Swarna Ramaswamy_Thesis

STRUCTURAL INVESTIGATIONS OF LIGAND GATED ION CHANNELS Swarna Ramaswamy, B.S Advisor: Vasanthi Jayaraman, Ph.D. Ion channels form an integral part of membrane proteins. In the nervous system including the central and the peripheral nervous system, ligand gated ion channels form a very important part of intercellular communications. They receive chemical signals and convert them to electrical signal, mainly by allowing ion passage across the cell membrane. Ion passage also translates into downstream signaling events. Faithful translation of these signals and transmittance is crucial for several physiological functions, implying that irregular ion channel function could lead to serious consequences. This thesis aims at gaining a better understanding of working of some of the excitatory neuro transmitter receptors. Signal transmission depends on the ability of the extracellular receptor segment and ion channel segment coordinating their movement to produce the one singular effect. My thesis work focuses on using various spectroscopic and molecular strategies to understand this process of how the extracellular segment controls the gating at the ion channel. I investigated these processes in two different classes of ligand gated ion channels. My work on Acid Sensing Ion Channel (ASIC), a proton sensitive ion channel found in central and peripheral nervous system aims to understand the proton sensors and structural changes. The resting state of the ion channel was still poorly understood, as well as the proton sensors. I used a combination of electrostatic simulations, mutational and spectroscopic investigations to identify the key proton sensing residues of the ion channel. The study also identifies the key conformational change that allows the extracellular membrane to communicate within the trimeric complex and allow ions to pass through. The next part of the study focuses on glutamate class of receptors, specifically the α-amino-5-methyl-3-hydroxy-4-isoxazolepropionate (AMPA) receptors. The modular nature of the ion channel allowed us to study specific domains of the protein, the ligand binding domain (LBD) for example. Using isolated LBD of AMPA receptors, we were able to study the dynamics of the protein using single molecule fluorescence experiments. The findings indicate that not only the average conformational change, but the dynamics of the protein also play a very important role in the ion channel gating