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Droplet Interface Bilayers for Mechano-Electrical Transduction Featuring Bacterial MscL ChannelsNajem, Joseph Samih 02 December 2015 (has links)
This dissertation investigates the behavior of the Escherichia Coli mechanosensitive (MS) channel MscL, when incorporated within a droplet interface bilayer (DIB). The activity of MscL channels in an artificial DIB system is demonstrated for the first time in this document. The DIB represents a building block whose repetition can form the basis to a new class of smart materials. The corresponding stimuli-responsive properties can be controlled by the type of biomolecule incorporated into the lipid bilayer, which is in the heart of this material. In the past decade, many research groups have proven the capability of the DIB to host a wide collection of natural and engineered functional biomolecules. However, very little is known about the mechano-electrical transduction capabilities of the DIB. The research present herein specifically seeks to achieve three direct goals: 1) exploring the capabilities of the DIB to serve as a platform for mechano-electrical transduction through the incorporation of bacterial MscL channels, 2) understanding the physics of mechano-electrical transduction in the DIB through the development of theoretical models, and 3) using the developed science to regulate the response of the DIB to a mechanical stimulus.
MscL channels, widely known as osmolyte release valves and fundamental elements of the bacterial cytoplasmic membrane, react to increased tension in the membrane. In the event of hypo-osmotic shocks, several channels residing in the membrane of a small cell can generate a massive permeability response to quickly release ions and small molecules, saving bacteria from lysis. Biophysically, MscL is well studied and characterized primarily through the prominent patch clamp technique. Reliable structural models explaining MscL's gating mechanism are proposed based on its homolog's crystal structure modeling, which lead to extensive experimentation. Under an applied tension of ~10 mN/m, the closed channel which consists of a tight bundle of transmembrane helices, transforms into a ring of greatly tilted helices forming an ~8 A water-filled conductive pore. It has also been established that the hydrophobicity of the tight gate, positioned at the intersection of the inner TM1 domains, determines the activation threshold of the channel. Correspondingly, it was found that by decreasing the hydrophobicity of the gate, the tension threshold could be lowered. This property of MscL made possible the design of various controllable valves, primarily for drug delivery purposes. For all the aforementioned properties and based on its fundamental role of translating cell membrane excessive tensions into electrophysiological activities, MscL makes a great fit as a mechanoelectrical transducer in DIBs.
The approach presented in this document consists of increasing the tension in the lipid bilayer interface through the application of a dynamic mechanical stimulus. Therefore, a novel and simple experimental apparatus is assembled on an inverted microscope, consisting of two micropipettes (filled with PEG-DMA hydrogel) containing Ag/AgCl wires, a cylindrical oil reservoir glued on top of a thin acrylic sheet, and a piezoelectric oscillator actuator. By using this technique, dynamic tension can be applied by oscillating one droplet, producing deformation of both droplets and area changes of the DIB interface. The tension in the artificial membrane will cause the MS channels to gate, resulting in an increase in the conductance levels of the membrane. The increase in bilayer tension is found to be equal to the sum of increase in tensions in both contributing monolayers. Tension increase in the monolayers occurs due to an increase in surface area of the constant volume aqueous droplets supporting the bilayer.
The results show that MS channels are able to gate under an applied dynamic tension. Interestingly, this work has demonstrated that both electrical potential and surface tension need to be controlled to initiate mechanoelectric coupling, a property previously not known for ion channels of this type. Gating events occur consistently at the peak compression, where the tension in the bilayer is maximal. In addition, the experiments show that no activity occurred at low amplitude oscillations (< 62.5um). These two findings basically present an initial proof that gating is occurring and is due to the mechanical excitation, not just a random artifact. The role of the applied potential is also highlighted in this study, where the results show that no gating happens at potentials lower that 80 mV. The third important observation is that the frequency of oscillation has an important impact of the gating probability, where no gating is seen at frequencies higher than 1 Hz or lower than 0.1 Hz.
Each of the previous observations is addressed separately in this research. It was found that the range of frequencies to which MscL would respond to in a DIB could be widened by using asymmetrical sinusoidal signals to stimulate the droplets. By increasing the relaxation time and shorting the compression time, a change in the monolayer's surface area is achieved, thus higher tension increase in the bilayer. It was also found that a high membrane potential assists in the opening of MscL as the droplets are stimulated. This is due to the sensitivity of MscL to the polarity of the signal. By using the right polarity the channel could be regulated to become more susceptible to opening, even at tensions lower than the threshold.
Finally, it was demonstrated, for the first time, that MscL would gate in asymmetric bilayers without the need to apply a high external potential. Asymmetric bilayers, which are usually composed from different lipids in each leaflet, generate an asymmetric potential at the membrane. This asymmetric potential is proven to be enough to cause MscL to gate in DIBs upon stimulation. / Ph. D.
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The pharmacology of the mechanosensitive channels of Escherichia coliNguyen, Thom Ngoc Minh January 2007 (has links)
Mechanosensitive (MS) channels are a class of ion channels which are gated by membrane stretch. The mechanosensitive channel of large conductance (MscL) of the bacterium E.coli has become a prototype MS channel for studying structure-function relationships in this class of ion channels. MscL homologues have commonly been found in Gram-negative and Gram-positive bacterial strains forming a sub-family of a larger family of MS class of ion channels encompassing prokaryotes (bacteria and archaea) as well as cell-walled eukaryotes (fungi and plants). MscL and its homologues have been found to play an important role in osmoregulation of bacterial cells. Though the MS channels of bacteria have been thoroughly studied, little is known about the pharmacology of these channels. This thesis has one general aim, that is, to identify compounds which are able to gate and/or alter the gating of the MS channels of bacteria in particular, the MscL of E. coli. Using the patch-clamp technique, potential compounds mostly identified via in-silico techniques were examined to observe the effects on MscL reconstituted in artificial lipid membranes and MscS in giant bacterial spheroplasts. The compounds were tested for the ability to spontaneously gate the MscL and MscS and/or alter the Boltzmann distribution parameters of the MscL, indicative of an effect on the gating of MscL. Compounds showing potential as MscL activators were then examined for in-vivo effects using different growth assays. The effects of parabens, gallates, eriochrome cyanine R, brilliant green, deoxycholic acid are reported. Of these compounds, parabens and eriochrome cyanine R showed the most encouraging results. Identification of MS channel gate ligands not only benefits structural studies as tetrodotoxin has for the voltage-sensitive sodium channel, these compounds could also V potentially serve as base compounds for novel antibiotics which would target the MS channels of bacteria. Since the MS channels of bacteria serve as safety valves for the bacterium, gating during exposure to a hypo-osmotic challenge such as rain to release excessive cellular turgor, a pharmacological agent that could impair the gating of the MS channels releasing essential cytoplasmic osmolytes, would cause the growth impairment or death of the bacterium. With the rise in multi-drug resistant bacteria, continual development of novel antibiotics is crucial.
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