Correspondence to: Eduardo Perozo, Department of Molecular Physiology and Biological Physics and Center for Structural Biology, University of Virginia Health Sciences Center, Box 449, Jordan Hall, Charlottesville, VA 22906-0011. Fax:(804) 982-1616 E-mail:eperozo{at}virginia.edu.
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Abstract |
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The molecular architecture of the NH2 and COOH termini of the prokaryotic potassium channel KcsA has been determined using site-directed spin-labeling methods and paramagnetic resonance EPR spectroscopy. Cysteine mutants were generated (residues 524 and 121160) and spin labeled, and the X-band CW EPR spectra were obtained from liposome-reconstituted channels at room temperature. Data on probe mobility (Ho-1), accessibility parameters (
O2 and
NiEdda), and inter-subunit spin-spin interaction (
) were used as structural constraints to build a three-dimensional folding model of these cytoplasmic domains from a set of simulated annealing and restrained molecular dynamics runs. 32 backbone structures were generated and averaged using fourfold symmetry, and a final mean structure was obtained from the eight lowest energy runs. Based on the present data, together with information from the KcsA crystal structure, a model for the three-dimensional fold of full-length KcsA was constructed. In this model, the NH2 terminus of KcsA forms an
-helix anchored at the membranewater interface, while the COOH terminus forms a right-handed four-helix bundle that extend some 4050 Å towards the cytoplasm. Functional analysis of COOH-terminal deletion constructs suggest that, while the COOH terminus does not play a substantial role in determining ion permeation properties, it exerts a modulatory role in the pH-dependent gating mechanism.
Key Words: KcsA, cytoplasmic domains, EPR spectroscopy, three-dimensional fold
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INTRODUCTION |
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Our knowledge of ion channel function has advanced dramatically since the discovery of KcsA, a small prokaryotic K+ channel first identified in the gram-positive bacterium Streptomyces lividans (
The KcsA crystal structure has led to an understanding of the physical basis of ion permeation and selectivity (140 pS in 250 mM K+) with rectifying properties at large negative potentials (
Gating mechanisms of eukaryotic channels are often subject to strict regulatory control by means of phosphorylation cascades, ligand binding, or direct interaction with other cytoplasmic proteins such as heterotrimeric G proteins (
In the current KcsA structure, features of the transmembrane and extracellular regions of the channel are clearly resolved (
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MATERIALS AND METHODS |
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Mutagenesis, Expression, and Spin Labeling of KcsA
Cysteine mutants were generated for residues 524 and 121160 in KcsA, covering most of the NH2- and COOH-terminal ends of the channel. Mutagenesis was performed by oligonucleotide mismatch site-directed mutagenesis using the Transformer kit (CLONTECH Laboratories, Inc.) and confirmed by dideoxy DNA sequencing. Mutant channels were expressed and purified as described (
Rb Influx and Stability Assays
The functional state of individual spin-labeled mutants was assessed by measuring the extent of 86Rb+ influx into proteoliposomes containing KcsA, as previously described (
The effects of spin labeling on the oligomeric stability of the mutant channels were evaluated from the changes in the energetics of the tetramer-to-monomer transition during thermal denaturation according to Gs were derived from the temperature dependence of denaturation in the presence of SDS using simple gel-shift assays. The mid-point of the denaturation curve was obtained after numerically fitting a Boltzmann function to the data and corresponded to the melting temperature of the tetramer.
Liposome Patch-Clamp Recordings
Liposome-reconstituted KcsA was patch clamped following the method of 24 h, at which time 20 µl of rehydration buffer were applied to each dried drop. Rehydration was allowed proceed for 5 h, yielding liposomes suitable for patch clamp. All patch-clamp measurements were done in symmetrical conditions: 200 mM KCl and MOPS buffer, pH 4.0, at room temperature. Single-channel currents were recorded with a Dagan 3900 patch clamp amplifier, and currents were sampled at 40 kHz with analogue filter set to 5 kHz (-3 dB). Pipette resistances were 510 M
.
