Novel Regulatory Pathways of Protein Channels

Since the proposal of the fluid mosaic model of a cell membrane, substantial scientific evidence has shown that the cell membrane is not simply an inert structure with the sole role of separating two chemically different environments. The cell membrane dynamically satisfies basic needs, such as water, ion and nutrient transport, without which the cell could not survive. It is a structure which actively participates in a great variety of physiological functions. The activity of the cell membrane is responsible for the contraction of our muscles and information processing in our brain. In order to participate in such a wide range of biological processes, the cell membrane incorporates an extensive variety of protein transporters in its structure. These transporters are highly regulated and contribute to the selective barrier function of the membrane. It is this regulation that enables certain complex physiological functions. The mechanisms of regulation of membrane transporters are obvious in the case of ion channels, which are transmembrane protein transporters facilitating controlled transport of specific ions across the membrane. Their regulation is mediated by specific physical or chemical stimuli, of which voltage, ligands, temperature, light and pressure are most common. However, recent reports indicate that regulation of such transporters may also be achieved by other environmental factors which are not easy to identify in the complex biochemical environment of the cell. Understanding these novel environmental factors and how they modulate the transport across membranes may be a crucial step to better understand the functionality of transmembrane transporters in health and disease. In this respect, the work presented here employs a highly regulated transmembrane transporter, lysenin, which is a pore-forming toxin extracted from red earthworms. Lysenin shares many of the fundamental features of ion channels, such as voltage and ligand regulation. In addition to these features, lysenin accumulates in lipid rafts (which are ubiquitous in animal cells). This model transporter offers opportunities to investigate novel regulatory pathways that are otherwise very difficult to identify in a living cell. In the work presented in this dissertation, I investigated how specific physical and chemical determinants of the membrane and surrounding solution, as well as the gating mechanism itself, may contribute to the emergence of unexpected cellular functionalities. In this endeavor, I showed that increasing the local density of lysenin channels in a target membrane substantially changed the voltage-induced regulation, and that this density can be simply manipulated by altering the membrane’s lipid composition. Next, I demonstrated that the macroergic molecule ATP plays an important role in adjusting the conductance of pore-forming transporters and modulates their biological activity. These observations expand the well-established role of ATP as a signaling molecule, which has been proposed and well-studied for the last several decades. Finally, based on experimental observations that lysenin is endowed with molecular memory, I hypothesized a gating mechanism capable of explaining such a novel and unexpected feature. For these investigations, I focused my work on understanding the influence of multivalent cations on lysenin, which are capable of modulating the voltage-induced gating by electrostatic screening of the voltage domain sensor. The proposed gating mechanism, in which the voltage domain sensor moves into the hydrophobic core of the membrane upon gating, is supported by experimental evidence showing that anion binding to the channel lumen presents qualitative and quantitative differences in voltage regulation, as opposed to binding to the voltage domain sensor. Therefore, the work presented here advances our knowledge with respect to how transmembrane transporters are influenced by frequently overlooked environmental factors, and how this may significantly contribute to the achievement of novel physiological functions. This level of understanding may prove crucial for determining potential connections between metabolic pathways and channelopathies that are commonly attributed to genetic defects of ion channels

Voltage Gating Interactions of the Protein Lysenin with Metal Ions in an Artificial Lipid Bilayer

Non-specific ion conductance channels can be formed in lipid membranes by the poreforming toxin lysenin. These channels are voltage regulated and are responsive to changes in metal ion concentration. In our research, we studied the effects of metal ion concentration on the lysenin channel’s voltage regulated gating, using both multivalent and monovalent metals. A model was developed to explain the apparent subunit cooperativity within the lysenin channel. The model allows for the complex reaction to changing concentration of metal ions, and offers knowledge of the lysenin channel’s internal workings

Intramembrane Congestion Effects on Lysenin Channel Voltage-Induced Gating

All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage sensitivity for smaller populations of channels by reducing the amount of sphingomyelin in the membrane. Given lysenin’s preference for targeting lipid rafts, this result indicates the potential role of the heterogeneous organization of the membrane in modulating channel functionality. Our work indicates that local congestion within membranes may alter the energy landscape and the kinetics of conformational changes of lysenin channels in response to voltage stimuli. This level of understanding may be extended to better characterize the role of the specific membrane environment in modulating the biological functionality of protein channels in health and disease

Gene Expression and Structural Skeletal Responses to Long-Duration Simulated Microgravity in Rats

In this study, we aim to examine skeletal responses to simulated long-duration spaceflight (90 days) and weight-bearing recovery on bone loss using the ground-based hindlimb unloading (HU) model in adolescent (3-month old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative defense genes and pro-bone resorption factors, where there is a progression and eventual plateau; furthermore, early transient changes in these pathways precede skeletal adaptations

