Correspondence to: Kenton J. Swartz, Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36 Room 2C19, 36 Convent Dr., MSC 4066, Bethesda, MD 20892. Fax:301-435-5666 E-mail:kjswartz{at}codon.nih.gov.
Released online: 28 December 1999
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Abstract |
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Voltage-gated K+ channels are tetramers with each subunit containing six (S1S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K+ channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of -helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting
-helical secondary structure. In addition, both the S1S2 and S3S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.
Key Words: secondary structure, scanning mutagenesis, amphipathic, Fourier transform, voltage-dependent gating
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INTRODUCTION |
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The voltage-gated K+ channels comprise a large family of tetrameric membrane proteins that open and close in response to changes in membrane voltage. Based on hydrophobicity analysis, each subunit in the tetramer contains six putative transmembrane segments, termed S1 through S6 (Figure 1 A). The central pore domain, which contains the K+ selective ion conduction pathway, is formed by the assembly of the S5 through S6 regions (-helices with the S5S6 linker, the most conserved region of all K+ channels, forming a short pore helix and the selectivity filter.
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The first four transmembrane segments (S1S4) of voltage-gated K+ channels are not present in the simple pore K+ channels, like KcsA and the inward rectifier K+ channels, and appear to underlie their unique voltage-sensing capabilities (
What are the secondary and tertiary structures of these four transmembrane segments? The inference from hydrophobicity analysis is that these hydrophobic segments are membrane-spanning -helices. This, however, remains to be established experimentally. Several studies examining the secondary structure of short peptides corresponding to isolated S2, S3, or S4 segments using circular dichroism, Fourier transform infrared spectroscopy, or 1H-NMR are consistent with these segments adopting
-helical structures (
In this paper, we explore the secondary structures of the four transmembrane segments in the voltage-sensing domains using alanine-scanning mutagenesis of the region spanning from the NH2-terminal edge of S1 to the COOH-terminal edge of S4 in the drk1 voltage-gated K+ channel (-helices. Our results support the presence of four transmembrane segments in the region on the NH2-terminal side of the pore domain (S5S6) and suggest that at least parts of all four segments adopt
-helical structure. We also found evidence for helical secondary structure in the two extracellular linkers between the four transmembrane segments. The distribution of gating perturbations on the four transmembrane segments, together with previously proposed electrostatic interactions, help constrain the packing arrangement within the voltage-sensing domain and suggest which region of the helical bundle may interface with the pore domain.
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MATERIALS AND METHODS |
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Mutagenesis and Channel Expression
Point mutations of drk1 K+ channels were introduced by generating mutant fragments by PCR and ligating into appropriately digested vectors. The construct used contained several previously introduced unique restriction sites (
Electrophysiology
Channels were studied using two-electrode voltage clamp recording between 1 and 5 d after cRNA injection using an OC-725C oocyte clamp (Warner Instruments). Oocytes were studied in a 160-µl recording chamber that was perfused with a solution containing (mM): 50 RbCl, 50 NaCl, 1 MgCl2, 0.3 CaCl2, and 5 HEPES, pH 7.6 with NaOH. Data were filtered at 2 kHz (eight-pole Bessel) and digitized at 10 kHz. Microelectrode resistances were between 0.2 and 1.2 M when filled with 3 M KCl. All experiments were performed at room temperature (~22°C).
Voltageactivation relations were obtained by measuring tail current amplitude for various strength depolarizations using 50 mM Rb+ as the charge carrier to slow deactivation. The reversal potential under these ionic conditions was approximately -25 mV. Holding voltages were chosen where no steady state inactivation could be detected, typically -130 to -80 mV. For most channels, inward tail currents were elicited by repolarization to -50 mV and tail current amplitude measured 13 ms after repolarization. More negative tail voltages were used for mutants with large negative shifts in the voltageactivation relationship. For mutants with large rightward shifts in the voltageactivation relationship, outward tail currents were elicited by repolarization to voltages between 0 and +30 mV. For a few mutant channels that displayed fast deactivation kinetics, conductancevoltage (G-V) relations were also examined using steady state outward K+ currents and calculating G according to G = I/V - Vrev. In these instances, tail voltageactivation relations and G-V relations were comparable.
