Address correspondence to Henry A. Lester, Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. Fax: (626) 564-8709; email: lester{at}caltech.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: MscL MscS patch clamping single channel substate
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The development of high-speed, low-noise recording methodology will allow a more detailed description of channel dynamics and lead to a better understanding of ion channel function. Recent advances in electrophysiological equipment allow high bandwidth studies, such as resolving the gating currents of Shaker K+ channels (Sigg et al., 2003) at a bandwidth of 350 kHz. Thus far, however, no data at comparable resolution have been acquired at the single-channel level or for mechanosensitive channel activity. MscL and MscS ion channels are appropriate for study with high-speed methodology, because their large conductance, 13 nS (Sukharev et al., 1993
; Hase et al., 1995
; Sukharev et al., 1999
) allows one to detect transitions despite the inevitable increase in noise as higher frequencies are included. For the most part, the studies of single MS channel activity are normally performed at a bandwidth of 10 kHz and give a limited detail on MS channel kinetics. Thus Ec-MscS kinetics are considered to be very simple with only one conductive state reported previously (Martinac et al., 1987
; Sukharev, 2002
).
Here we report and exploit techniques that use high temporal resolution but also limit the noise in single-channel recording instrumentation. Single-channel data on Ec-MscL and Ec-MscS were acquired at a time resolution of 3 µs, a 1020-fold higher resolution than previously used for studies on these (Sukharev et al., 1999
; Sukharev, 2002
; Anishkin et al., 2003
) or other channels. The acquisition of Ec-MscL and Ec-MscS currents at an effective bandwidth of
250 kHz has allowed us to observe and analyze previously undefined features of single-channel kinetics.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ec-MscS Reconstitution
Protein was provided by Doug Rees group (Bass et al., 2002). For the reconstitution of Ec-MscS, 10 µl of azolectin dissolved in methanol (100 mg/ml) was transferred to a small cavity formed on a Teflon surface and allowed to dry until the majority of methanol was evaporated. 3 µl of a 15 mg/ml solution of Ec-MscS protein in 0.05% foscholine-14 was then mixed with the lipid by repetitive pipetting (protein to lipid ratio of
1:3,000), and the mixture was dehydrated overnight with Drierite. The pellet formed from this dehydration was rehydrated with 5 µl of rehydration buffer for 2 h. If necessary the rehydrated pellet was stored at 4°C in a Petri dish on a wet paper towel for several weeks. 1 µl of this rehydrated lipid was applied to the glass coverslide of the electrophysiological recording cell and allowed to dry for 10 min. 0.5 µl of 400 mg/ml MgCl2 solution was then spread on the coverslide and dried for 10 min. Then 400 µl of rehydration buffer was added to the recording cell, and blister formation was allowed to proceed for 30 min (Clayton et al., 2004
).
Instrumentation for Low-noise and High Frequency Acquisition
Several modifications were made to the electrophysiological recording rig to enable the acquisition of high temporal resolution and low-noise current traces. A specially modified headstage (Axon Instruments) was used that had a low-noise field-effect transistor substituted at the input stage (FET model no. Axon 4600005; Axon Instruments). The headstage was modified mechanically to house a special Teflon pipette holder as previously described (Parzefall et al., 1998). To decrease stray capacitance, thick-wall, hard borosilicate (WPI 1B150F-4; World Precision Instruments) or quartz (Sutter QF150-75-7.5; Sutter Instrument) glass pipettes were used. These were pulled to form a tip opening of
1 µm. The Axopatch 200B amplifier was modified to bypass the internal filters, and the differentiator was modified so that the gain was decreased 10-fold in order to increase the bandwidth.
