Dynamic AFM of Patch Clamped Membranes
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
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