Analysis of Channel Gating
To evaluate the gating properties of mutant channels, we considered the channel as existing in two states, closed and open, with a single transition between them. Voltageactivation relations were fit with single Boltzmann functions according to:
where I/Imax is the normalized tail current amplitude, z is the equivalent charge, V50 is the half-activation voltage, F is Faraday's constant, R is the gas constant, and T is temperature in Kelvin. Curve fitting was performed using Levenberg-Marquardt minimization procedures (Origin software; MicroCal Software, Inc.). The difference in Gibbs free energy between closed and open states at 0 mV (G0) was calculated according to:
G0 = 0.2389 zFV50.
The change in G0 caused by each mutation (
G0) was then calculated as:
G0 =
G0mut -
G0wt.
Analysis of Periodicity
Fourier transform methods (G0 for mutations in various regions according to: P(
) = [X(
)2 + Y(
)2],
where P() is the Fourier transform power spectrum as a function of angular frequency
, n is the number of residues of a segment, Vj is |
G0| at a given position j and<<V>>is the average value of |
G0| for the segment. Corrections of the Fourier transform power spectra, such as smoothing the outliers and ramp correction with least squares, did not significantly change the power spectra. All power spectra are therefore shown without correction.
To quantitatively evaluate the -helical character from power spectra, the
-periodicity index (
-PI)1 (
While an ideal amphipathic helix should give a peak in the power spectrum at ~100°, transmembrane helices in membrane proteins of known structure tend to show peaks significantly shifted to higher frequencies (-PI values >2 have been considered indicative of
-helical secondary structure (
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RESULTS |
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We began by supposing that the four transmembrane segments (S1S4) of the voltage-sensing domains in voltage-gated K+ channels adopt -helical structures. In this case, at least some of the helices would be amphipathic, interacting with lipid on one face and protein on another. Since the region in question undergoes a conformational change in response to changes in membrane voltage, a mutation to alanine might be expected to alter the channel's gating properties if it lies at a proteinprotein interface, but have little consequence if located at a proteinlipid interface because alanine is hydrophobic. If all residues in a hypothetical amphipathic helix were mutated to alanine (one at a time), then the positions with large effects on gating might cluster on one face and demarcate the proteinprotein interface and the positions with only small effects on gating might cluster and identify the proteinlipid interface. The actual situation is likely to be more complicated because there may also be water filled crevices projecting from the internal or external aqueous environments, and therefore a proteinwater interface is also possible on the transmembrane surface. Some helices may interact with other helices in the voltage-sensing domain through one face and interact with the helices in the pore domain (S5 and S6) through another face. Thus, there may also be two different types of proteinprotein interfaces with mutations in each having different manifestations. We were encouraged, however, by a recent study by
Perturbation in Channel Gating by Mutations in S1 through S4
With the above strategy in mind, we alanine scanned the voltage-sensing domain of the drk1 voltage-gated K+ channel (G0) for each channel, and then calculated the difference in
G0 between wild-type and mutant channels (
G0). Table 1 summarizes the gating properties for all 127 mutations.
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Distribution of Gating Perturbations
The absolute values of G0 for all the mutant channels are shown in Figure 3 superimposed with a Kyte-Doolittle hydrophobicity analysis (
G0| values tend to cluster into four groups separated by stretches of relatively small |
G0| values. The distribution of perturbation energy correlates nicely with the hydrophobicity profile. The energy maxima correspond to the peaks in the hydrophobicity index, while the energy minima correspond to the valleys in the index. Thus, mutations in the linkers between transmembrane segments where the residues tend to be more hydrophilic typically have much smaller effects on gating. The results are quite remarkable and support the notion that four transmembrane segments are present within this region of voltage-gated K+ channels. While the largest perturbations are seen in S4 (up to 6.6 kcal mol-1), there are also sizable changes in
G0 for the other three transmembrane segments. It is interesting that there is a trend in the perturbation energies across the region studied with the smallest effects occurring in S1 and the largest effects occurring in S4.
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Periodicity of Gating Perturbations
Figure 4 shows a plot of the actual G0 values for all of the mutations, revealing periodicity in the data. Most notably, positions with negative values of
G0, where mutations cause a relative stabilization of the open state, tend to cluster together in patches. Similar clustering is observed for positions with positive values of
G0, where mutations produce a relative stabilization of the closed state. For example, mutations in the NH2-terminal half of S4 generally result in large negative values of
G0, while mutations in the COOH-terminal half of S4 mostly cause large positive
G0 values. There are similar patterns evident in S1, S2, and S3.