Other modifications decreased the interference produced by power supplies and other sources in the environment. The Faraday cage was an Al garden shed (House of Redwood) scraped to remove paint at joints, and fitted with an Al floor. The switching power supply was removed from the Axopatch 200B amplifier, placed outside the Faraday cage in a separate enclosure, and powered by an isolating transformer (0211T35ST; Topaz Electronics). The microscope bulb was powered by a 12-V battery. After a gigaohm seal was formed and the pipette tip positioned, the power supply of the piezoelectric micromanipulators (Burleigh PCS-5000) was shut off. The internal wiring that fed the piezos was connected to the common ground. In general, ground loops were eliminated. Additional shielding was employed by forming a small cover of aluminum foil around the recording chamber.
While these modifications dramatically decreased apparent peak-to-peak and rms noise, they did not completely abolish peaks in the noise spectrum from external sources (Fig. 1 B). When the reset circuitry of the headstage, as shipped by supplier, was tuned for maximum suppression of capacitive reset transients, it produced a noticeable interference pattern that had a characteristic frequency ramping from 500 kHz down to 300 kHz. This interference pattern clearly correlated to the accumulated charge on the feedback capacitor. We therefore tuned the compensation circuitry for a suitable compromise between compensation of reset transients and diminished interference by the reset circuitry.
|
Single-channel Recordings
Prior to recordings, the response of the instrumentation was tested by capacitively feeding a 10-kHz square wave into the system. This was achieved by bringing an electrode connected to a triangle generator near the recording chamber (we verified that the transition from positive to negative slope occurred in <0.5 µs). The measurements were also performed after forming a gigaohm seal, essentially recreating conditions under which ion channel data were recorded. Waveform amplitude and the distance from the electrode to the recording chamber were adjusted so that the amplitude of the injected current was equal to that of the studied transitions (40 and 120 pA).
Single channel patch-clamp recordings were performed as previously described (Clayton et al., 2004), with 100 mV applied across the membrane (intracellular medium is negative) in symmetrical 250 mM KCl, 5 mM HEPES (pH 7.1). Suction was applied manually with a 5-ml syringe connected via plastic tubing to the pipette holder and monitored with a pressure transducer (243PC15M; Omega Engineering) powered by a custom made box that also provided an LED readout display. Currents were acquired with amplifier modified as described above and anti-alias filtered with an external 8-pole Bessel unit (AP220-5-FP; Avens Signal), with the anti-aliasing filter set at a bandwidth of 400 kHz. The ideal 1090% rise time of such a filter is 1 µs; however, we found that a 1.5-k
resistor used to stabilize a cable connection introduced additional filtering, so that the 1090% rise time measured with the square-wave stimulus was
2 µs, corresponding to an effective bandwidth of
250 kHz. Signals were digitized with a Digidata 1322A modified to run at 1 MHz (Axon Instruments), and recorded with pCLAMP 8 software (Axon Instruments). To reserve the full digitization bandwidth of 1 MHz for the measured current, the suction was monitored visually and was kept at a level corresponding to Popen of
0.5 (1 PSI for Ec-MscL and 0.6 PSI for Ec-MscS) during data acquisition. All recordings were done at room temperature.
Collection and Analyses of Inter-substate Transitions
Elementary transition events were collected using a template search routine from Clampfit 9. The template used in the collection procedure recognized currents that were constant for 1020 µs before, and 1020 µs after, the transition. For capacitively coupled test pulses of 40120 pA (the amplitude of the single-channel transitions measured here), noise introduced an additional uncertainty of 0.51 µs when signals were retrospectively synchronized (see below), so that the measured 1090% rise time was 3 µs (Table I, I = 120 pA). To study Ec-MscS sojourns at 2/3 conductance, additional steps were taken to reliably identify transitions, due to low signal-to-noise ratio. Traces recorded at the full bandwidth of
250 kHz were first subjected to 100 kHz Gaussian digital filter, and the template search routine was then employed to identify downward transitions. Subsequently, fragments of original unfiltered traces were extracted starting 50 µs before the onset of the transition and with the duration of 350 µs for the purpose of dwell time identification. The fragments starting 30 µs before the transition and with a duration of 60 µs were used for sub-µs alignment as described below. A similar procedure was used to extract MscS substate-2/3
full conductance transitions.
|
![]() |
(a Boltzman function), where A1 and A2 are the amplitudes before and after the transition, t0 and are the transition midpoint and the slope at midpoint correspondingly. The slopes at the midpoint were collected and converted to 1090% transition times (T10-90 = 2*ln(9)*
). Transition amplitudes were also collected for the analysis of MS ion channel activity.