EPR Spectroscopy and Data Analysis
X-band CW EPR spectra were obtained in a Bruker EMX spectrometer equipped with a loop-gap resonator under the following conditions: 2 mW incident power, 100 kHz modulation frequency, and 1 G modulation amplitude. Power saturation curves were obtained for each spin-labeled mutant after equilibration in N2, air (21% O2), and N2 in the presence of 10 mM Ni-Edda. Data were analyzed and converted to the accessibility parameter according to
Power spectra of residue-specific parameters were obtained by applying a discrete Fourier transform to a given data set ( values was calculated according to
was obtained as the value of the resultant M(
) evaluated at
= 100°, taking an arbitrary residue as a reference point (
= 0). Windowed periodicity analysis was carried out by calculating the periodicity index (
PI) parameter (
Structure Calculation
Simulated annealing and restrained energy minimization calculations were carried out using InsightII and Discover (Biosym/MSI), with the CVFF force field and a dielectric constant of 4. An extended polyalanine model was constructed and appended, as residues 120160, on the last position of the crystal structure coordinates (residue 119; (the values for tyrosine are rß = 3.05 and r1-2 = 3.82 Å). Intersubunit distances were estimated from a combination of two approaches. A convolution of a Lorentzian broadening function (
-C
(i + 3, i + 4, and i - 3). To maintain canonical secondary structure, target values were set to between 4 and 4.2 Å and between 5 and 6.5 Å.
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The initial model was then subjected to a series of minimization steps followed by SA/RMD in the absence of explicit fourfold symmetry. During this protocol, atoms corresponding to the crystal structure (residues 23119) remained fixed, only contributing van der Waals contacts. Through the initial energy minimization step, the van der Waals interactions were scaled down to 10-6, and gradually increased to 1.0 over 1,500 steps. Simulated annealing was performed by applying 40 ps of molecular dynamics at 1,000°K, followed by cooling to 300°K over a 30-ps period (time step 1 fs), and subsequently subjected to energy minimization. The full-length KcsA model was assembled by attaching an -helix corresponding to the NH2-terminal sequence to the first residue in the crystal structure (A23), the helix was tilted
14° relative to the membrane plane, as derived from membrane depth measurements (see Fig 2 D). This was followed by a local annealing and minimization step that included only residues 2024. The statistics of the final model set are summarized in Table 2. Data analyses, structure display, and figure drawing were carried out using InsightII (Biosym), MOLMOL (
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RESULTS |
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Overall Approach for Structure Determination
The underlying notion driving protein structural determinations using SDSL and EPR spectroscopy is that by obtaining a large number of structural measurements, qualitative in nature, and analyzing their patterns and spatial relations, global conclusions can be made regarding overall structure and conformation (
EPR spectral line shapes contain information about the motional freedom of the spin label and how it is affected by local steric restrictions. In the absence of a formal line shape analysis for each spectrum, the parameter Ho-1 (the inverse of the width of the central resonance line) has been successfully used as an empiric measure of probe mobility (
O2) is indicative of a residue exposed to the membrane, high NiEdda (
NiEdda) accessibility reflects exposure to the aqueous environment (
Functional and Structural Consequences of Site-directed Spin Labeling
Individual cysteine mutants were prepared in a background containing a histidine tag at either end of the channel. In this study, unique cysteine mutations were introduced at positions 521 in the NH2 terminus of the channel, and residues 121160 in its COOH terminus (Fig 1 A). In most cases, cysteine mutants expressed at or near wild-type levels (residues 144 and 146 expressed at 50% of wild type); in all cases, mutants were purified as SDS-resistant tetramers and were stable at room temperature. This has been the case for the majority of cysteine mutants throughout the transmembrane segments of KcsA (
A more quantitative analysis of the effects of attaching a spin label on each of the cysteine mutants was performed based on its effects on channel function and oligomeric stability. We have previously shown that macroscopic KcsA activity can be accurately estimated by measuring the extent of Ba2+-sensitive 86Rb+ influx at acidic pH (
The relative energies of destabilization due to the incorporation of the spin-label probe can be calculated from the characteristic melting temperature of the tetramer-to-monomer transition obtained from simple gel-shift assays in SDS PAGE (Gs of destabilization for NH2 and COOH termini, respectively, are plotted against residue number. Spin-labeling produces destabilization energies
1 kcal/mol per subunit in all tested residues along the NH2-terminal end of KcsA, which we take as having essentially the same oligomeric stability as wild-type KcsA. In contrast, residues at the COOH terminus of the channel exhibit three distinct behaviors according to their position along the sequence. Cysteine mutagenesis and spin labeling had little or no structural consequences at the far end of the COOH terminus (residues 142160), with destabilization
Gs at or near 1 kcal/mol per subunit. The midregion of the COOH terminus (residues 126141) was moderately destabilized and had
Gs
4 kcal/mol per subunit. The largest effects were observed at the end of the second transmembrane segment (TM2), with destabilization
Gs up to 9 kcal/mol per subunit. The sensitivity of this segment of the molecule agrees with the effects on tetrameric stability upon COOH-terminal deletion (
Structure of the NH2 Terminus
Examination of the spectral data set derived from the NH2 terminus reveals a remarkable similarity in the line shape of the different spin-labeled mutants (Fig 2 A). This is reflected in a mobility profile showing very low contrast throughout the length of the segment (Fig 2 B, top), with most of the Ho-1 values falling within the motional regime equivalent to that found on surface residues (
5% of the total signal amplitude from the vast majority of positions.
