Dynamic AFM of Patch Clamped Membranes
- 482 Downloads
Table 1 Summary of Patch-Clamp Scanning Force Microscope Applications
Identification and classification of all types of ion channels based on biophysical properties Whole cell measurements allow clarification of the relative importance and physiological role of a specific type of ion channel in a given cell type. Capacitance measurements yield current density and assessment of endocytotic and exocytotic activity Instantaneous manipulation of membrane voltage or current.
The number of channels in a patch or whole cell is not known. Response time of pressure pulses is limited. Local variation of membrane tension during pressure steps is unknown. Natural membrane structure is altered during patch formation. Lack of consistent sealing protocols might cause variability in biophysical parameters. Mechanical properties estimated using micropipet aspiration are confounded by the mechanics of patch adhesion.
High-resolution topographic images of biological samples (membranes and proteins). Images of ion channels yield important substructure (subunits, pore diameter, binding sites). Dynamic observation (directly image conformational changes). Ability to apply small forces and make elasticity measurements. Ability to chemically modify the tip and make images or force spectroscopy. Use as a nanotool to cut, push, or pull in a controlled way.
Low lateral resolution when imaging unsupported biological samples. Difficulty locating object of interest within bilayers or on cell surface. Undefined contact area between tip and sample (pressure) when doing elasticity measurements. Can be overcome by using linker molecules of known stiffness to pull on supported membranes. Limited understanding of tip sample interactions.
Table 1 (contd.) Summary of Patch-Clamp Scanning Force Microscope Applications
Use of micropipet as support for imaging of biological membranes. Comparison of independent measures of channel density and surface area (electrical and topographic estimates). Correlation of dynamic topological detail of ion channels with ionic currents (i.e., correlate conductance with number of subunits, pore size, conformational changes, and binding events). Use of patch-clamp to identify channels of interest for AFM imaging. Clarification of patch morphology by imaging substructure on inner or outer membranes of patch. Tool of choice for the study of mechanoelectric transduction (i.e., application of force directly to MSC while monitoring channel conductance). Tool of choice for studying electromechanical transduction (i.e., flexoelectricity, OHC electromotility) Estimates of membrane mechanics using well-defined probe sample adhesion points (especially if using linkers to specific proteins in the membrane).
KeywordsAtomic Force Microscopy Contact Force Atomic Force Microscopy Image Lateral Resolution Patch Pipette
- 1.Hille, B. (1992) Ionic Channels of Excitable Membranes. Sinauer Associates Inc., Sunderland, MA.Google Scholar
- 7.Besch, S. D. and Sachs, F. (2000) A Compact, High Speed, Air Pressure Servo. In press.Google Scholar
- 8.Mcbride, D. W. and Hamill, O. P. (1995) A fast pressure-clamp technique for studying mechanogated channels, in Single-Channel Recording (Sakmann, B. and Neher, E., eds.), Plenum, New York, pp. 329–340.Google Scholar
- 9.Martinac, B. (1993) Mechanosensitive ion channels: biophysics and physiology. Thermodynamics of Cell Surface Receptors (Jackson, M., ed.), CRC Press, Boca Raton, FL, pp. 327–351.Google Scholar
- 10.Sachs, F. (1992) Stretch sensitive ion channels: an update, in Sensory Transduction (Corey, D. P. and Roper, S. D., eds.), Rockefeller University Press, Society General Physiology, NY, pp. 241–260.Google Scholar
- 12.Sachs, F. and Morris, C. (1998) Mechanosensitive ion channels in non specialized cells, in Reviews of Physiology and Biochemistry and Pharmacology (Blaustein, M. P., et al., eds.), Springer, Berlin, pp. 1–78.Google Scholar
- 13.Sokabe, M. and Sachs, F. (1992) Towards a molecular mechanism of activation in mechanosensitive ion channels, in Advances in Comparative and Environmental Physiology, vol. 10 (Ito, F., ed.), Springer-Verlag, Berlin, pp. 55–77.Google Scholar
- 16.Guharay, F. and Sachs, F. (1984) Stretch-activated single ion channel currents in tissue-cultured embryonic chick skeletal muscle. J. Physiol. (Lond.) 352, 685–701.Google Scholar
- 31.Horber, J. K., Haberle, W., Ohnesorge, F., Binnig, G., Liebich, H. G., Czerny, C. P., et al. (1992) Investigation of living cells in the nanometer regime with the scanning force microscope. Scan. Microsc. 6, 919–929.Google Scholar
- 36.Fritzsche, W., Takac, L., and Henderson, E. (1997) Application of atomic force microscopy to visualization of DNA, chromatin, and chromosomes. [Review] [65 refs]. Crit. Rev. Eukaryotic Gene Exp. 7, 231–240.Google Scholar
- 42.Lal, R. (1996) Imaging molecular structure of channels and receptors with an atomic force microscope. Scan. Microsc. 10(Suppl.), 81–95.Google Scholar
- 73.Horber, J. K., Mosbacher, J., and Haberle, W. (1995) Force microscopy on membrane patches, in Single-Channel Recording (Sakmann, B. and Neher, E., eds.), Plenum, New York, pp. 375–393.Google Scholar
- 76.McEwen, B. F., Song, M. J., Ruknudin, A., Barnard, D. P., Frank, J., and Sachs, F. (1990) Tomographic three dimensional reconstruction of patch clamped membranes imaged with the high voltage electron microscope. XII Intl. Cong. El. Microsc. 522–523 (Abstract).Google Scholar
- 86.Petrov, A. G. (1999) Lyotropic State of Matter: Molecular Physics and Living Matter Physics. Gordon & Breach Publishing Group, Amsterdam.Google Scholar
- 94.Snyder, K., Besch, S. D., Zhang, P. C., and Sachs, F. (2000) Hardware and software modifications of the Quesant AFM for use in biology. In press.Google Scholar
- 97.Beyder, A., Snyder, K. V., and Sachs, F. (2000) New AFM cantilever for low force applications in liquids. In press.Google Scholar
- 98.Ashmore, J. F. (1987) A fast motile response in guinea-pig outer hair cells: the cellular basis of the cochlear amplifier. J. Physiol. (Lond.) 388, 323–347.Google Scholar