Retrospective alignment of selected transitions with sub-µs resolution was performed using software developed in house, and realigned fragments were averaged. In some cases, remaining interference was eliminated as follows: the averaged traces were fit with the sum of two sine waves at frequencies corresponding to the major interference peaks in the power spectrum (125 and 250 kHz; Fig. 1) and the resultant waveform was subtracted. Ec-MscS dwell times at substate-2/3 were accumulated with the use of the single-channel search routine of Clampfit 9, applied to the extracted fragments.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To exploit these advances in instrumentation, we required patch-clamp seals of higher resistance (>3 G) than the usual spheroplast recordings. Therefore we employed reconstituted vesicle preparations for both MscL and MscS recordings. The most stable recordings were obtained from unilamellar blisters on artificial liposomes. These were produced in adequate numbers from multilamellar vesicles by a rehydration/dehydration cycle (Sukharev et al., 1993
), modified and performed directly in the recording chamber (Clayton et al., 2004
).
Ec-MscL channel demonstrates rich gating kinetics as evidenced by traces of activity at a bandwidth of 250 kHz
Fig. 2 presents fragments of Ec-MscL activity recorded at 250 kHz. The top panel shows a sample of typical Ec-MscL activity, and the middle and the bottom panels show an individual full conductance transition at full bandwidth and further digitally filtered at 10 kHz, respectively. Comparison of the middle and bottom panels shows that at the higher bandwidth, the channel activity is captured in greater detail. Traces at 10 kHz (bottom panel) make the majority of transitions appear smooth, and there are few, if any, datapoints present at substate conductances. In contrast, recordings performed at the full bandwidth of
250 kHz (middle panel) show that all of the transitions are significantly faster than those on 10-kHz traces, and the substates are represented by multiple datapoints (acquisition interval was 1 µs). Visual inspection of Ec-MscL traces reveals the existence of a large number of different subconductive states, more than the five (Sukharev et al., 1999
) or seven (Sukharev et al., 2001
) that have been previously reported.
|
|
The individual distributions of the transition amplitudes and their durations are also presented in Fig. 3 B, and complementary plots are aligned with the corresponding axes. Both distributions are smooth, lacking distinguishable subpeaks. This plot also shows that the average observed 1090% transition time (6.2 ± 0.2 µs) is significantly greater than that of the test pulses (3.0 ± 0.2 µs). These results suggest that, on average, Ec-MscL transitions are longer than the response of instrumentation; however, their fine details still escape resolution at the effective bandwidth of 250 kHz. We note that the presence of a large number of substate conductances resulted in continuous distributions of their amplitudes and durations (Fig. 3 B). The plot and average time for downward transitions was essentially identical to the data for upward transitions (unpublished data).
Ec-MscS Has Conductance Substates
Fig. 4 A presents samples of Ec-MscS activity recorded at 250 kHz with 100 mV applied across the membrane. The top panel shows 12 s of typical single-channel activity, illustrating the simpler kinetics of Ec-MscS as compared with Ec-MscL (Fig. 3 A). The middle and bottom panels show both an individual transition from the closed to the fully open state, and a sojourn in a subconductance state of
2/3 full conductance (denoted "substate-2/3"). The middle subpanel shows data at full bandwidth and the bottom subpanel shows the same fragments further filtered at 10 kHz with a Gaussian digital filter. The sojourn in substate-2/3 is resolved more completely at 250 kHz than at 10 kHz. Control recordings of liposomes formed following a reconstitution procedure performed with a buffer containing only Foscholine-14 (0.05%) and without Ec-MscS protein did not show any activity (unpublished data).