Although a featureless mobility profile could be interpreted as an indication of a lack of secondary structure, O2 and NiEdda accessibility profiles for the NH2 terminus of KcsA reveal a strongly periodic behavior (Fig 2 B, middle and bottom). Frequency analysis in Fourier space was used to extract the components of angular periodicity for both O2 and
NiEdda accessibility profiles (Fig 2 C). In each case, the power spectra showed the main frequency peaks to be at or near 100°, a clear indication of the
-helical nature of this segment. When the individual accessibility values were projected on a helical wheel, the resultant vectors for
O2 and
NiEdda were almost 180° out of phase (Fig 2 C, bottom). The opposite orientations of these two accessibility moments strongly suggest that the NH2 terminus is an
-helix positioned at the lipidwater interface, and the sharp drop in NiEdda accessibility around residues 15 and 16 may suggest a point of full insertion into the membrane. We have calculated the approximate depths of insertion for each of the NH2-terminal residues from the ratio between
O2 and
NiEdda (
-helix with a pitch of 1.5 Å and an insertion angle of
14° relative to the plane of the bilayer. This information allowed us to propose a specific topological model of the NH2 terminus relative to the membrane shown at the bottom of Fig 2 D.
Structure of the COOH Terminus
In contrast to the NH2 Terminus, the COOH terminus of KcsA exhibits a wide range in the dynamic behavior of individual residues. Fig 3 A shows the EPR spectral data set obtained from residues 121160. *Spectra were obtained under conditions that ensured the absence of dipolar coupling (see below).
The Ho-1 profile shows that the COOH terminus can be divided into two regions of different overall dynamics: a segment of low and intermediate mobility encompassing residues 121147 and a highly dynamic region containing the last 13 residues of KcsA (Fig 3 B, top). The presence of a relatively large number of motionally restricted positions along the entire length of the COOH terminus implies that this region of KcsA is involved in extensive intra- or intersubunit interactions. In fact, there is a clear correlation between the dynamic behavior of a given residue and its solvent accessibility. Analysis of the NiEdda accessibility profile (Fig 3 B, bottom) reveals that the same set of residues displaying restricted mobility are also solvent inaccessible, as expected from protein regions that are not exposed to the aqueous milieu. Furthermore, residues with the highest motional freedom, at the end of the COOH terminus, have the highest NiEdda accessibilities. In contrast, the O2 accessibility profile is largely featureless and appears low throughout the entire segment (Fig 3 B, middle).
Frequency analysis performed on the accessibility profiles reveals two different behaviors. On one hand, there is a strong -helical periodicity in the COOH terminus
NiEdda profile, as derived from the dominant peak in the power spectrum of Fig 3 C (thick line). The
O2 profile, on the other hand, displays a major peak at 65° with no significant components near the expected frequencies for
-helices or ß-sheets. A likely explanation for this discrepancy is that the COOH terminus of KcsA may form a structure with crevasses or cavities large enough to allow free diffusion of O2, but not of NiEdda. Therefore, only the NiEdda accessibility profile has enough contrast to reveal structurally related periodicities. In effect, when individual accessibility moments are calculated (Fig 3 C, bottom), the direction of the resultant vectors for
O2 and
NiEdda coincide.