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To look more closely for evidence of helical secondary structure, we examined each transmembrane segment individually using Fourier transform methods and helical wheel diagrams. The Fourier transform power spectrum calculated from the |G0| values for the S1 segment is shown in Figure 5 B. The spectrum is for 23 residues beginning with K185 and ending with L207, just before a highly conserved proline at 208. The strongest peak in the spectrum occurs at 106°, a frequency within the characteristic range for an
-helix. Previous studies suggest that
-PI > 2.0 are a strong indication of
-helical secondary structure (
-PI calculated from the spectrum in Figure 5 B was 2.3. The periodicity within the data for S1 can also be seen in the helical wheel (Figure 5 C) and net (D) diagrams. Six residues (K185 I192, A188, F194, I195, N205) have |
G0|
1 kcal mol-1 and most are clustered on one side of the wheel. The remaining residues have |
G0| < 1 kcal mol-1 and cover a larger fraction of the wheel circumference. Individual |
G0| values plotted as vectors on the helical wheel (Figure 5 C) have a sum of 4.8 kcal mol-1, a value larger than any individual |
G0| value. The sum vector therefore points to the side where residues have the largest |
G0| values, most likely identifying the face involved in proteinprotein interactions. It is notable that this side of the helical wheel contains many of the highly conserved residues observed in sequence comparisons (Figure 1 B) (
-helix and that it primarily contacts two distinct environments.
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Figure 6 shows power spectra and both helical wheel and net diagrams using |G0| values in S2. The first power spectrum covers a stretch of 18 residues, from V228 to L245, and ends several residues before a highly conserved proline residue at position 248. As with S1, the spectrum shows a strong peak near the expected frequency for an
-helix, in this case at 102° (Figure 6 B, left). The
-PI for this spectrum is 1.8. The
-PI for a shorter window in S2 (containing 13 residues) is 2.1 (see Figure 9). The results from a previous tryptophan scan of S2 in the Shaker K+ channel also showed that residues having large and small effects on channel gating cluster on opposite sides of a helical wheel (
-PI = 2.4) and the alanine scan of drk1 giving a peak at 103° (
-PI = 1.6). The spectra for S2 in the drk1 K+ channel are somewhat more complex than for S1, with multiple peaks observed at lower frequencies (Figure 6 B). The helical wheel and net diagrams in Figure 6C and Figure D, show the distribution of mutations in S2. Many of the residues with |
G0|
1 kcal mol-1 (shaded circles) overlap those presented by
G0| values were plotted as vectors on the helical wheel with a sum of 5.3 kcal mol-1, larger than any individual |
G0| value. The vector sum points to the side with the largest |
G0| values, again most likely identifying the face involved in a proteinprotein interface. This side also contains the most highly conserved residues (Figure 1 B;
-helix.
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Two power spectra calculated for S3 are shown in Figure 7 B. As with S1 and S2, a conserved proline residue (P268) is present in S3, but in this case the proline is located at the center of the segment, as collectively defined by the hydrophobicity analysis and the distribution of gating perturbations (Figure 1 and Figure 3). We therefore initially examined the power spectrum for only 15 residues beginning with F253A and ending at L267A, just before the conserved proline at 268. This power spectrum shows a major peak at 122°, near the edge of the -helical frequency, with an
-PI of 1.8. The
-PI for a 13-residue window in this region is 2.2 (see Figure 9). The spectrum for a COOH-terminally extended segment past the conserved P268 (F253 to L275) is more complex. A peak is still observed within the helix frequency (120°), but it is no longer dominant, and there are many other peaks observed at both lower and higher frequencies. The helical wheel diagram presented in Figure 7 C shows that the mutations with |
G0|
1 kcal mol-1 (shaded circles) distribute over a large portion of the wheel. However, the net diagram in Figure 7 D shows a cluster of residues on one face of the helix where mutations have only small effects on gating. The sum of the individual |
G0| vectors was 2.6 kcal mol-1, slightly smaller than the largest individual |
G0| (3 kcal mol-1 for A265). The sum vector points to the side containing conserved residues (Figure 1 B;
-helical structure, they are ambiguous for the COOH-terminal part of S3.
The amino acid sequence of the S4 segment is unique in that it contains a repeating triad of two hydrophobic residues and one basic residue (arginine or lysine). There is strong evidence that the basic residues in S4 move in response to changes in membrane voltage and determine the voltage dependence of the channel (G0 = +0.1 kcal mol-1 for R296A and
G0 = +0.9 kcal mol-1 for K305A). Thus, there are only three positions in the S4 segment of drk1 (R299, R302, and R308) where mutations produce large perturbations that could, either partially or completely, be due to changes in gating charge. To minimize the contribution of changes in voltage dependence to the evaluation of periodicity, we compared spectra containing all |
G0| values for a given stretch of residues with those where the |
G0| values for the basic S4 residues were set to the mean value of |
G0|. This procedure effectively removes these residues from the power spectrum calculation.
