intersect at the same
1/2 but have variation in their slopes. Statistical simulations of nonhomogeneous channel populations illustrated that the slope of the doseresponse curves should be lower than that of an ideally homogeneous population. Furthermore, this slope reduction increases with the scatter of intrinsic parameters (
E and
A) characterizing each individual channel. If the source of nonhomogeneity is a deviation of the intrinsic parameters, then the values extracted directly from the activation curves represent lower bound estimates for
E and
A. If however the deviations of the doseresponse curves are more related to the stochastic nature of crossing the transition barrier (which can produce variations in Po under nonequilibrium pressure ramps) during the gating transition then these average values of
E and
A may closer reflect the true intrinsic parameters for MscS. Further analysis is required to fully explain the doseresponse curve variability in the MscS channel population.
Voltage Dependence of MscS Activation
The same patch clamp experiments, with linear ramps of saturating pressure, were repeated at different voltages from 100 mV to +100 mV. The midpoint for MscS activation remained essentially constant at both high positive and moderate negative voltages as shown by the raw traces in Fig. 2 A. A similar character of currents was observed in both symmetrical KCl and NaCl-based buffers. In several independent experiments, the maximal conductance for each individual trace was normalized to that observed at +20 mV and plotted as a function of voltage (Fig. 2 B). The maximal conductance at high positive pipette voltages (+60, +80, and +100 mV) increases shallowly. At negative pipette voltages, beyond 40 mV, a steep decrease of the integral patch conductance was obvious (Fig. 2 B). Traces were treated as described in the previous section (Fig. 1, B and C) and the effective parameters E and
A were obtained for each voltage. The plot of
E versus voltage showed MscS activation to be only weakly dependent on applied electric field (Fig. 2 C). From the slope of the fit, gating charge (q) was determined to be +0.8 per MscS heptamer or about +0.1 per subunit. This small value for q indicates that the charges within the electric field, most of which lie on the lipid facing helices, do not displace significantly in the direction of electric field during MscS activation. Given that a net charge of +3.0 resides on each TM1TM2 pair, the transmembrane movement of these helices would be no more than 0.7 Å.
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Inactivation is Favored by Depolarization
Experiments similar to those above, with a prepulse pressure slightly higher than p1/2, were conducted at different pipette voltages. A comparison of these traces showed a clear increase in the rate of inactivation at negative potentials (Fig. 6 A). As previously described, the inactivation phase of each trace was fitted with an exponent, and characteristic times were determined. A plot of versus voltage (Fig. 6 B) exhibited a shallow increase between 40 and +60 mV, indicating that moderate positive pipette voltages oppose inactivation. A steep decrease of
below 40 mV revealed the onset of strong MscS inactivation observed under depolarizing conditions. The "break" point in this dependence corresponds to the voltage (40 mV) where substates become dominant (compare Fig. 6 B and Fig. 3 A). This coincidence suggests that the leftmost part of the
(V) dependence likely reflects the rate of transitions between the substates (S) and the inactivated state (I) but not a direct inactivation from the open (O) state. The slope thus reflects the decrease of the activation barrier for the S
I transition on voltage. Estimation shows that this process corresponds to an outward transfer of approximately two positive charges per heptamer across the entire electric field. If one elementary charge per subunit (seven charges total) were moving outward, the effective displacement of charges within the membrane from the S state to the transition state between the S and I states (top of the barrier) would be
5 Å. This displacement would be smaller if more charges were engaged.
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DISCUSSION |
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The major traits of MscS observed in situ can be reiterated as follows. The transition from the closed to fully open state (CO) displays a steep dependence on membrane tension with an apparent mechanotransduction area of
18 nm2. The fully open state is stable at high tensions (and at moderate voltages), however at tensions below the activation midpoint (
1/2), the channel tends to inactivate within tens of seconds. Thus, under slow application of pressure, the resultant channel activity is much lower than with an abruptly applied stimulus of the same amplitude. The inactivated state of MscS is nonconductive and long lived. The complete return from the inactivated (refractory) to the closed state (I
C) takes at least 3 min. The main closed-to-open transition (C
O) is essentially voltage independent. However, at negative pipette (depolarizing) voltages <40 mV and strong positive pipette (hyperpolarizing) voltages >+80 mV, MscS tends to occupy subconducting states (Fig. 3 A). Depolarizations beyond 40 mV strongly promote transitions to the subconducting states (O
S) followed by inactivation (S
I). While channel activation (C
O) was always observed as an instant conductance increase (at a given recording bandwidth), signifying a fast and concerted conformational change (Shapovalov and Lester, 2004
), inactivation broken up into subtransitions appears to be a less cooperative event. The rate of inactivation exponentially increases with depolarizing voltage and decreases with tension. The gating characteristics of MscS are similar in KCl and NaCl buffers.