Additional information about how this helical region may assemble in three dimensions comes from the study of the extent and distribution of spinspin interactions. These interactions originate from trough-space dipolar coupling between the unpaired electrons, and reveal themselves as a Lorentzian-type broadening of the standard spectral line shape (
The extent of inter-nitroxide spinspin interactions is used as the primary data to obtain distance information in spin-labeled proteins. Current methods include convolution or deconvolution of the spectral broadening induced by the dipolar coupling ( parameter, an operationally defined value calculated from the normalized amplitude ratio between the under-labeled and fully labeled spectra (
values, but as the interspin separation increases, the
parameter decreases to one (no spinspin coupling). Fig 4 B shows that the
profile of the COOH terminus presents a roughly pyramidal distribution, in which residues at the center of the 121160-residue segment are closer to each other than residues at either end. This particular distribution of intersubunit proximities cannot be explained by a parallel arrangement of the helices in the bundle, and strongly suggests a definitive interhelical angle in which the narrowest point of the bundle is located at or near residue T141. Interestingly, there is a noticeable increase in the
values at the very end of the segment (particularly residue N158). Coupled with the existence of two distinct regions of mobility (Fig 3 B), this finding suggests the presence of a separate intersubunit association at the end of the COOH terminus. Although each individual
value is an approximate estimate of intersubunit proximity, the unique pattern of proximities along the full set of COOH-terminal residues can be used to reliably determine an overall three-dimensional fold for this region of KcsA.
The Molecular Architecture of Full-Length KcsA
A more specific assignment of the -helical components for both NH2 and COOH termini, was obtained from
PI plots shown in Fig 5 A. The
PI is a weighted-area ratio of the
-helical frequency components (80°120°) relative to the entire power spectrum, and for values
2, it defines statistically significant
-helical regions (
PI derived from the
O2 profile is significantly
-helical in most of the analyzed stretch, dropping below
2 in a short region next to the expected point of insertion to the first transmembrane segments TM1 (Fig 5 A). For the COOH terminus, analysis of the
NiEdda profile reveals three
-helical regions joined by two nonhelical "linkers" regions of different length (Fig 5 B). Additionally, when the periodicity index is computed specifically for frequencies associated with coiled-coil structures (100°140°), a statistically significant helical region emerges at the end of the COOH-terminus. Prediction algorithms based on primary sequence (coils;
-helical up to position 20 to 21, where a sort stretch of two to three residues connects to TM1. The COOH terminus contains three distinct helices: the first one is an extension of TM2 to position 122 to 123, the second one is at least 16 residues long, and the final one includes the last 810 residues of this domain.
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To simplify our modeling, once assigned, secondary structure elements were considered essentially rigid and a pattern of proximity ranges among these secondary structure elements was estimated based on the location and extent of the intersubunit dipoledipole interactions. This set of constraints was used to compute a number of three-dimensional folds using simulated annealing methods (SA/RMD;
After appending the NH2-terminal residues to the channel core (as modeled in Fig 2 D), and replacing all COOH-terminal alanines and spin-label pseudo-atoms for wild-type side chains, a final full-length KcsA model was constructed by applying a global minimization step to the resulting aggregate structure. This differs from the standard SA/RMD approach (-helix at the membranewater interface that protrudes away from the channel core, and does not interact with any other part of the channel (except perhaps near its insertion point to TM1). Direct mapping of the EPR data onto the accessible surface of the average structure (Fig 6 B) demonstrates an excellent correspondence between the surface patterns of the mobility, accessibility, and proximity parameters with the proposed overall fold. In general, residues involved in quaternary contacts display low mobility and low solvent accessibility, while residues near the symmetry axis show extensive spinspin interactions. Residues with the highest motional freedom at both extremes of the molecule also displayed the largest levels of NiEdda accessibility.
