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

Novel Roles for Peroxynitrite in Angiotensin II and CaMKII Signaling

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) oxidation controls excitability and viability. While hydrogen peroxide (H2O2) affects Ca(2+)-activated CaMKII in vitro, Angiotensin II (Ang II)-induced CaMKIIδ signaling in cardiomyocytes is Ca(2+) independent and requires NADPH oxidase-derived superoxide, but not its dismutation product H2O2. To better define the biological regulation of CaMKII activation and signaling by Ang II, we evaluated the potential for peroxynitrite (ONOO(-)) to mediate CaMKII activation and downstream Kv4.3 channel mRNA destabilization by Ang II. In vitro experiments show that ONOO(-) oxidizes and modestly activates pure CaMKII in the absence of Ca(2+)/CaM. Remarkably, this apokinase stimulation persists after mutating known oxidation targets (M281, M282, C290), suggesting a novel mechanism for increasing baseline Ca(2+)-independent CaMKII activity. The role of ONOO(-) in cardiac and neuronal responses to Ang II was then tested by scavenging ONOO(-) and preventing its formation by inhibiting nitric oxide synthase. Both treatments blocked Ang II effects on Kv4.3, tyrosine nitration and CaMKIIδ oxidation and activation. Together, these data show that ONOO(-) participates in Ang II-CaMKII signaling. The requirement for ONOO(-) in transducing Ang II signaling identifies ONOO(-), which has been viewed as a reactive damaging byproduct of superoxide and nitric oxide, as a mediator of GPCR-CaMKII signaling