|
To investigate the substate-2/3 in greater detail, we analyzed a set of 370 sojourns in this substate. The distribution of the identified dwell times is presented in Fig. 5 B. As can be seen, the dwell times are exponentially distributed and have a time constant of 19 ± 4 µs. We have also performed alignment of the extracted fragments that enclose downwards transitions (unpublished data). The post-transition region of the average waveform yielded an exponential relaxation toward the fully open state with a time constant of 21 ± 4 µs, showing good agreement between these two analyses of the same events. It should be noted that in order to achieve a reasonable rate of false positives during transition identification, we have used a template that had a 10-µs long post-transition interval with constant amplitude. This procedure may overestimate the average dwell time of substate-2/3. Other recordings suggest that the MscS channel has additional, longer-lived substates of partial conductance (Martinac et al, 1987; Lee, L. W., personal communication); these sojourns have not been analyzed systematically.
|
As can be seen, the 1090% Ec-MscS transition times (5.4 ± 0.2 µs, average of the upwards and downwards) exceed those of the test pulses of corresponding amplitude (3.0 ± 0.2 µs). Inspection of the selected transitions shows that in many cases, multiple datapoints can be observed at intermediate conductance during the transition. While the time spent in these transients is typically smaller than the resolution achieved, it indicates that the majority of Ec-MscS transitions are in fact composite. We also noted that 10% of the transitions did have 1090% times near the resolution limit of 3 µs.
We have also compared full conductance Ec-MscS openings and closings. Average 1090% transition times are statistically equivalent at 5.4 ± 0.2 µs, as can be seen in Table I.
We searched for "anticipations" or "regrets"; that is for temporally correlated brief changes in the current before or after MscS transitions (Sigworth, 1986). As a first step, we performed retrospective alignment of fragments containing identified transitions in each direction (Fig. 5 C). Temporal distortions caused by the instruments, such as residual <1% creep right after the transition, may be a complicating factor when one tries to compare channel openings or closings to the step response. Therefore, we superimposed and added the aligned and averaged opening transitions to the corresponding closing transitions (Fig. 5 C, bottom subpanel). As can be seen from the figure, the transitions are symmetric and no statistically significant difference in their time course can be inferred from their average. The uncertainty in these measurements is
5 pA, or 4% of the full amplitude of the transition. This implies that, on a time scale of
3 µs and 4% of the transition amplitude, no anticipations precede channel opening or closing, and no regrets follow opening or closing. This conclusion is summarized in Fig. 5 E. The only possible exceptions would be a deviation before opening that is symmetric in time with a deviation before closing, and vice versa.
Substate-2/3 to Fully Open State Transitions of Ec-MscS Are Faster than 3 µs
We have analyzed substate-2/3 full conductance transitions of Ec-MscS in a manner similar to that described above. Visual observation of selected transitions showed no evidence of intermediate states. To test the limits of our knowledge about these transitions, we performed additional analyses (Fig. 6). Average 1090% transition times both to and from fully open state are listed in Table I. As can be seen, average transition times match in both directions, and for both Ec-MscS transitions and test pulses, at 3 ± 0.2 µs on average. We have also performed retrospective alignment of extracted transitions as described above. Fig. 6 shows the superimposition of averaged Ec-MscS transitions and responses to test pulses (top panel) as well as difference of these averages (bottom panel). No significant variance can be observed in a subtraction plot.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The high conductance of the MscL and MscS channels suggests a correspondingly large diameter, with opportunities for side chain and domain arrangements while the channel is partially conducting. Yet the MscL protein is smaller than most channel proteins. There is no reason to suspect that lower-conductance channels (such as those found in excitable cells and epithelia) occupy a smaller spectrum of states, but most of these states are presumably nonconducting and revealed only by gating currents (Sigg et al., 2003). Therefore, the present results are best viewed in a general context: the high conductance of these channels allows us to detect unusually early states in the transitions from fully closed to fully open. Data such as Fig. 3 emphasize the general point that conformational transitions in single protein molecules can follow several alternative trajectories, on time scales in the microsecond range.