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To explore some of the implications of this model, we used the average structure (Fig 6 A) to calculate molecular accessible surfaces and cavities, and to perform a simple electrostatic analysis of KcsA (not shown). In the present model, the internal cavity along full-length KcsA extends almost 100 Å along the axis of symmetry, as the COOH-terminal bundle is proposed to reach some 50 Å into the cytoplasm (Fig 6 A). The inner surface of full-length KcsA (
An initial electrostatic analysis of the whole molecule indicates the presence of distinct regions of potential along the cytoplasmic surface of KcsA. The isopotential contours at ±1 kT/e reveals a complex interplay among the large number of charged residues present in both NH2 and COOH termini. There is a large positive potential that surrounds the channel precisely at the level of the membranewater interface. This may suggest a role for the NH2 terminus in the physical anchoring of the channel to the membrane, since positive residues in this region could form ionic interactions with negatively charged lipids located at the inner leaflet of the bilayer. Closer inspection of the critical region that extends the -helical conformation of TM2 beyond the current crystallographic model highlights the presence of a cluster of charged residues that include R117, E118, E120, R122, and H124 (Fig 7 B, right). This cluster might form a complex intersubunit charge network, representing a prime candidate for the location of the putative "pH sensor" responsible for triggering channel openings at low pH. Clearly, because our present model is limited to backbone features, further experiments are needed to determine the precise role of these charged residues on pH-dependent activation.
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Functional Implications of COOH-terminal Deletions
Fig 7 A clearly demonstrates that the COOH-terminal domain appears to have no significant influence on ion conduction in KcsA. At pH 4.0 and in symmetrical K+ solutions, deletion of most of the COOH-terminal end by chymotrypsin cut (125) does not affect single-channel currents and appears to have minimal effects on dwell times (not shown). In 200 mM K+, single-channel conductances obtained from I-V relations (Fig 7 B) were essentially identical (
100 pS), although the I-V curve for the
125 channel flattens out at positive potentials. In the absence of the highly charged COOH terminus, this effect might be due to an increased sensitivity to blockers, or may even originate from changes in the local electrostatic field near the permeation pathway.
The role of the COOH terminus on pH-dependent gating was studied with three sequential deletion constructs: 140 (deletion to the middle of the helix bundle),
125 (deletion to the COOH-terminal linker), and
120 (deletion to the end of TM2). The pH dependence of 86Rb+ influx was analyzed for each deletion construct, as shown in Fig 7 C. While construct
140 showed minimal effects on pH dependence and apparent pKa, further deletions revealed both a progressive shift in the apparent pKa towards higher pH and a decrease in the pH dependence of channel activation (empirically estimated from the Hill number). This behavior seems to correlate well with the dynamics of the different regions of the COOH-terminal bundle (Fig 3 B). Although these results clearly point to a role of the COOH terminus in modulating pH-dependent gating (probably by stabilizing the closed state), all of the tested KcsA deletion constructs were able to fully close at pH 9.0, firmly demonstrating that the COOH-terminal end does not form part of the activation gate.
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DISCUSSION |
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Using site-directed spin labeling and electron paramagnetic spectroscopic methods, we have derived the solution three-dimensional fold of the NH2- and COOH-terminal regions of the KcsA K+ channel embedded in a lipid bilayer. This information was used to propose a model of full-length KcsA in which the NH2 terminus of the channel is an interfacial helix stretching some 3540 Å away from its transmembrane core, while the COOH-terminal end forms a helical bundle extending 4050 Å into the cytoplasm.
