Morris, Cathy,Gu, Cicely Xi.2009-03-232009-03-2319991999Source: Dissertation Abstracts International, Volume: 61-04, Section: B, page: 1822.9780612481008http://hdl.handle.net/10393/8806http://dx.doi.org/10.20381/ruor-16002Mechanosensitive (MS) channels are those whose open probability (P open) changes in response to mechanical stimulation. Principally, there are two types of mechanical effects on the channel activity, stretch-activation and stretch-inactivation. Mechano-responses can be detected with patch-clamp recordings in cells from bacteria to human, indicating the ubiquitous distribution of MS channels. Mechanosensitivity has recently been shown to be associated with a variety of unrelated channel types, suggesting that susceptibility to membrane tension could be an inherent property of many integral membrane proteins. In this thesis, I tested the hypothesis that an arbitrarily chosen channel with no known mechanotransducer function could be mechanosusceptible under conditions when (1) studied by patch-clamp recordings and (2) heterologously expressed where the protective machinery of its native environment is absent. In order to test this hypothesis, a simple well characterized voltage-dependent K+ channel, Shaker-Inactivation-Removed (Shaker-IR) was expressed in Xenopus oocytes in which the endogenous MS channels were blocked by Gd3+ (100 muM GdCl3) to facilitate the observation of mechano-response of Shaker-IR. Characteristic conductance-voltage (G-V) curves were obtained for Shaker-IR by two-electrode voltage clamp recordings, although Gd3+ shifted the voltage gating by 23 mV. Shaker-IR currents (ISh) were studied in cell-attached and inside-out excised patches with or without mechanical stimulations. In macropatches, various mechanical effects on Ish were recorded at different activation levels (i.e. at different membrane potentials). At the foot of the G-V curves where Popen was low, stretch-activation dominated the MS Shaker-IR channel activity. At the intermediate range of the curves, only minor stretch effects were detectable. At the top of the curves where Popen was high, sustained stretch frequently caused stretch-inactivation. In small patches, stretch-activation and stretch-inactivation were evident as single channel events. Single channel recordings revealed that at a given membrane potential, Popen changed steeply with the applied pressure, in a dose-dependent manner. As in macro-patch recordings, both stretch-activation and stretch-inactivation were induced in the same patch, depending on the pre-stretch Popen. Before reaching a conclusion that the observed mechanical effects were indicative of the mechanosusceptibility of a voltage-dependent channel, it was necessary to rule out the possibility that these effects might be caused by stretch-induced changes in membrane area or by "breakthrough currents" from the oocytes' endogenous MS channels. These possibilities were excluded by the following arguments: (1) Mechanical stimuli produced no apparent increase in capacitance of the patched-membrane, while the increase of the channel activity upon stretch was often one to two orders of magnitude. (2) Stretch-inactivation (not expected from increased area of the recording patch) occurred and could be observed in the same patch that showed stretch-activation. (3) Both the activation and inactivation could be elicited by either suction or blowing. (4) None of these responses were observed in no-Shaker-IR control oocytes. (5) The effects of mechanical stretch depended on the membrane potential, i.e. on the pre-stretch Popen of the Shaker-IR channel. In conclusion, membrane stretch can interfere with the voltage gating of Shaker-IR, consistent with the assumption that mechanosusceptibility may be an inherent property of many integral membrane proteins. Since mechanosusceptibility is easy to demonstrate, it might play a role in the physiology of cells by (a) modifying the activity of ion channels and (b) creating a need for mechanoprotection for channels when they are not used to monitor bilayer tension.252 p.Biology, Cell.Thesis