ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of

Dec 5, 2012 - evanescence field and minimizes contribution from the water background. In this approach, FTIR measurements of the KcsA potassium channe...
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ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K+ and Na+ in the Open and Collapsed Conformations of the KcsA Potassium Channel Yuji Furutani,†,§ Hirofumi Shimizu,‡ Yusuke Asai,† Tetsuya Fukuda,† Shigetoshi Oiki,*,‡ and Hideki Kandori*,† †

Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan Division of Biomolecular Sensing, Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan ‡ Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui, Yoshida, 910-1193 Fukui, Japan §

S Supporting Information *

ABSTRACT: The potassium channel is highly selective for K+ over Na+, and the selectivity filter binds multiple dehydrated K+ ions upon permeation. Here, we applied attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to extract ion-binding-induced signals of the KcsA potassium channel at neutral pH. Shifts in the peak of the amide-I signal towards lower vibrational frequencies were observed as K+ was replaced with Na+. These ion species-specific shifts deduced the selectivity filter as the source of the signal, which was supported by the spectra of a mutant for the selectivity filter (Y78F). The difference FTIR spectra between the solution containing various concentrations of K+ and that containing pure Na+ demonstrated two types of peak shifts of the amide-I vibration in response to the K+ concentration. These signals represent the binding of K+ ions to the different sites in the selectivity filter with different dissociation constants (KD = 9 or 18 mM). SECTION: Biophysical Chemistry and Biomolecules

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configuration). Such strong interactions between ions and the channel are not observed elsewhere in the KcsA structure. To understand bindings of different ion species to different conformations of the selectivity filter, here we present an approach using ATR-FTIR spectroscopy.12−15 The ATR mode allows the measurement of vibrational singals from the evanescence field and minimizes contribution from the water background. In this approach, FTIR measurements of the KcsA potassium channel are performed in two electrolyte solutions, and the difference spectra are analyzed. The rigorous selectivity function of the KcsA channel for K+ over Na+ was exploited to extract FTIR signals for the ion and channel interactions. The difference in the FTIR spectra of the KcsA channel in a K+containing solution and Na+ containing solution should help to address the vibrational frequencies relevant to the selectivity function. The KcsA channel was reconstituted into the membrane (the molar ratio of the channel/lipid [asolectin] was 1: 50). A suspension of the KcsA channels16 was overlaid on a diamond ATR crystal and was allowed to dry under a gentle stream of

he potassium channel exhibits a high throughput rate of ion flux with a high selectivity for K+ over Na+, the latter having smaller size in terms of the ionic radius.1 The mechanism underlying the highly selective K+ permeation has long been an issue of debate. The crystal structure of the KcsA potassium channel2−5 provided insights into the molecular mechanism of the selectivity (Figure 1).6−8 There is a structural part that is narrow (3 Å in diameter) and short (15 Å in length), named the selectivity filter, and the backbone carbonyl oxygens line the inner surface of the filter and form the four K+ binding sites. On the other hand, the amide nitrogens of the backbone do not form the hydrogen bonds with the carbonyl oxygens of the selectivity filter. Fully hydrated ions in the bulk solution must shed most of their water molecules upon entering (Figure 1b−e),1,4,9−11 and K+ ions are solvated by eight backbone carbonyls with the cage configuration.3,4 The high-resolution atomic structure revealed the ion distribution pattern in the selectivity filter. At high K+ concentration, the average of two ions distributed among the four binding sites. At low K+ concentrations and in the presence of nonpermeating Na+, the selectivity filter, especially the main chain of Gly77, undergoes structural changes into a collapsed, nonconducting conformation, to which structure the ions bind differently (Figure 1d,e).3 Also, it has been recently demonstrated that a Na+ ion is coordinated with four carbonyl oxygens (the plane © 2012 American Chemical Society

Received: October 24, 2012 Accepted: December 5, 2012 Published: December 5, 2012 3806

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Figure 2. The K+-minus-Na+ difference FTIR spectra recorded at pH 7 and 293 K. (a) The solid line shows the K+-minus-Na+ difference spectra of a KcsA sample, while the dotted line shows the spectrum obtained without a KcsA sample. The latter spectrum mainly comes from the OH bending mode of bulk water hydrating K+ or Na+ ions. (b) The spectrum is calculated by subtracting the dotted line from the solid line in (a). One division of the y axis is a 0.005 absorbance unit.