In macroscopic averaged recordings, voltage-gated K+ channels manifest a gating charge motion, representing a conformational change, with a time constant <10 µs after a voltage step (Sigg et al., 2003). Acetylcholine receptor channels begin to open <10 µs after agonist appears nearby (Lester et al., 1980
). Mechanosensitive transduction channels in hair cells begin to open within <40 µs during a stimulus (Corey and Hudspeth, 1979
). This paper, like other attempts (Benndorf, 1995
; Parzefall et al., 1998
), extends high-frequency analyses of ion channels to the single-channel level.
Complex Ec-MscL Channel Kinetics Suggest Many States
Our results confirm and extend the complex nature of MscL channel kinetics. At lower frequencies, we and others (Sukharev et al., 1999; Shapovalov et al., 2003
; Chiang et al., 2004
; Clayton et al., 2004
) find peaks in the all-points amplitude histogram at several intermediate amplitudes between the fully closed and fully open channel, suggesting that some conductance states are frequently visited. Interestingly, the observed number of Ec-MscL substates increases as more electrophysiological data becomes available; from five (Sukharev et al., 1999
) to seven (Sukharev et al., 2001
) and finally nine (Chiang et al., 2004
) in the latest analysis of Ec-MscL activity in spheroplasts. We have analyzed all-points histograms like those of Chiang et al. (2004)
, and we agree that there are at least nine conductive substates during the single-channel activity of Ec-MscL. Moreover, we find a broad, featureless, smooth spectrum of intersubstate transition amplitudes and times (Fig. 3 B). The 1090% rise times increase as the transition amplitude increases, again suggesting many unresolved substates. We believe that, even on time scale of 3 µs, the majority of Ec-MscL transitions are composite. Other recent data show that some transitions are asymmetric, involving just one or two of the five subunits (Shapovalov et al., 2003
). These observations suggest that kinetic analyses based on a Markov model with discrete states may be inappropriate to describe Ec-MscL activity, as that would require a large number of substates with little basis for assigning the parameters. It may be more appropriate to employ schemes that emphasize continuous models (Levitt, 1989
).
Properties of Elementary Transitions: Neither "Anticipation" Nor "Regret"
Although it is not yet clear which kinetic formalism will prove most fruitful for analyzing high-frequency data such as these, the experiments do support a major assumption of many formal kinetic schemes for ion channels, that conformational transitions show no history dependence. This observation derives from the observation that no detectable changes in conductance precede or follow upward and downward transitions; that is, the transitions show neither "anticipation" nor "regret". In this analysis, applied to MscS, we added averaged upward and downward transitions and found no statistically significant deviation from zero (Fig. 5, C and D) (Sigworth, 1986). The result also implies that the transitions are symmetrical within statistical error. Summarizing these symmetries by a single parameter, the 1090% times of the majority of full MscS transitions, although longer than the effective resolution, were similar for upward and downward transitions of the same magnitude for Ec-MscS (Table I). Although the 1090% rise times for MscL transitions were broadly distributed, these distributions were similar for upward and downward transitions.
Ec-MscS Gating Is Also More Complex Than Previously Known
The majority of full conductance Ec-MscS transitions are slower than the instruments' resolution limit and display multiple datapoints at intermediate conductances during the time course of the transition. We conclude that full conductance Ec-MscS transitions are composite, though less complex than MscL transitions. A transition such as this, whose time course can be partially resolved, becomes subject to the question, "does the opening transition occur via the same set of intermediate states as the closing transition?" The observation of resolvable but symmetrical transitions is compatible with the idea that the channel protein does traverse the same set of states for openings as for closings. A contrasting mechanism would involve, for instance, closing into an absorbing state such as (a) inactivation in voltage-gated channels or (b) cross-linking in MscL Cys mutants (Sukharev et al., 2001; Shapovalov et al., 2003
).