Figure 8 shows four power spectra for the S4 segment. The top left spectrum was calculated using all |G0| values for a stretch of 19 residues beginning at Q293 and ending at T311 (starting from the equivalent of R1 in the Shaker K+ channel and ending with the equivalent of K7). This spectrum is very complex, with the closest peak to the
-helix frequency occurring at 134°. The spectrum for the same stretch after minimizing the contribution of the basic residues is still rather complex, but now the dominant peak occurs at lower frequency (Figure 8 B, top right). The power spectrum calculated from all |
G0| values for the COOH-terminal 13 residues in S4 (R299 to T311) contains a strong peak at 109°, within the frequency range expected for an
-helix, with an
-PI of 1.9 (Figure 8 B, bottom left). After minimizing the contribution of the basic residues, the spectrum for this region still has a peak in the
-helical frequency range (110°), but now this peak is more dominant, as indicated by the larger
-PI value of 2.9 (Figure 8 B, bottom right). The similarity of the spectra with and without contributions from the basic residues for the COOH-terminal part of S4 suggests that the observed periodicity reflects an underlying
-helical secondary structure. The helical wheel and net diagrams in Figure 8C and Figure D, show that mutations with |
G0|
1 kcal mol-1 (shaded circles) are distributed with little evidence of clustering. Moreover, the sum of the individual |
G0| vectors was 3.4 kcal mol-1, significantly smaller than several individual |
G0| values. These results suggest that the COOH-terminal part of S4 is
-helical, but say little about the structure of the NH2-terminal part of S4.
Figure 9 shows a sliding window analysis of -PI beginning at the NH2 terminus of S1 and ending at the COOH terminus of S4. A 13-residue window is shown in A and a 17-residue window is shown in B. A sliding window hydrophobicity analysis is also shown for comparison. The comparison between the
-PI and the hydrophobicity index is quite remarkable for two reasons. First, there is an excellent correlation between the
-PI and hydrophobicity index for all four transmembrane segments, especially for S1, S2, and S3. For the 13-residue window,
-PI values greater than 2 are observed for S1, S2, and S3 and a value of 1.9 is seen for S4. This high degree of correspondence between
-PI and hydrophobicity makes it highly probable that all four transmembrane segments are in fact
-helical in structure. A second remarkable aspect of the graph is that within the two extracellular linkers (S1S2 and S3S4) there are peaks in the
-PI that correspond to troughs in the hydrophobicity index. For the 17-residue window, peak
-PI values of 2.1 and 1.6 are observed for the S1S2 and S3S4 linkers, respectively. In contrast, for the S2S3 linker, a corresponding minimum is observed for both the
-PI and hydrophobicity index. Figure 10 and Figure 11 show power spectra and helical wheel diagrams for the S1S2 and S3S4 linkers, respectively. Both spectra show dominant peaks within the
-helical frequency range and both helical wheels show clustering of residues with small versus moderate effects on gating. These results raise the possibility that there is an additional
-helix in each of the two extracellular linkers.
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DISCUSSION |
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The goal of this study was to probe the secondary structure of the transmembrane segments in the voltage-sensing domain of the drk1 K+ channel using alanine-scanning mutagenesis. The premise of the approach is that rearrangement of the segments in the voltage-sensing domains is important for the conformational changes involved in gating. There is considerable support for this notion for S2, S3, and S4 from mutagenesis (G0) produced by the mutations. The first observation was that the pattern of perturbations in gating energy nicely paralleled both the hydrophobicity index and the degree of conservation (Figure 1 and Figure 3), supporting the notion that there are four transmembrane segments present on the NH2 terminal side of the pore domain. Closer examination of the periodicity in the energetic perturbations within individual transmembrane segments suggests that at least portions of all four segments (S1S4) adopt
-helical structures. In addition, we found evidence for
-helical structure in the two extracellular linkers. We will discuss each of these regions in detail, pointing out a number of interesting features for each.