Figure 1. X-ray structure of the membrane spanning region (a) and the selectivity filter of the KcsA channel with K+ (b−d) or Na+ (e,f) ions (the PDB IDs for KcsA in the high-K+, low-K+, and Na+ conditions are 1K4C, 1K4D, and 2ITC, respectively). For clarification, only two diagonal subunits are shown in different colors (cyan and green), and the ion selectivity filter is closed up. The selectivity filter is lined by the carbonyl groups in the main chains of the TVGYG sequence (stick drawing). The K+ and Na+ ions are colored green and orange, respectively. In (d−f), the KcsA(high-K+) structure is superimposed on the KcsA(low-K+) and KcsA(Na+) structure (transparently colored light gray). (c,f) Top view of the KcsA tetramer with the side chains of W68 and Y78. W68 and Y78 are located within a hydrogen-bonding distance (3.0 Å in KcsA(high-K+) and 3.3 Å in KcsA(Na+)).

Figure 3. The K+-minus-Na+ difference FTIR spectra of the wild-type (black) and Y78F mutant (red) KcsA proteins recorded at pH 7 and 293 K. The black line is a spectrum reproduced from the spectrum in Figure 2b. One division of the y axis is a 0.002 absorbance unit.

N2. The surface was continuously perfused with electrolyte solution, and the solution containing either K+ or Na+ (200 mM at pH 7.0) was alternated every 20 min (see the Supporting Information, Figure S1). All of the experiments were performed at neutral pH, and the activation gate of the KcsA channel was kept closed. The ATR-FTIR measurements at 293 K were repeated throughout the perfusion time course. The spectrum gradually changed upon the solution exchanges and settled down to a steady state within 5 min after the onset. FTIR spectra during the steady state were averaged (an average of 6900 scans), and the difference between the K+ solution and Na+ solution was obtained (the K+-minus-Na+ difference FTIR spectra; Figure 2). The bare difference spectrum (Figure 2a, solid line) contains signals that originated from differences of the electrolyte solutions, including distortion from the water and buffer absorption changes caused by the exchanging of K+ with Na+ (dotted line). This distortion was subtracted in the

following spectra, as shown in Figure 2b. The spectra were reproducibly recorded in different samples. In the K+-minus-Na+ difference FTIR spectra, typical bands for the amide-I and -II vibrations were detected. The spectra were expanded in Figure 3 (black line). Positive bands at 1680, 1669, and 1659 cm−1 and negative bands at 1650, 1639, and 1627 cm−1 were resolved. These bands correspond to the characteristic frequencies of the amide-I mode, that is, mainly CO stretching vibrations of the peptide backbone. On the other hand, the bands at 1560 (−; negative signal), 1552 (+; positive signal), 1535 (+), 1518 (−), and 1509 (−) cm−1 were observed in the possible frequency region of the amide-II mode, that is, coupled C−N stretching and N−H bending vibrations of the peptide backbone. Also, the band at 1518 cm−1 is characteristic of a tyrosine ring mode.17 This pattern of 3807

