No abstract

Acid (4), and a role for ASIC3 has been shown in pseudo-chronic hyperalgesia induced by repeated acid injections in muscle (5). In the central nervous system ASIC1a is the most abundant and most ubiquitously expressed of all ASICs. Evidence exists for a role of ASIC1a in modulating synaptic transmission, memory, and fear conditioning (6, 7). Recently it was shown that ASIC1a mediates cell injury induced by conditions associated with acidosis in the mammalian nervous system (8). On the cellular level, we and others have shown that ASIC activity modulates action potential signaling (9, 10). The ASICs themselves are targets of various regulatory mechanisms, one of them being serine proteases such as trypsin. Extracellular trypsin can cleave ASIC1a in the extracellular loop and thereby shift the pH dependence of channel activation and inactivation to more acidic pH (11). This regulation may adapt the ASIC1a pH dependence to situations in which the extracellular pH is constitutively lowered, as e.g. ischemia. In addition, the trypsin-modified ASIC1a channel shows a reduced permeability toward divalent cations (12).Identification of a protease cleavage site on ASIC1a would be a starting point for the search for endogenous proteases related to trypsin that may regulate ASIC function in vivo. With regard to ASIC function, the mechanism by which protons control ASIC gating is currently unknown. Knowledge of channel parts involved would make it possible to test hypotheses about gating mechanisms. The observation that trypsin cleaves ASIC1a in the extracellular loop and induces significant functional changes suggests that cleavage occurs at a functionally important site of the channel protein. The goal of this study was therefore first to verify that the cleavage event and the functional changes induced by trypsin are correlated and second to identify the cleavage site(s). The data show a correlation between cleavage and functional effect in ASIC1. The mutagenesis approach showed that trypsin cleaves ASIC1a in the N-terminal part of the extracellular loop, between a highly conserved domain and a channel domain that is critical for ASIC1a inhibition by the venom of the spider Psalmopoeus cambridgei. This channel portion is important for channel inactivation properties and undergoes conformational changes during channel inactivation. EXPERIMENTAL PROCEDURES Construction of Chimeras, Site-directed Mutagenesis, and Expression in Xenopus oocytes-TheASIC1a clone used in this study corresponds to expressed sequence tag clone Image 6849487 (National Institutes of Health) and was entirely sequenced. Its sequence is identical to the published mouse ASIC1a sequence BC067025. On the amino acid level its sequence is identical to the rat ASIC1a sequence and downstream of Val-186 to that of rat ASIC1b, except for a S476N substitution in the intracellular C terminus. Other cDNA constructs were kindly provided by M. Lazdunski (rat ASIC3) and S. Gründer (rat ASIC1b).Chimeras were made with ASIC1a and either ASIC1b or ASIC3. To make the cons...

Force spectroscopy has been used to measure the adhesion of Saos-2 cells to a glass surface at different phases of the cell cycle. The cells were synchronized in three phases of the cell cycle: G 1 , S, and G 2 M. Cells in these phases were compared with unsynchronized and native mitotic cells. Individual cells were attached to an atomic force microscope cantilever, brought into brief contact with the glass surface, and then pulled off again. The force-distance curves obtained allowed the work and maximum force of detachment as well as the number, amplitude, and position of discrete unbinding steps to be determined. A statistical analysis of the data showed that the number of binding proteins or protein complexes present at the cell surface and their binding properties remain similar throughout the cell cycle. This, despite the huge changes in cell morphology and adhesion that occur as the cells enter mitosis. These changes are rather associated with the changes in cytoskeletal organization, which can be quantified by force spectroscopy as changes in cell stiffness.

No abstract

The use of force spectroscopy to study the adhesion of living fibroblasts to their culture substrate was investigated. Both primary fibroblasts (PEMF) and a continuous cell line (3T3) were studied on quartz surfaces. Using a fibronectin-coated AFM cantilever, it was possible to detach a large proportion of the 3T3 cells from the quartz surfaces. Their adhesion to the quartz surface and the effects of topography on this adhesion could be quantified. Three parameters characteristic of the adhesion were measured: the maximum force of detachment, the work of adhesion, and the distance of detachment. Few PEMF cells were detached under the same experimental conditions. The potential and limitations of this method in measuring cell/surface interactions for adherent cells are discussed.