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of end the protein. We observed a substantial decrease in Rm when the antibody was applied to the ROMK1-coated tip. This seems to indicate that anti-ROMK1 interferes with a func- Physiology: Oberleithner et al. Proc. Natl. Acad. Sci. USA 94 (1997) 14149 ning electron microscopy on AFM tips. We gratefully acknowledge the continuous support of Profs. Gerhard Giebisch and Stefan Silbernagl during the course of these experiments. We thank Drs. D. Hornby and S. White from Sheffield University for providing the purified ROMK1 channel protein. We are grateful to Mrs. A. Rakowska for preparing Fig. 1. The study was supported by the Deutsche Forschungsgemeinschaft, SFB 176 (A6). 1. 2. 3. FIG. 6. Single tracing of the stochastically occurring fluctuations in apparent molecule height measured with MOST. Upward deflections indicate molecule expansion, downward deflections indicate molecule contraction in reference to the baseline ( zero line). The AFM tip was coated with the ROMK1 protein. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Pflugers Arch. 391, 85 100. Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V. & Hebert, S. C. (1993) Nature (London) 362, 31 38. Wang, W., Schwab, A. & Giebisch, G. (1990) Am. J. Physiol. 259, F494 F502. Henderson, R. M., Schneider, S., Li, Q., Hornby, D., White, S. J. & Oberleithner, H. (1996) Proc. Natl. Acad. Sci. USA 93, 8756 8760. Wang, W. & Giebisch, G. (1991) Proc. Natl. Acad. Sci. USA 88, 9722 9725. McNicholas, C. M., Wang, W., Ho, K., Hebert, S. C. & Giebisch, G. (1994) Proc. Natl. Acad. Sci. USA 91, 8077 8081. Wang, W. & Giebisch, G. (1991) J. Gen. Physiol. 98, 35 61. Macica, C. M., Yang, Y., Hebert, S. C. & Wang, W. H. (1996) Am. J. Physiol. 271, F588 F594. Florin, E.-L., Moy, V. T. & Gaub, H. E. (1994) Science 264, 415 417. Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K. & Schindler, H. (1996) Proc. Natl. Acad. Sci. USA 93, 3477 3481. Radmacher, M., Fritz, M., Hansma, H. G. & Hansma, P. K. (1994) Science 265, 1577 1579. Thomson, N. H., Fritz, M., Radmacher, M., Cleveland, J. P., Schmidt, C. F. & Hansma, P. K. (1996) Biophys. J. 70(5), 2421 2431. Radmacher, M., Cleveland, J. P., Fritz, M., Hansma, H. G. & Hansma, P. K. (1994) Biophys. J. 66, 2159 2165. Lee, W.-S. & Hebert, S. C. (1995) Am. J. Physiol. 268, F1124 F1131. Boim, M. Ho, A., K., Shuck, M. E., Bienkowski, M. J., Block, J. H., Slightom, J. L., Yang, Y., Brenner, B. M. & Hebert, S. C. (1995) Am. J. Physiol. 268, F1132 F1140. Bleich, M., Schlatter, E. & Greger, R. (1990) Pflugers Arch. 415, 449 460. Hurst, A. M. & Hunter, M. (1989) Pflugers Arch. 415, 115 117. Oberleithner, H., Kersting, U. & Hunter, M. (1988) Proc. Natl. Acad. Sci. USA 85, 8345 8349. Fakler, B., Schultz, J. H., Yang, J., Schulte, U., Brandle, U., Zenner, H. P., Jan, L. Y. & Ruppersberg, J. P. (1996) EMBO J. 15, 4093 4099. Coulter, K. L., Perier, F., Radeke, C. M. & Vandenberg, C. A. (1995) Neuron 15, 1157 1168. Schneider, S., Folprecht, G., Krohne, G. & Oberleithner, H. (1995) Pflugers Arch. 430, 795 801. Oberleithner, H., Schneider, S. & Bustamante, J. O. (1996) Pflugers Arch. 432, 839 844. tionally important amino acid sequence at the C-terminal end of the K channel. The AFM recordings of the height f luctuations with ROMK1-coated tips suggest that the protein mounted to the AFM tip stochastically expands and contracts. A representative example of this structural activity of a ROMK1 protein is shown in Fig. 6. We assume that a single ROMK1 molecule is approximately a sphere with a diameter of 5 nm (this estimation is based on 40 kDa, the molecular mass of the ROMK1 protein, and 70 nm3, the respective volume, calculated as described (4, 21, 22). The individual fluctuations in height observed with MOST were maximally 0.5 nm. Thus, the structural changes occurring stochastically while the ROMK1 protein is active are about 10% of the total protein height. The minimal height change we can detect with our AFM set-up, taking into account background noise, is about 0.2 nm, which is about 4% of the total height of a single ROMK1 protein. The resolution in time of the structural activity of the ROMK1 protein was about 1 msec; i.e., the protein height was measured once per msec. Such a resolution in time was recently used for observing conformational changes of lysozyme molecules (11). The perspective of measuring structural changes of a membrane protein is the millisecond range will possibly allow the direct correlation of the function of an ion channel or any other membrane protein, analyzed by electrophysiological techniques, and the structural change of the respective protein, analyzed by MOST. We thank Prof. Georg Krohne (Division of Electron Microscopy, Theodor Boveri Institute, University Wurzburg) for performing scan

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