Uncovering the molecular mechanisms of cardiac ion channels’ regulation by lipids and pore formation in membranes using computer simulations

Membranes are complex cellular structures consisting of many different lipid types, a variety of bound proteins, and other molecules. Growing evidence suggests that membranes and lipids play significant bioactive roles in modulating protein function across several cellular processes. Molecular dynamic (MD) simulations have proven to be a valuable method to study lipid organization and membrane protein activity. In this thesis, I used MD simulations to study how lipids regulate two types of membrane proteins: ion channels and pore-forming proteins. Previous simulations and experimental studies showed that polyunsaturated fatty acids (PUFAs) activate KCNQ1 channels while blocking hERG channels. However, some questions regarding how the channel state or PUFA structural properties influence their molecular mechanisms remained unclear. In part of my work, I built a cardiomyocyte membrane model to study the molecular mechanism underlying the interactions between PUFAs and two voltage-gated potassium channels involved in the cardiac action potential: KCNQ1 and hERG. My results revealed that when KCNQ1 voltage sensor domain (VSD) was in the resting state or ‘down’ conformation, the PUFAs established short-lasting interactions that were different from the long-lasting interactions previously observed in the KCNQ1 intermediate state, where the VSD is in the ‘up’ conformation. Additionally, my studies showed that the number of double bonds in the PUFA acyl tail and the size of the polar head regulates their affinity for KCNQ1. Moreover, MD simulations of the hERG channel in the cardiomyocyte membrane unveiled the PUFA interacting site on hERG at the interface between the VSD and the PD in the open and closed states. I anticipate that this detailed molecular understanding of how PUFAs interact with KCNQ1 and hERG will aid in developing future drugs that utilize these mechanisms. As part of this work, I also studied the pore-forming mechanism of the N-terminal peptide StII1-30, derived from the actinoporin StII. My results revealed that this peptide followed a toroidal pore formation mechanism. Additionally, I unveiled the role of curved lipids as cofactors in the formation of toroidal pores. This work has the potential to lead to strategies for the rational use of these peptides as immunotoxins for immunotherapy in cancer tumors. The overall work in this thesis enhances our understanding of lipid-protein interactions in voltage-gated ion channels and the mechanism underlying pore formation by lytic peptides

Emerging Diversity in Lipid-Protein Interactions

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions

Pore-forming proteins: From defense factors to endogenous executors of cell death

Pore-forming proteins (PFPs) and small antimicrobial peptides (AMPs) represent a large family of molecules with the common ability to punch holes in cell membranes to alter their permeability. They play a fundamental role as infectious bacteria's defensive tools against host's immune system and as executors of endogenous machineries of regulated cell death in eukaryotic cells. Despite being highly divergent in primary sequence and 3D structure, specific folds of pore-forming domains have been conserved. In fact, pore formation is considered an ancient mechanism that takes place through a general multistep process involving: membrane partitioning and insertion, oligomerization and pore formation. However, different PFPs and AMPs assemble and form pores following different mechanisms that could end up either in the formation of protein-lined or protein-lipid pores. In this review, we analyze the current findings in the mechanism of action of different PFPs and AMPs that support a wide role of membrane pore formation in nature. We also provide the newest insights into the development of state-of-art techniques that have facilitated the characterization of membrane pores. To understand the physiological role of these peptides/proteins or develop clinical applications, it is essential to uncover the molecular mechanism of how they perforate membranes