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