Cationic Polymers Inhibit the Conductance of Lysenin Channels

The pore-forming toxin lysenin self-assembles large and stable conductance channels in natural and artificial lipid membranes. The lysenin channels exhibit unique regulation capabilities, which open unexplored possibilities to control the transport of ions and molecules through artificial and natural lipid membranes. Our investigations demonstrate that the positively charged polymers polyethyleneimine and chitosan inhibit the conducting properties of lysenin channels inserted into planar lipid membranes. The preservation of the inhibitory effect following addition of charged polymers on either side of the supporting membrane suggests the presence of multiple binding sites within the channel's structure and a multistep inhibition mechanism that involves binding and trapping. Complete blockage of the binding sites with divalent cations prevents further inhibition in conductance induced by the addition of cationic polymers and supports the hypothesis that the binding sites are identical for both multivalent metal cations and charged polymers. The investigation at the single-channel level has shown distinct complete blockages of each of the inserted channels. These findings reveal key structural characteristics which may provide insight into lysenin’s functionality while opening innovative approaches for the development of applications such as transient cell permeabilization and advanced drug delivery systems

ZnO Nanoparticles Modulate the Ionic Transport and Voltage Regulation of Lysenin Nanochannels

Background: The insufficient understanding of unintended biological impacts from nanomaterials (NMs) represents a serious impediment to their use for scientific, technological, and medical applications. While previous studies have focused on understanding nanotoxicity effects mostly resulting from cellular internalization, recent work indicates that NMs may interfere with transmembrane transport mechanisms, hence enabling contributions to nanotoxicity by affecting key biological activities dependent on transmembrane transport. In this line of inquiry, we investigated the effects of charged nanoparticles (NPs) on the transport properties of lysenin, a pore-forming toxin that shares fundamental features with ion channels such as regulation and high transport rate. Results: The macroscopic conductance of lysenin channels greatly diminished in the presence of cationic ZnO NPs. The inhibitory effects were asymmetrical relative to the direction of the electric field and addition site, suggesting electrostatic interactions between ZnO NPs and a binding site. Similar changes in the macroscopic conductance were observed when lysenin channels were reconstituted in neutral lipid membranes, implicating protein-NP interactions as the major contributor to the reduced transport capabilities. In contrast, no inhibitory effects were observed in the presence of anionic SnO2 NPs. Additionally, we demonstrate that inhibition of ion transport is not due to the dissolution of ZnO NPs and subsequent interactions of zinc ions with lysenin channels. Conclusion: We conclude that electrostatic interactions between positively charged ZnO NPs and negative charges within the lysenin channels are responsible for the inhibitory effects on the transport of ions. These interactions point to a potential mechanism of cytotoxicity, which may not require NP internalization

Functionalized Liposomes for Tumor Targeting as Advanced Tools for Cancer Therapy

Liposomes are self-closed, spherical structures composed of a phospholipid bilayer membrane that encapsulates an aqueous cavity. Liposomes are the most promising tools for controlled drug delivery, and are currently approved by the FDA as drug carriers for cancer treatment. Liposomes are able to retain large quantities of drugs and their PEGylation prevents their removal from circulation by macrophages. Although small liposomes have the capability to self-accumulate at tumor sites by extravasating the leaky blood vessels, this process is slow and uncontrollable. New methods of targeting tumors are necessary to increase the efficiency of liposome-based cancer treatments. Two liposome-based treatment systems are presented. In the first example, specific interactions between the over-expressed folate receptors in cancer cells and the folate-conjugated liposomes are utilized to target cancer cells without causing harm to normal tissues. In the second example, Anginex-conjugated liposomes are used to target endothelial cells as a method for specifically inhibiting vascular endothelial cell proliferation and halting angiogenic processes associated with the formation of new blood vessels at tumor sites. This work opens up new avenues for developing more effective treatment approaches for curing cancer

Modulation of Membrane Transport via Lysenin Channels Controlled by Electrochemical Gradients

Lysenin, a pore forming toxin extracted from the red earthworm E. fetida, self assembles as a large conductance pore in artificial and natural membranes containing sphingomyelin. Unlike many other toxins, the inserted channels are highly regulated by voltage, i.e. open and close in response to external voltage stimuli. This exquisite feature provides opportunities to control the transport of ions and molecules across artificial and natural cell membranes via diffusional transmembrane voltages produced with selective transporters. Additionally, lysenin channels present an inherent ionic selectivity, which may be further used to elicit bias potentials sufficient to induce conformational changes of the channel and conductance control. Our work shows that the permeability of an artificial lipid membrane containing lysenin channels may be easily modulated by adjusting the transmembrane gradient of the ionic concentration and in the absence of any other selective transporter. A more complete channel closure was observed upon addition of valinomycin, a highly selective K+ transporter, which produced a much larger diffusional potential and completely obliterated the membrane permeability function