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the difference spectra is interpreted to mean that the amide-I modes in the K+ solution (K+-induced vibrations) are shifted to the lower-frequency region in the Na+ solution (Na+-induced vibrations), and the amide-II modes at 1552 and 1535 cm−1 in the K+ solution turn into the 1560 and 1509 cm−1 bands in the Na+ solution. Emergence of the band at 1518 cm−1 in the difference spectra may suggest that a hydrogen bond of a Tyr residue was modified upon bindings of different ion species. Accordingly, the ion species-specific vibrations of the KcsA channel were resolved in the FTIR spectrum. Purely spectroscopic arguments on the above results imply that the secondary structure might be changed by replacing K+ with Na+.18 The crystal structure of the KcsA channel in the two different electrolyte solutions revealed, however, that the global architecture of the channel, including the content of the secondary structure,19 does not change (Figure 1a and Figure S2, Supporting Information). Locally, the selectivity filter collapses in Na+ solution, and this structural change spreads around the selectivity filter (Figure 1f),3,8 while the content of the secondary structure remains unchanged (Figure 1a). Thus, the most plausible source of the difference comes from the selectivity filter, where the vibrational shifts originate from either the binding of two different ion species with different fashions or the conformational changes (i.e., the open and collapsed conformations). To address the origin of these difference spectra more specifically, we examined the Y78F mutant, in which the tyrosine residue in the selectivity signature sequence (TVGYG) was replaced with phenylalanine, and functional measurements of the channel activity were reported to exhibit similar gating behavior to that of the WT.20 The Y78F mutant gave small but substantial changes in the spectrum. In Figure 3, the K+-minus-Na+ difference FTIR spectra for the WT and Y78F are superimposed (see the Supporting Information, Figures S3 and S4). The 1518 cm−1 band disappeared in the Y78F mutant, indicating that the band originated from Tyr78 of the selectivity filter. In contrast, other amide-II peaks including the negative band at 1509 cm−1 remain intact in the Y78F mutant. Overall, the amide-I signal changed significantly, while the amide-II signal was nearly unaltered except for the band at 1518 cm−1. The changes of amide-I and amide-II should be correlated if these signals originate from the changes in the secondary structure. Thus, the lack of the correlated changes between the amide-I and -II vibrations in the Y78F mutant suggest that the amide-I signal represents the intimate ion and carbonyl interactions in the selectivity filter. The above results have narrowed down to the selectivity filter as the origin of the amide-I signals. In the experimental conditions for the difference spectra, multiple K+ ions bind to the open-filter conformation in the pure K+ solution, while in the pure Na+ solution, the selectivity filter assumes the collapsed conformation, to which Na+ ions are bound. Thus, the K+-minus-Na+ difference FTIR spectra should potentially be accompanied by signals of conformational change in the filter structure and/or the binding of ions on either of these two conformations. To distinguish these possibilities, the difference FTIR spectra were measured under an experimental condition in which the selectivity filter takes the collapsed conformation in two different ionic environments. In fact, the selectivity filter is in the collapsed conformation at low K+ (3 mM; 1K4D) as is the case in the pure Na solutions (Figure 1d,e). Thus, the

Figure 4. (a) The K+-minus-Na+ difference FTIR spectra recorded at pH 7 and 293 K at the different K+ concentrations (3−200 mM) with a constant ionic strength obtained by adding NaCl (total 200 mM). One division of the y axis is a 0.005 absorbance unit. (b) The K+minus-Na+ difference spectra normalized at an amide-II band at 1560 cm−1. (c) The peak-to-peak intensity at 1659 and 1627 cm−1 in (a) (closed circle) and the peak intensity at 1680 cm−1 after the normalization in (b) (open circle) are plotted against the K+ ion concentration with standard error bars from three independent experiments. The data are fitted by Hill equations (red and blue lines). The apparent KD values are estimated to be 9.0 ± 0.7 and 18 ± 1.4 mM, and the Hill coefficients are 1.8 ± 0.2 and 1.4 ± 0.1, respectively.

difference spectra in these electrolyte solutions should exclusively represent the binding of different ion species. In Figure 4, the difference FTIR spectra were measured between the solutions containing the variable K+ concentrations and the pure Na+, and the amide-I spectra are shown. In these series of experiments, the ionic strength was made constant by the addition of Na+. Among the traces, the blue trace of Figure 4a shows the difference spectra between the solutions containing [3 mM K+ + 197 mM Na+] and that of [200 mM Na+]. Addition of 3 mM K+ elicited the small but substantial difference spectrum. This means that the vibrational signal originates from the ion binding to the collapsed conformation, 3808