Cytoplasmic helical bundles are emerging as a common structural motif in a number of ion channels and other signaling membrane proteins. Not counting the difference in the number of subunits, the present KcsA structure is reminiscent of the large-conductance mechanosensitive channel MscL found in a number of bacteria (
Recent experiments have shown that deletion of the first 20 residues in KcsA is associated with a dramatic reduction in expression levels (125160) produces an increase in resting 86Rb+ fluxes at neutral pH with a simultaneous decrease in oligomeric stability, pointing to the importance of the COOH terminus in the stabilization of the closed state (
The location and packing arrangement of the COOH-terminal bundle in the present full-length structure leads to important questions regarding the likely permeation path followed by ions in full-length KcsA. While the details of the possible pH-dependent conformational changes in these cytoplasmic domains remain to be established, it is clear that the predicted water-filled cavity and associated openings underneath the membrane-embedded channel core represent a potential entryway for ions into the transmembrane pore. Indeed, experiments shown in Fig 7 A argue that the presence of the COOH-terminal end has no significant effect on the permeation properties of KcsA, and thus imply that the ion permeation path does not extend to the COOH-terminal bundle. Given that the current structure was obtained at neutral pH (and thus corresponds to the closed conformation of the channel;
Although originally considered extracellular (
The fact that, even in the absence of the entire COOH terminus, KcsA is still able to gate in a pH-dependent manner points to a stretch of charges located at the end of TM2 as a potential site for the pH sensor of KcsA. COOH-terminal deletions appear to have only marginal effects on the pH dependence and apparent pKa of KcsA activation for most of the length of the bundle, yet significant changes in pH dependence do occur as the regions deleted approach the charge cluster at the end of TM2 (120 and
125). While the current structure does not contain information regarding the orientation of specific side chains, the overall arrangement of the helices in this region suggests possible intersubunit electrostatic interactions that can be drastically affected by changes in pH or by binding of a specific charged ligand or lipid. Under these conditions, local rearrangements may propagate to the transmembrane regions of the molecule, triggering the types of gating-related movements seen for TM1 and TM2 (
It is important to note that due to the particular data set used in the structure calculations, the accuracy of the present structure is limited in three important ways. Secondary structure assignment depends on the periodicity analysis of a finite stretch of sequence, which makes dealing with short segments of secondary structure problematic. Additionally, because of the fourfold symmetry of fully labeled channels, actual internitroxide distances cannot be computed and have been estimated only within approximate ranges. Finally, because all of the long-range constraints in the structure were derived from intersubunit interactions, the resolution along the membrane normal is limited, and this will affect the actual dimensions of the COOH-terminal bundle as well as the precise conformation of the loop linking TM2 and the COOH-terminal helix. While some of these caveats are intrinsic to the current SDSL approach, we have tried to minimize these problems by concentrating on "pattern analysis" and on the reliability of the individual measurements. In fact, the entire SDSL analysis of the COOH-terminal end was independently performed twice with essentially identical results. Slight differences in probe mobility and NiEdda accessibility produced no major changes in our proposed three-dimensional KcsA model, at least given the accuracy of the available data.
In spite of these limitations, the proposed fold offers a first glimpse at full-length KcsA, setting the stage for further experimental tests of specific predictions related to the physiological control of KcsA activity. Clearly, given the pattern of intersubunit spinspin interactions in the linker region between the end of TM2 and the start of the COOH-terminal bundle, this is the least defined region in our model. Additional information derived from intrasubunit distance measurements or the determination of longer-range intersubunit distances (i.e., using pulsed EPR) will certainly help define the true conformation of this region. We expect that the use of SDSL and EPR spectroscopy to derive three-dimensional folds in oligomeric proteins will be further improved by calculating more precise distances from tandem dimer constructs and by including a potential term derived from EPR-derived solvent accessibility measurements as a restraint. This term can be applied as part of a refinement step once a family of structures has been generated using the present methods. Such approach might find a general use in structural dynamics studies of membrane proteins and other systems in which traditional structural methods are still difficult to apply.
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Footnotes |
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1 Abbreviations used in this paper: EPR, paramagnetic resonance; Kv channel, voltage-dependent channel; PI, periodicity index; SDSL, site-directed spin labeling; TM2, second transmembrane segment.
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Acknowledgements |
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We thank Drs. Robert Nakamoto and Pornthep Sompornpisut for critical reading of the manuscript, Dr. Boris Martinac for advice on liposome patch clamping, Drs. Yeon-Kyun Shin and Wenzhong Xiao for sharing SA/MD protocols, and the members of the Perozo lab (Y.-S. Liu, C. Ptak, A. Shen, and P. Sompornpisut) for insightful discussions.
This study was supported by the National Institutes of Health (grants GM54690 and GM57846) and the McKnight endowment fund for neuroscience.
Submitted: 9 August 2000
Revised: 29 December 2000
Accepted: 3 January 2001
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