The long-range goal of this work is to understand how cells generate and transmit signals from mechanical and electrical inputs. More specifically, this proposal examines the properties of ion channels using simultaneous atomic force microscopy (AFM), patch clamp electrophysiology and Ca2+ imaging. The proposal also deals with method developments in the above areas and in nanofabrication to expand the capabilities of the combined technique. The coupling of electrical and mechanical forces will be characterized in cell membranes and in specific ion channels. Mechanically gated channels. The activation of mechanosensitive ion channels is brought about by changes in membrane tension. In eukaryotic cells, the forces are distributed between the extracellular matrix, the bilayer, and the cytoskeleton. These systems display time-dependent relaxations that obscure the force that activates the channels. The atomic force microscope makes a fine mechanical stimulator capable of producing known forces over known distances at microscopic dimensions. When the AFM is combined with a patch-clamp, it is possible to correlate the cell's mechanical and electrical properties. Time dependent point scans will characterize stress relaxation rates, force-volume images will be created using contrast derived from compliance, mechanical transduction currents and changes in local Ca2+. To define physical properties relevant for transduction, stimulation stress, strain and velocity are varied. To establish the role of particular extracellular matrix elements, cantilevers with specific ligands are used to pull in known directions on these elements with the AFM. Experiments will test whether local deformation changes bilayer tension or whether it behaves as a global fluid. Other experiments will measure how global stress of osmotic swelling influences these properties. Cloned mechanosensitive channels with reactive groups will be individually linked to the cantilever and the channel gating measured as a function of local strain. Voltage gated channels. The estimates of S4 movement are well within the resolution of the AFM. The AFM cantilever will be connected to cloned, cysteine mutant, K+ channels that with linkers bearing maleimide or other sulfhydryl reagents. Using gating current protocols, experiments will measure the distance that different sites in the channel can move. The movement will be correlated with gating currents and channel opening. The equivalence between the electrical forces and the mechanical forces will be measured by comparing the distances moved as a function of voltage and mechanical force. The local electrical field around S4 will be measured by the electrically generated force as a function of the valence at known positions of S4.
| Spagnoli, Chiara; Beyder, Arthur; Besch, Stephen et al. (2008) Atomic force microscopy analysis of cell volume regulation. Phys Rev E Stat Nonlin Soft Matter Phys 78:031916 |