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and K+ as low as 3 mM could be replaced in the binding site in exchange for Na+, indicating the high-affinity binding of K+. As the K+ concentration increased, the spectral amplitudes of 1669 and 1659 cm−1 were simply augmented until the highest amplitude in [pure K+]/[pure Na+]. In this titration experiment, the selectivity filter undergoes structural changes from the collapsed to the open conformation as the K+ concentration increases, while the filter remains in the collapsed conformation in the pure Na+ solution. We found that the spectral pattern in [3 mM K+ (+ 197 mM Na+)]/[pure (200 mM) Na+] (blue line) was roughly similar to that in [pure K+]/[pure Na+] (red line). Thus, the vibrational signal elicited by the binding to the collapsed conformation at low K+ concentration is shared at high K+ concentration in the open-filter conformation. This result suggests that the amide-I shift mainly originates from the binding of K+ or Na+ ions to the filter irrespective of its conformation. The positive peaks in the amide-I vibrations in the K+ solutions exhibit a higher frequency than the negative peaks in the Na+ solution, especially at the predominant positive and negative peaks at 1659 and 1627 cm−1. These results indicate that K+ interacts weaker with the carbonyl oxygens in the selectivity filter of either the open or collapsed conformation compared to that for Na+. To examine fine changes in the difference spectra for the variable K+ concentrations, the amplitudes of the spectra were normalized, as shown in Figure 4b. The predominant peaks were mostly superimposable, but we found a lower-frequency shift of the positive band from 1688 to 1680 cm−1 as the K+ concentration was increased. What is the origin of this shift? One may think that these signals originate from the changes in the conformation of the selectivity filter; the 1688 cm−1 vibration originates from the collapsed conformation, and the 1680 cm−1 originates from the open-filter conformation. However, this simple interpretation is not valid in the difference spectra. In the [3 mM K+/pure Na+] difference spectra, the selectivity filter takes on the collapsed conformation in both solutions, still exhibiting a distinct positive peak at 1688 cm−1. This result indicates that the 1688 cm−1 peak represents the binding of K+ to the collapsed conformation, and as K+ concentration increases, this signal disappears as the filter turns to the open conformation. At high K+, the binding site with lower affinity appears in the open filter, binding to which site elicits the signal at 1680 cm−1. To characterize the concentration dependency of the FTIR signal, the peak-to-peak amplitude between the negative (1627 cm−1) and positive (1659 cm−1) signals in the amide-I was plotted as a function of the K+ concentration (Figure 4c). The apparent binding affinity (KD) for K+ was 9.0 ± 0.7 mM. This value is comparable to those reported by using the electron density in the crystal structure,4 and the fitting of the data with a function for the competitive binding of Na+ would give the similar KD value for K+. It should be noted that the KD value for Na+ was reported to be much higher than that for K+.8 Furthermore, the peak shift from 1688 to 1680 cm−1 was evaluated. Here, the amplitude of the 1680 cm−1 band was plotted as a function of K+ concentration. The fitted KD value was found to have a slightly higher value (18 ± 1.4 mM) than that of 1659 cm−1. Thus, we conclude that there are at least two vibrational modes for the K+ binding in the selectivity filter, a higher-affinity mode (KD = 9.0 mM) irrespective of the filter conformation and a lower-affinity mode (KD = 18 mM) related to the open conformation.

Crystallographically, four potassium binding sites exist in the open filter, while there are two in the collapsed conformation (Figure 1). They are not equivalent and display different affinities.4 In this study, we found at least two distinct vibrational modes that exhibited the different concentration dependency. At this stage of the present study, assigning the amide-I signals to the specific carbonyl oxygens is not feasible, and the vibrational signals have been related to the binding sites. In the filter structure, the binding of a K+ ion to the outermost and innermost sites (the S1 and S4 sites) is commonly observed for both the high and low K + concentrations, suggesting that S1 and S4 are involved in the high-affinity sites. That is, the 1669 and 1659 cm−1 bands may have originated from the carbonyl groups in the S1 and S4 sites. On the other hand, the 1688 and 1680 cm−1 signals indicate that the former represents K+ binding to the collapsed conformation at low K+ concentrations. The 1680 cm−1 signal that represents K+ binding with a lower affinity to the sites appears upon opening of the filter from the collapsed conformation (the S2 and S3 sites). However, the conclusive assignment of these modes to the specific carbonyl groups needs further ATR-FTIR study with specific isotope-labeled samples and normal-mode analysis. In the observed amide-I signals, the vibrational frequency shifted toward the higher frequency upon binding of K+ relative to that of Na+, indicating that K+ induced the weaker interactions with the carbonyl oxygens in the selectivity filter. What is the origin of these shifts? With the smaller ionic radius of Na+, four carbonyl oxygens surround a Na+ ion in the planar configuration, which may exhibit stronger interaction compared to K+ coordinated with the cage configuration. These different modes of interactions are likely the origin of the different vibrational frequency. It should be noted that the binding affinity is determined by the sum of the interaction energy with either eight carbonyl oxygens for K+ or four carbonyl oxygens for Na+, and these quantitative arguments still remain elusive at this stage. In this study, the K+-minus-Na+ difference FTIR spectra captured the binding of K+ and Na+ in various experimental conditions, and we focused on the origin of the signals. Further ATR-FTIR experiments with other alkali cations or quantum chemical calculations will deepen the understanding of the molecular mechanism of the potassium ion channel in the future.

