Summary
Mechanosensitive ion channels play an important role for the perception of mechanical signals such as touch, balance, or sound. Here, a new experimental strategy is presented providing well-defined access to single mechanosensitive ion channels in living cells. As a representative example, the investigation of mechanosensitive transduction channels in cochlear hair cells is discussed in detail including all essential technical aspects. Three different techniques were combined: atomic force microscopy (AFM) as a device for local mechanical stimulation, patch clamp for recording the current response of mechanosensitive ion channels, and differential interference contrast (DIC) microscopy equipped with an upright water-immersion objective lens. A major challenge was to adapt the mechanical design of the AFM setup to the small working distance of the light microscope and the electrical design of the AFM electronics. Various protocols for the preparation and investigation of the organ of Corti with AFM are presented.
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Acknowledgments
I would like to thank S. Fink for providing Fig. 9, H.P. Zenner and F. Lehmann-Horn for providing laboratory space and equipment, K. Loeffler for technical assistance, J.K.H. Hoerber for supporting the development of the AFM/patch clamp setup, F. Grauvogel for providing Figs. 4 and 5, and A. Koitschev of the Department of Otolaryngology of the University of Tuebingen for proofreading the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to M.G. Langer (LA1227/1-3) and the Bundesministerium fûr Bildung und Forschung (BMBF) to M.G. Langer (“Bio-AFM”; 0312017A).
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Langer, M.G. (2007). Mechanosensitive Ion Channels Investigated Simultaneously by Scanning Probe Microscopy and Patch Clamp. In: Molnar, P., Hickman, J.J. (eds) Patch-Clamp Methods and Protocols. Methods in Molecular Biology™, vol 403. Humana Press. https://doi.org/10.1007/978-1-59745-529-9_9
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DOI: https://doi.org/10.1007/978-1-59745-529-9_9
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