The presented phenomenology of MscS gating is different from what was depicted in early studies. The very first report by Martinac et al. (1987) described a 1-nS channel activated at pipette pressures of 4060 mm Hg, considerably lower than those we used to activate MscS (140300 mm Hg). The open probability for these channels markedly increased at depolarizing voltages, which we do not observe for MscS. The channel was active in KCl, but practically inactive in NaCl, a property later ascribed to MscK (Li et al., 2002
). We acknowledge that at the time of the study it was difficult to separate the activities of these two different species, MscS and MscK. The characteristic propensity of MscS to time-dependent inactivation, however, suggests that the quasi-equilibrium traces recorded for minutes presented by Martinac et al. belonged mostly to MscK. The inactivation of the small (1 nS) channels taking place specifically at intermediate pressures was well documented by Koprowski and Kubalski (1998)
, however, these experiments covered only a narrow range of potentials (+30 to 30 mV), which precluded observation of the steep voltage dependence at stronger depolarizations described above.
The presented phenomenology of MscS also helps us to interpret the crystal structure (Bass et al., 2002). The crystal structure of MscS revealed a de-lipidated protein complex with TM1 and TM2 helices splayed at an unusual 30° angle relative to the membrane normal. The protein surface displayed conspicuous gaps between the TM1TM2 pairs and the pore-forming TM3 helices, which should be either hydrated or filled with lipid (if such a conformation occurs in the native membrane). It is possible that the native resting conformation of MscS packs the TM1TM2 pairs more tightly against the central pore, forming a more compact barrel. Indeed, in the absence of direct interactions between TM1TM2 and TM3, it is unclear how tension developing in the lipid bilayer could be transmitted from the lipid-facing protein surface to the pore. Our preliminary steered molecular dynamic (SMD) simulations indicated that the swinging motion of the TM1TM2 bundle about the extracellular hinge (G90 and adjacent residues) is essentially unrestrained. A gentle pressure, constricting the barrel from the outside, could then restore the contacts between TM2 and TM3 (unpublished data).
Despite initial conclusion that the crystal structure represents the open state (Bass et al., 2002), our computational analysis suggested that the pore in the crystal structure is largely dehydrated and must be nonconductive (Anishkin and Sukharev, 2004
). The estimations showed that the pore constriction, with a water-accessible lumen of
7 Å in diameter, must be expanded by at least 8 Å to conduct at 1 nS as measured in experiments. The dehydrated pore and the uncoupled state of the peripheral helices from the pore-forming helices suggest that the channel, in this particular conformation, would not only be nonconductive but also irresponsive to tension applied to its periphery, the functional definition of an inactivated state. We believe that the overall structure, represented in the crystal, is a conformation which likely resembles the inactivated state of MscS. Based on these considerations and the phenomenology above, we propose the following kinetic scheme and mechanism of MscS gating.
The scheme presented in Fig. 7 A shows positions of the main states in the area-charge plane. The solid arrows represent transitions observed frequently at moderate pipette voltages. Dashed lines depict transitions, involving substates, favored by moderate to high negative pipette voltages. Dotted lines represent possible transitions, which appeared infrequently, and have therefore not been characterized. An example of a dotted transition is the "silent" inactivation of the channel directly from the closed state. This transition seems to occur during prolonged exposures of the channel population to subthreshold tensions. We also cannot eliminate the possibility of a small number of channels returning to the open state (O) from the inactivated state (I). However, the probability of such a transition appears to be extremely low. The most common pathway from the inactivated state back to the closed state was a slow return favored by low tension and positive pipette voltages. Direct transitions from the fully open to the inactivated state were observed most frequently in traces at low to moderate positive voltages. Although we observed this pathway as a straight transition (OI), the frequency response of our recordings was insufficient to exclude a composite (O
S
I).
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The data presented in Figs. 2, 3, and 6 show that depolarizing voltages favor transitions to subconducting states followed by the inactivated state. The fact that the channel enters the inactivated state (I) typically through a substate (S) led us to infer that substates are structural intermediates between the open and inactivated states. We therefore placed these states on the area-charge plane in sequence with increasing q. We presume that under depolarizing conditions, the outwardly directed electric field acts on the charged residues residing in TM1 and TM2, pushing the helices toward the extracellular side and thus favoring their detachment from the core TM3 helices. This uncoupling, associated with an upward swinging motion, generates gating charge and stipulates voltage dependence. At the same time, the TM3 helices collapse back to a narrow-pore conformation, which eventually causes dehydration of the constriction and return to a nonconductive state. This mechanism of combined activation and inactivation performed by a single gate, and involving hinged motion of the inner helices, is similar to the inactivation mechanism proposed for the voltage gated channel NaChBac (Zhao et al., 2004). The substates may represent intermediate states in this pathway with only a few subunits having detached TM1TM2 pairs and a partially collapsed pore. The fully inactivated state is achieved when this process of helix decoupling completes, resulting in formation of the characteristic crevices on the cytoplasmic side of the channel seen in the crystal structure. The conformations of an "asymmetric" substate and the inactivated state are schematically illustrated in Fig. 7 B. Whether these crevices are filled with water or lipid is unclear, but the slow return from the inactivated to the closed state may be limited by slow lipid diffusion out of the confined crevice. Furthermore, this return would be aided by the charges on the TM1 and TM2 helices as the bacterium reenergizes its membrane.
