Activation of phospholipase C beta enzymes by G beta-gamma and corresponding regulation of downstream ion channels

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Project Summary Cells respond to many extracellular stimuli via G protein coupled receptors (GPCR). Extracellular signaling molecules bind GPCR’s and catalyze the release of intracellular membrane anchored G proteins, Ga and Gbg, which act on downstream targets. Ion channels are a downstream target of numerous GPCR signaling cascades, connecting the cellular response to these stimuli to membrane excitability. GPCR-dependent regulation of ion channels plays an essential role in many physiological processes including nociception, regulation of heart rate, and inflammation; therefore, it is essential to understand this regulation. Ion channels can be regulated by GPCR signaling directly by G proteins or by G protein-regulated second messengers, including phosphatidylinositol- 4,5-bisphosphate (PIP2). PIP2 is degraded by the G protein-dependent b family of phospholipase C (PLC) enzymes, which cleave PIP2 to produce IP3 and DAG. Numerous families of ion channels are regulated by PIP2 in a PLCb-dependent manner, including inwardly rectifying K+ (Kir) channels and voltage-dependent K+ channels. PLCb enzymes are activated by both Gaq and Gbg, linking their function to both Gaq and Gai-coupled receptors. While the regulation by Gaq is well-understood, much is unknown regarding the Gbg-dependent activation. In order to understand the GPCR-dependent regulation of downstream ion channels and the associated physiological processes, it is necessary to understand the G protein regulation of PLCb enzymes, which are the key signaling intermediate. To this end, I propose to study the Gbg-dependent activation of PLCb enzymes via the following two aims: (1) Investigate the minimal requirements for Gbg-dependent activation of PLCb enzymes and the regulation of downstream ion channels using a cell-free reconstituted system, (2) Characterize the interaction between Gbg and PLCb including localization of the binding site and characterization of the Gbg- dependent conformational changes using cryogenic electron microscopy and bioluminescence resonance energy transfer. Successful completion of these aims will directly connect PLCb regulation to its effect on ion channels, expanding the knowledge of these signaling cascades. This project will be conducted under the supervision of Dr. Roderick MacKinnon at Rockefeller University. Dr. MacKinnon has extensive experience studying the function and regulation of ion channels as well as training postdoctoral researchers to succeed as independent researchers. Together, the lab and Rockefeller University generate an environment with the resources and intellectual input necessary for completing the proposed project. The accompanying training plan includes a timeline describing the completion of the proposed experiments and allocates time for personal and career development including frequent meetings with Dr. MacKinnon, attending conferences to present the findings, mentoring students, and grant writing to acquire funding for an independent research program.