This project will study the kinetics of membrane phosphoinositide metabolism and signaling by G protein coupled receptors to modulate ion channels. The focus includes intracellular membranes and compartments as well as the plasma membrane. When such ubiquitous signaling goes wrong either in neurodegenerative diseases, schizophrenia, Parkinsonism, or depression, or by action of drugs of abuse, there are major alterations in states of mind, affect, feeling of well-being and many other broad mental functions. The same signals are targets of many of the drugs of biological psychiatry. They need to be understood. These studies will use primary neurons and cultured transfected tsA cells as a model. The methodology will involve single-cell biophysics, electrophysiology, fluorescence resonance energy transfer, photometry, molecular biology, and confocal microscopy. The focus will be on the phosphatidyl inositide phospholipids as regulators and signals for channel function. These lipids are synthesized in many intracellular membranes, regulated by receptors, and in turn, regulate ion channels. The overall goal will be to understand this kind of signaling i mechanistic terms including mathematical kinetic description. Phosphoinositides will be measured in multiple cellular compartments by transfected optical indicators, and they will be perturbed in several compartments by translocating or activating transfected enzymes using a drug, light, or voltage as the triggers. This project will define the membrane compartments and dynamic kinetics underlying metabolism, interconversions, and membrane trafficking of cellular phosphoinositides. Using enzymatic perturbations, the hypothesis will be tested that there are multiple intracellular pools of precursors contributing to the plasma membrane synthesis of PI(4,5)P2. The trafficking of ion channels and lipids will be followed to understand organization of cell compartments and how organelle lipids regulate channel functions. All quantitative results will be analyzed by an integrated realistic explanatory kinetic model for neurons and expression cells.

Public Health Relevance

The molecular actions studied here are the neuro-hormonal communication between nerve cells that modifies how nerve circuits respond to their inputs and what outputs they will make. Such signals affect the mental state, mood, arousal, and psychiatric balance of the brain, and are the target of many drugs of abuse. The focus is on changes of the metabolism of phospholipids of the cell membrane, a regulatory system that, when it goes wrong, underlies many defects of brain development.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Method to Extend Research in Time (MERIT) Award (R37)
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Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
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Stewart, Randall R
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University of Washington
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Yeon, Jun-Hee; Park, Cheon-Gyu; Hille, Bertil et al. (2018) Translocatable voltage-gated Ca2+ channel ? subunits in ?1-? complexes reveal competitive replacement yet no spontaneous dissociation. Proc Natl Acad Sci U S A 115:E9934-E9943
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