The kinetic data presented in Fig. 6 was insufficient to conclude the entire gating charge associated with inactivation because we only measured the increase in rate of the OI transition on voltage, but not the equilibrium probability distribution. However, the data indicated that this increase in the rate of inactivation must be accompanied by a decrease in the transition barrier equivalent to the energy of transferring two positive charges across the entire electric field. If we assume that only one charge per subunit is engaged in inactivation, then all seven charges per complex have to move by
5 Å across the bilayer. If three charges per subunit are involved (the net charge of each peripheral pair of helices is +3), only 1.7 Å of displacement is necessary to reach the transition state leading to inactivation. This estimate seems to be consistent with the expected moderate scale of the TM1TM2 helices swing.
Fig. 4 illustrates full activation of MscS on a steep application of pressure but strongly attenuated responses to slow ramps. The fact that the midpoint of activation does not change with the speed of ramp suggests that opening is not impeded by channel kinetics within the studied time frame. The lower total activity observed with a slow ramp is apparently due to MscS inactivation at intermediate tensions near the midpoint. Since this process was observed at all voltages, it can be assumed that the inactivated state of the channel is the lowest energy state at intermediate tensions. At negative pipette voltages, a low-barrier passage is opened to the inactivated state through the substates. How then does high tension stabilize the open state? Although we cannot exclude the possibility that high membrane tensions kinetically trap the open state of MscS, it is more likely that under such conditions, the open conformation represents a true energetic minimum. If the effective in-plane area of the open state were larger than that of the inactivated state (as denoted in the area-charge plane, Fig. 7 A), at high tension, the energy of the open state would be lower than that of inactivated state. The concerted expansion of TM1, TM2, and TM3 associated with the CO transition, during which the protein retains a tight packing of these transmembrane helices, brings a substantial increase of the channel footprint in the lipid bilayer. The area increase for this transition is estimated to be 18 nm2 from doseresponse curves (Fig. 1). An activating tension of 7 dyne/cm would result in an energetic stabilization of the open state of
30 kT. In contrast, the O
I transition likely results in a decrease of the effective in-plane area of the channel. The decoupling of TM1TM2 pairs from the core helices and their subsequent spreading creates slits and crevasses critically changing the protein outline in the plane of the membrane. Lipids may penetrate into the slits, taking a part of the protein area and thus reducing the effective lipid-excluded area of the protein. In this way, spreading of helical pairs and "intermixing" with the surrounding lipids explains the effective decrease of the in-plane area as a result of transitions into the subconducting and inactivated states. With increasing tension, the inactivation rate decreases (Fig. 5 B, inset) and the slope suggests that the transition state leading to inactivation has 10.6 nm2 smaller in-plane area than the open state. This implies that the area difference between the end states (O and I) is even larger.
The ability of MscS to reversibly inactivate may be one of the features that allow it to respond differently to various environmental parameter changes. Indeed, the total channel population responds in full when stimulated by a high pressure applied abruptly, but largely inactivates during the slow passage through a narrow region of pressures below the midpoint. The quick reaction to an abrupt stimulus and "dumping" of a slowly applied force resembles a "dashpot": a velocity-sensitive viscous element that would connect the gate of the channel to the tension-receiving protein interface (Fig. 7 B). This dashpot functionality may be ascribed to the postulated contacts between the pore-forming (TM3) and peripheral helices (TM1 and TM2) capable of disengaging and allowing lipid penetration.
This mechanism that makes MscS susceptible to a sudden pressure onset, but allows it to "ignore" slowly applied stimuli, may be important in environmental situations where rehydration of cells occurs gradually and nonselective release of osmolytes is not desirable. Instead of simply jettisoning the solutes through opening, the channel inactivates giving time to other osmoregulatory systems to release some of the solutes in a more selective manner or exchange them (Wood, 1999). Because the time course of pressure buildup depends on water permeability, there is a possible functional interplay of MscS with aquaporins previously discussed in Booth and Louis (1999)
. It would be interesting to test the osmotic rescuing function of MscS in aqpZ strains. Previously, MscS and MscL have been shown to be redundant in E. coli (Levina et al., 1999
). However, the evolutionary preservation of the two channel species suggests that they are not interchangeable in certain environmental situations. In fact, after a gradual change of pressure, all MscS may be inactivated, and only noninactivating MscL would be capable of fulfilling the "safety valve" function. The described kinetics now suggests several enticing possibilities for the study of MscS under different dynamic conditions of osmotic shock.
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ACKNOWLEDGMENTS |
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This work was supported by National Aeronautics and Space Administration and National Institutes of Health research grants to S. Sukharev.
Olaf S. Andersen served as editor.
Submitted: 22 October 2004
Accepted: 9 December 2004
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REFERENCES |
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