Susy C. Kohout - US grants

DESCRIPTION (provided by applicant): The plasma membrane of every cell serves as a signaling hub, controlling the flux of information across it. As part of the membrane, phosphatidylinositol phosphates (PIPs) are lipid second messengers and are involved in almost all facets of cell physiology, including synaptic transmission, cell proliferation, cell differentition, migration and many others. Their concentrations are tightly regulated by a series of kinases and phosphatases. One such phosphatase, PTEN, is one of the most frequently mutated genes in sporadic human tumors with mutations occurring in glioblastomas, lymphoma, thyroid and melanomas. When PTEN is not functioning properly, PIP levels increase and could activate out of control growth and migration. While cancer is the most commonly cited, several human diseases are linked to PIP metabolizing enzymes such as peripheral neuropathy, Alzheimer's, autism and bipolar disorder. As a result, the proteins regulating PIP signaling have been extensively studied. The voltage sensing phosphatase (VSP) is homologous to PTEN and is a new member of the large class of proteins that regulate the membrane concentrations of PIPs. Classically, PIP regulating enzymes are cytosolic. VSP, on the other hand, is an integral membrane protein and is activated by voltage. The discovery of VSP reveals a gap no one knew existed because voltage regulation of PIP regulating proteins has not been considered. Understanding the effect of VSP on PIP regulatory pathways is crucial because those pathways are fundamental to understanding cells and their signaling. ! This project has the long term objective of probing the molecular mechanism and physiological roles of VSP. To address this long term goal, the immediate specific aims target factors that activate VSP such as lipids. Many membrane proteins are modulated by the lipid composition in the membrane. The expectation that VSP is also modulated by lipids is not unusual. What makes VSP more complicated than a regular membrane protein is the fact that VSP will change the concentrations of those same lipids. This could result in an internal feedback loop causing VSP to essentially self-regulate. This is critical because while other kinases and phosphatases exist to maintain PIP concentrations, those mechanisms may be slower than voltage activation. This proposal will expand our understanding of VSP on a molecular level and how it fits into the broader PIP signaling pathway.

This research will investigate how brain cells called neurons use both electrical and chemical signals to communicate. Specifically, one component in neurons, called the voltage sensing phosphatase or VSP, uses an electrical charge to directly change the chemistry inside the neuron. However, how VSP then changes neuron communication is still unknown and a greater understanding of this process may clarify how brains process information such as vision, hearing and touch, by communication between neurons. The results from this research may also be applicable to other tissues apart from the brain that are known to use electrical signals to regulate cellular function. Graduate and undergraduate students involved in this project will be trained on how to think critically, solve problems and conduct experiments. Aspects of the project will be shared with the public through hands-on experiences for children and adults. To reach a broader audience within the rural communities of Montana, video conferencing will also be used to share the project and the underlying science behind it.

Membrane potential and phosphatidylinositol phosphates (PIPs) are critical signals in neurons, regulating synaptic transmission, ion channels, growth and migration. VSP is the only known protein to directly link both signals, using a voltage sensing domain to activate a phosphatase domain that then dephosphorylates PIPs in a voltage dependent manner. VSP could significantly influence neuronal signaling because it is activated on a faster time scale than other phosphatases. However, the impact of VSP on neuronal function is still unknown. This research aims to determine whether VSP dimerization serves as a regulatory mechanism for VSP activity and specificity. First, the dimer will be disrupted to determine the impact of VSP dimers versus monomers on function. Second, the consequences of VSP heterodimerization on catalysis will be determined. Methods will include electrophysiology, fluorescence, imaging, biochemical techniques, and molecular biology. This research is critical for understanding the molecular mechanism behind VSP, increasing the field’s understanding of voltage sensing proteins and serving as a basis for engineering new voltage-gated tools such as sensors and enzymes.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.