The long term goal of this project is to understand the molecular mechanisms underlying the fundamental physiological process of mechano-electrical transduction. The main focus of the proposal is on the dynamic properties (i.e., latency of response, turn on, turn off and adaptation) of MS membrane ion channels. These properties not only relate to the varied physiological roles of this channel, but also provide insight into the underlying gating mechanism. Two broad classes of MS gating mechanisms have been recognized; namely, direct and indirect. This classification is made according to how mechanical energy is coupled to the gating mechanism. The pressure-clamp/patch- clamp technique provides the opportunity to carry out rapid relaxation/perturbation measurements necessary to determine such dynamic properties as response latencies and thereby provide one means to distinguish between the two classes of mechanisms. A major experimental effort will be to characterize and compare the dynamic properties of the MS cation nonselective channel in Xenopus oocytes and the MS K+ selective channel in snail neurons. The investigators will determine if these channels are representative of the two classes. MS channels have also been classified as stretch activated (SA) and stretch inactivated (SI) according to whether mechanical gating opens or closes the channel. However, a complication in this classification is that SA channels also adapt (i.e., appear to inactivate) in the presence of maintained mechanical stimulation.
One aim of this proposal will be to determine whether a common mechanism can explain the apparently opposite gating of SA and SI channels. Another aim of this proposal is to identify the role of the cytoskeleton in MS channel gating and the implication that this has in regard to patch-clamp recording. This aspect will be studied by investigating the effects of specific cytoskeletal protein disrupting and stabilizing agents on dynamic MS channel properties. The possible role of the cytoskeletal protein dystrophin in MS channel gating will be determined by comparing results obtained from control and dystrophic (mdx) mouse muscle. To resolve a recent controversy concerning the significance of MS channels measured in snail neurons with patch clamp, the relationship between MS channel currents measured in the patch and whole cell MS currents will be determined to test whether MS channels underlie whole cell MS responses. The results of this study will provide insight into the physiological and possible pathological roles of MS channels in cell function.