The observation that 10% of the transitions did have 1090% times near the resolution limit of 3 µs is consistent with, but does not prove, the existence of a single state of intermediate conductance, with an exponentially distributed lifetime of 24 µs. This intermediate state would be reached from either the open or closed state.
High-speed recordings of Ec-MscS activity also allowed us to observe a previously unknown substate at 2/3 of full conductance, having an average dwell time of no longer than 20 µs. We do not believe that this substate-2/3 is also the postulated state usually visited during transitions between zero and full conductance; its lifetime would lengthen the transitions beyond the measured value. Instead, the substate-2/3 is probably a "dead-end" state reached only from the fully open state. A linear free-energy analysis of the partial and full transitions would clarify this point.
The Ec-MscS substate-2/3 full conductance transitions were the only transitions in this study that showed no detectable fine structure: they resembled the test pulses in their shape and duration (Fig. 6). Thus it can be said that the quest to resolve an elementary single-channel transition (Hamill et al., 1981
) is still incomplete. The level of the accessible detail on Ec-MscS transitions that, at the effective resolution of 3 µs, appear as elementary is still apparently limited by the attained resolution of the electrophysiological setup and not by the properties of the channel itself.
Simulations and Measurements Are Still Greater Than Two Orders of Magnitude Apart
The observation that the majority of Ec-MscL and Ec-MscS transitions are slower than 3 µs has implications for simulations of channel dynamics. The general approach of MD simulations is to use the crystal structure as a starting point and to perform simulations of the channel openings by using available computing power. Though these experiments on membrane-embedded channels are by nature computationally challenging, recent advances in methodology and increases in available computing power have allowed simulating MscL channel dynamics on the timescale of 10 ns. These simulations do not yet realistically reveal major reorientation of the transmembrane helices, an event deemed necessary in order for large conductance channels to form a pore of 30 Å (Elmore and Dougherty, 2001
). To facilitate a reorientation in the transmembrane helices on a 10-ns timescale, unphysiologically large forces needed to be applied (Colombo et al., 2003
). However, the majority of transition durations observed in this study are on the order of 310 µs, demonstrating that there is a significant (3001,000-fold) discrepancy between the timescale of simulation and experimental determination. The only distinct transition class observed during this study that was faster than or equal to 3 µs was the MscS substate-2/3
full conductance transition, representing a conductance change of only 40 pS. Although the present timescale of MD simulations is appropriate for modeling more moderate rearrangements of channel structure, new computational algorithms should be developed to exploit the ability to measure microsecond timescale gating events in mechanosensitive channels.
High Time Resolution, Low-noise Measurements of Current Transitions
Single-channel instrumentation has conventionally been optimized for the 5100-pS ion channels common in excitable tissue and epithelia. High-conductance channels (>200 pS) are also widespread among prokaryotes (Korchev et al., 1998; Miles et al., 2002
; Contreras et al., 2003
; this paper) as well as among eukaryotes, where they occur in mitochondria (Mirzabekov et al., 1993
), glial and other plasma membranes (Guibert et al., 1998
; Liang et al., 2003
), connexons (Contreras et al., 2003
), and nuclear membranes (Danker et al., 2001
). We hope that others will study these high-conductance channels with resolution similar to the present experiments. Also, single DNA molecules are being studied with high-conductance channels and with fabricated nanopores (Deamer and Branton, 2002
; Sauer-Budge et al., 2003
; Vercoutere et al., 2003
). From the experimental viewpoint, we believe that straightforward modifications to the techniques presented here can bring the temporal resolution of single-channel recording to <1 µs.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by a grant from the National Institutes of Health (GM-062532).
Olaf S. Andersen served as editor.
Submitted: 3 May 2004
Accepted: 23 June 2004
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|