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EXPERIMENTAL SECTION Sample Preparation of KcsA; Expression, Purif ication, and Reconstitution into Liposomes. The KcsA sample was prepared according to the method described previously.16 The gene of the wild-type KcsA channel was inserted into a pQE-82L vector (QIAGEN, Valencia, CA), which was used to transform E. coli BL21 cells. Protein expression was induced by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside. E. coli cells expressing KcsA channels were broken up by sonication, and the membrane fractions were solubilized in buffer (20 mM potassium phosphate, 200 mM KCl, 20 mM 2-mercaptoethanol, 50 mM imidazole) containing 1% n-dodecyl-β-D-maltoside (DDM; Dojindo, Kumamoto, Japan). Histidine-tagged channels were purified with a Co2+-based metal chelate chromatography resin. Purified channels were eluted by 100−400 mM imidazole at a protein concentration of 0.5−3 mg/mL. The purified sample was mixed with lipid [asolectin] at a molar ratio of 1:50. For reconstitution into lipsomes, DDM was removed 3809

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by size exclusion column chromatography. The protein concentration was ultimately adjusted to 1 mg/mL. Perf usion-Induced Attenuated Total Ref lection (ATR) FTIR Spectroscopy. A 5 μL aliquot of KcsA suspension was placed on the surface of a diamond ATR crystal (Smiths Detection, DurasamplIR II; nine effective internal reflections with 45° of the incident angle). After drying in a gentle stream of N2, the sample was filled by flowing perfusion buffer (20 mM HEPES at pH 7, 200 mM NaCl). Before measuring the ion-exchangeinduced difference spectra, the sample was perfused with the same buffer at a flow rate of 0.5 mL/min for 100 min to remove excess buffer and any impurities concentrated upon drying. ATR-FTIR spectra of the KcsA sample were recorded at 293 K and 2 cm−1 resolution by a Bio-Rad FTS-6000 spectrometer equipped with a liquid-nitrogen-cooled MCT detector.13,14 A background spectrum of the film was first recorded during perfusion with buffer in the presence of 200 mM NaCl for 15 min (an average of 1150 interferograms). The buffer was then switched to one containing x mM KCl and 200 − x mM NaCl (x = 3, 5, 10, 20, 50, 100, and 200), and after a 5 min delay for equilibration, a K+-minus-Na+ difference spectrum (Figure S1a, blue, Supporting Information) was recorded for 15 min (an average of 1150 interferograms). Then, after a new background was taken, the buffer was switched back to that with 200 mM NaCl, and after a 5 min delay, an equivalent Na+-minus-K+ difference spectrum was recorded (Figure S1a, red, Supporting Information). The cycling procedure was repeated six times for each K+ concentration, and the difference spectra were calculated as averages of the K+-minus-Na+ spectra (in total. 6900 scans). It should be emphasized that the calculation of the (K+-minus-Na+) − (Na+-minus-K+) spectra (the red-minusblue spectra in Figure S1, Supporting Information) found significantly reduced vapor bands at 1800−1400 cm−1 and long-term spectral distortion, which appear on either the positive or negative side regardless of the cations present. The experiments were independently repeated three times for each K+ concentration. The flow rate was maintained at 0.5 mL/min.