The mutations in S1 had the smallest effects on G0 compared with the other three transmembrane segments with the largest change of +2.3 kcal mol-1 occurring for I192A. Nevertheless, the periodicity in the data for S1 was clear from both the Fourier transform power spectrum and helical wheel diagrams (Figure 5). A clean peak in the power spectrum occurred at 106°, a frequency corresponding to an
-helix, with an
-PI of 2.3. The sum of the individual |
G0| vectors (4.8 kcal mol-1) was much larger than any individual |
G0| vector and pointed to the face containing the majority of residues having large effects. This suggests that S1 is an
-helix with two main faces interacting with different environments. The face identified by the vector sum likely represents a proteinprotein interface. The opposite face contains 10 residues with only minor effects on gating and these residues are all hydrophobic, suggesting that they likely interact with lipid. The highly conserved proline (P208) at the COOH-terminal border of S1 may serve as a helix breaker and thus demarcate the end of the S1 helix. If the NH2 terminus of the S1 helix begins at K185, where our analysis starts, the helix would contain 23 residues and have sufficient length to cross the hydrophobic core of the lipid membrane (~35 Å). Since the hydrophobicity index is still rather large at K185, it is possible that the S1 helix may begin several residues before K185.
Our results for the S2 segment also support a helical structure, consistent with previous results in the Shaker K+ channel (-PI values of 1.8 and 1.6, the sliding 13-residue window analysis gave an
-PI of 2.1. Multiple lower frequency peaks, especially in the stretch containing 23 residues, imply that the S2 segment may have a somewhat more complex environment than S1. As with S1, the sum of the individual |
G0| vectors was larger (5.3 kcal mol-1) than any individual |
G0| vector, pointing to the face containing the majority of positions where mutants have large effects. These results suggest that S2 is an
-helix with the large vector sum identifying the proteinprotein interface. This face contains two acidic residues (E229 and E239) that are equivalent to residues in the Shaker K+ channel (E283 and E293) thought to form electrostatic interactions with basic residues in S4 (
It is remarkable that our results for an alanine scan of S2 in the drk1 K+ channel are so similar to those for a tryptophan scan of S2 in the Shaker K+ channel (-helix, and in addition contain several coincident minor peaks. The similarity in the results with the two scans strongly supports the premise that the large perturbations occur at proteinprotein interfaces and that the weak perturbations occur at proteinlipid or proteinwater interfaces. For positions where the native residues are hydrophobic, and equally tolerant of alanine or tryptophan, it seems likely that the solvent is lipid.
Our results for S3, together with the hydrophobicity and sequence analysis, suggest that either the structure of this region or its environment is considerably more complex than seen for S1 and S2. We first considered only the results obtained with the first 15 residues in S3 because there is a very highly conserved proline residue (P268) only 15 residues in from the likely start of the S3 segment. P268 corresponds, not to minima in the |G0| or hydrophobicity index patterns, but to maxima in both. The power spectrum for this stretch of 15 residues contains a large peak at 122°, near the
-helix frequency range, but pushing the high frequency end of that range. The calculated
-PI for the spectrum shown in Figure 7 is 1.8, but values as high as 2.2 are observed in the sliding 13-residue window analysis (Figure 9). The unusually high frequency of 120° would be consistent with the NH2-terminal part of S3, adopting an
-helical structure that is more tightly coiled than a typical helix, possibly indicating that S3 is a 310 helix. This seems unlikely since a 310 helix of this length would be highly unusual, if not unprecedented. Alternately, this may simply reflect that the environment of the helix is rather complex, possibly suggesting that S3 has proteinprotein contacts on multiple faces. In this regard, it is interesting that residues having large effects on gating are rather distributed on a helical wheel. This is manifest in a rather weak vector sum for |
G0| (2.6 kcal mol-1), a value actually less than any individual |
G0| value. In any case, a helix containing 15 residues cannot span the width of the membrane and the minima in both the |
G0| and hydrophobicity index patterns occur some distance past P268 towards the COOH terminus. However, the power spectrum for a COOH-terminally extended region past P268 and containing 23 residues was very complex. Many more peaks are now observed with the 120° peak being of equal or smaller size than many other peaks in the spectrum. One possibility is that the entire S3 segment is
-helical, but there is a break or kink at P268 that results in a phase shift between the NH2- and COOH-terminal portions of the segment. Alternatively, it may simply be that the environments surrounding the NH2- and COOH-terminal portions of the S3 helix are very different. In this regard, it is interesting to note that before P268 there is a cluster of mutations that give positive
G0 values. On the COOH-terminal side of P268, several mutants significantly perturb channel gating, but with no clustering with respect to relative stabilization of closed or open states. It is important to stress that our results are equally compatible with the COOH-terminal region of S3 adopting a completely different secondary structure (i.e., nonhelical).