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REFERENCES

(1) Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates, Inc: Sunderland, MA, 2001. (2) Doyle, D. A.; Morais Cabral, J.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Cohen, S. L.; Chait, B. T.; MacKinnon, R. The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity. Science 1998, 280, 69−77. (3) Zhou, Y.; Morais-Cabral, J. H.; Kaufman, A.; MacKinnon, R. Chemistry of Ion Coordination and Hydration Revealed by a K+ Channel−Fab Complex at 2.0 Å Resolution. Nature 2001, 414, 43−48. (4) Morais-Cabral, J. H.; Zhou, Y.; MacKinnon, R. Energetic Optimization of Ion Conduction Rate by the K+ Selectivity Filter. Nature 2001, 414, 37−42. (5) Thompson, A. N.; Kim, I.; Panosian, T. D.; Iverson, T. M.; Allen, T. W.; Nimigean, C. M. Mechanism of Potassium-Channel Selectivity Revealed by Na+ and Li+ Binding Sites within the KcsA Pore. Nat. Struct. Mol. Biol. 2009, 16, 1317−1324. (6) LeMasurier, M.; Heginbotham, L.; Miller, C. KcsA: It’s a Potassium Channel. J. Gen. Physiol. 2001, 118, 303−314. (7) Eisenman, G.; Latorre, R.; Miller, C. Multi-Ion Conduction and Selectivity in the High-Conductance Ca+2-Activated K+ Channel from Skeletal Muscle. Biophys. J. 1986, 50, 1025−1034. (8) Lockless, S. W.; Zhou, M.; MacKinnon, R. Structural and Thermodynamic Properties of Selective Ion Binding in a K+ Channel. PLoS Biol. 2007, 5, e121. (9) Iwamoto, M.; Oiki, S. Counting Ion and Water Molecules in a Streaming File Through the Open-Filter Structure of the K Channel. J. Neurosci. 2011, 31, 12180−12188. (10) Berneche, S.; Roux, B. A Microscopic View of Ion Conduction Through the K+ Channel. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8644−8648. (11) Jensen, M. O.; Borhani, D. W.; Lindorff-Larsen, K.; Maragakis, P.; Jogini, V.; Eastwood, M. P.; Dror, R. O.; Shaw, D. E. Principles of Conduction and Hydrophobic Gating in K+ Channels. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5833−5838. (12) Rich, P. R.; Iwaki, M. Methods to Probe Protein Transitions with ATR Infrared Spectroscopy. Mol. Biosyst. 2007, 3, 398−407. (13) Kitade, Y.; Furutani, Y.; Kamo, N.; Kandori, H. Proton Release Group of pharaonis Phoborhodopsin Revealed by ATR-FTIR Spectroscopy. Biochemistry 2009, 48, 1595−1603. (14) Furutani, Y.; Murata, T.; Kandori, H. Sodium or Lithium IonBinding-Induced Structural Changes in the K-Ring of V-ATPase from Enterococcus hirae Revealed by ATR-FTIR Spectroscopy. J. Am. Chem. Soc. 2011, 133, 2860−2863. (15) Nyquist, R. M.; Ataka, K.; Heberle, J. The Molecular Mechanism of Membrane Proteins Probed by Evanescent Infrared Waves. ChemBioChem 2004, 5, 431−436. (16) Iwamoto, M.; Shimizu, H.; Inoue, F.; Konno, T.; Sasaki, Y. C.; Oiki, S. Surface Structure and Its Dynamic Rearrangements of the KcsA Potassium Channel upon Gating and Tetrabutylammonium Blocking. J. Biol. Chem. 2006, 281, 28379−28386. (17) Barth, A. The Infrared Absorption of Amino Acid Side Chains. Prog. Biophys. Mol. Biol. 2000, 74, 141−173. (18) Bandekar, J. Amide Modes and Protein Conformation. Biochim. Biophys. Acta 1992, 1120, 123−43. (19) Le Coutre, J.; Kaback, H. R.; Patel, C. K. N.; Heginbotham, L.; Miller, C. Fourier Transform Infrared Spectroscopy Reveals a Rigid αHelical Assembly for the Tetrameric Streptomyces lividans K+ Channel. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6114−6117. (20) Splitt, H.; Meuser, D.; Borovok, I.; Betzler, M.; Schrempf, H. Pore Mutations Affecting Tetrameric Assembly and Functioning of the Potassium Channel KcsA from Streptomyces lividans. FEBS Lett. 2000, 472, 83−87.

ASSOCIATED CONTENT

S Supporting Information *

Figures S1, S2, S3, and S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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Letter

AUTHOR INFORMATION

Corresponding Author

*Phone and Fax: 81-52-735-5207. E-mail: [email protected] (H.K.); Phone: 81-776-61-8306. Fax: 81-776-61-8101. E-mail: [email protected] (S.O.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Masayuki Iwamoto and Takashi Sumikama for valuable discussions. This work was partly supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) or the Japan Society for the Promotion of Science (JSPS) to H.K. (22247024, 20108014), Y.F. (22770159, 22018030, 21026016), H.S. (22121507, 22018007, 20050010, 20679002), and S.O. (23370067, 21107508, 21657038, 20247016). We thank Pacific Edit for reviewing the manuscript prior to submission. 3810

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