Another interesting aspect of the COOH-terminal part of S3 is that it seems to form the binding site for hanatoxin, a gating modifier of the drk1 K+ channel. Three residues (I273, F274, and E277) located on the COOH-terminal side of P268 are particularly important for interaction with hanatoxin (
The power spectrum for the COOH-terminal 13 residues of the S4 segment contains a peak at 109°, suggesting -helical structure. This result does not appear to be dominated by the repeating pattern of basic residues because minimizing the contribution of these residues in the analysis does not alter the position of the main peak at the
-helical frequency (see RESULTS). In fact, removing the basic residues from the Fourier transform analysis greatly improves the
-PI (from 1.9 to 2.9). The spectrum for a longer stretch (containing 19 residues) was much more complex, with no dominant peaks at frequencies indicative of an
-helix. These results suggest that the COOH-terminal portion of S4 is
-helical, but are ambiguous when it comes to the NH2-terminal part. However, the repeating triad of two hydrophobic residues and a basic residue extends through the entire S4 segment, suggesting a similar secondary structure throughout. Perhaps the simplest explanation is that the environments of both the NH2- and COOH-terminal parts of the S4 helix are rather different, possibly involving different combinations of proteinprotein, proteinwater, and even small proteinlipid interfaces. The observation that mutations producing the largest effects on gating are more or less evenly distributed around the helical wheel suggests that proteinprotein interfaces may dominate most of the transmembrane surface of S4. In addition, we find that mutations in the NH2-terminal part of S4 produce negative values of
G0, whereas mutations in the COOH-terminal part produce positive values of
G0. This suggests that the NH2- and COOH-terminal portions of S4 may play different roles in the voltage-gating process such that mutations in the NH2-terminal part cause a relative stabilization of the open state and mutations in the COOH-terminal part cause a relative stabilization of the closed state. Perhaps the two parts of S4 interact with distinct structural elements of the protein.
One of the most unanticipated results of the present study was that the analysis of both the S1S2 and S3S4 linkers shows evidence of helical secondary structure. This was first apparent from the sliding window analysis where peaks in the -PI are observed at minima in the hydrophobicity analysis (Figure 9). The helical character of these linkers is also evident in both the power spectra and helical wheel diagrams (Figure 10 and Figure 11). If the extracellular linker between S1 and S2 is an
-helix, then it most likely rests on the surface of the protein for two reasons (Figure 12A and Figure B). First, we observed strong helical character for long enough stretches (23 residues) in both S1 and S2 to completely span the hydrophobic core of the membrane. Second, in contrast to most of the membrane-spanning helices, the entire circumference of the S1S2 linker helix contains many hydrophilic residues. Thus, for the S1S2 linker helix, the likely interfaces are proteinprotein and proteinwater, with no significant proteinlipid interfaces. The conceptual frame of reference for the S3S4 linker is less clear because of the uncertainties with the secondary structure of the COOH-terminal part of S3. The S3S4 linker helix may lie on the surface, as we postulate for the S1S2 linker. Alternately, this helix may begin soon after the conserved proline (268) in S3 and thus represent an extension of S3 and possibly lie partially within the membrane (Figure 12A and Figure B).
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The present results are consistent with S1 to S4 each adopting -helical secondary structures, albeit with the above-noted complexities. What is the tertiary structure of the four transmembrane helices, and where do they interface with the pore domain? Our helical wheel diagrams and vector plots identify the most likely faces where proteinprotein interactions take place and where proteinsolvent interfaces form. Several previously proposed electrostatic interactions add additional constraints (
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Footnotes |
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1 Abbreviation used in this paper: -PI,
-periodicity index.
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Acknowledgements |
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We thank Rod MacKinnon for helpful discussions, Rosalind Chuang for making some of the mutants, and J. Nagle and D. Kauffman in the National Institute of Neurological Disorders and Stroke Sequencing Facility for DNA sequencing. We thank Chris Miller and Kwang Hee Hong for sharing unpublished tryptophan-scanning results in the Shaker K+ channel.
D.H. Hackos was supported by the PRAT program, National Institute of General Medical Sciences, National Institutes of Health.
Submitted: 27 August 1999
Revised: 25 October 1999
Accepted: 29 October 1999
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