Pertussis toxin (PTX) sensitive Gi/o proteins are traditionally thought to only exert inhibitory effects on neurons through inhibition of adenylyl cylases and voltage-gated Ca2+ channels, as well as activation of G protein-gated inwardly rectifying K+ channels. These actions cause either decreased neurotransmitter release or reduced membrane excitability. However, emerging evidence suggest that the Gi/o proteins have other effectors and among them, two members of the Transient Receptor Potential Canonical (TRPC) family, TRPC4 and TRPC5, are particularly noteworthy because they respond to the activation of Gi/o signaling with membrane depolarization and intracellular Ca2+ concentration rise, owning to their function as non-selective cation channels. However, the activation of TRPC4/C5 by Gi/o proteins depends on other factors, with coincident activation of phospholipase C (PLC) being the most effective. Thus, these channels act by integrating signals from Gi/o and Gq/11-PLC? (or tyrosine kinase- PLC?) pathways. Consistent with the high expression levels in the nervous systems, TRPC4/C5 channels have been implicated in neurotransmission, neurite outgrowth and neurodegeneration. However, to what extent the Gi/o signaling pathway is involved and how it integrates with PLC signaling in these processes are poorly understood. This project aims to elucidate how Gi/o signaling triggered by metabotropic neurotransmitter receptors act in concert with the PLC pathway to regulate growth and function of brain neurons. Using TRPC knockout mice, we have found that TRPC4 is critical for integrating multiple neurotransmitter inputs, acting through both Gi/o and Gq/11 pathways, to alter excitation of lateral septal neurons. We also show that TRPC4 plays a key role in dendritic branching of hippocampal neurons through integration of signals from Gi/o proteins and neurotrophin receptors. Building upon these findings, we will first investigate how TRPC4 integrates Gi/o and neurotrophin signals to regulate dendritic arborization using cultured hippocampal neurons (Specific aim I). We found this regulation to be dependent on TRPC4 expression and Gi/o protein activation via metabotropic glutamate receptors and neurotrophin receptors. We will then examine the mechanisms and implications of coincident stimulation of Gi/o and Gq/11 pathways on postsynaptic response using mouse lateral septal neurons in brain slices as examples to elucidate Gi/o-TRPC4 coupling in neurotransmission (Specific aim II). We found that a number of G protein-coupled receptors activate native TRPC4 channels in this brain region through co-stimulation of Gq/11 and Gi/o proteins and the resulting channel activity strongly impacts excitability of lateral septal neurons. We will combines molecular and cellular biology, pharmacology, genetic mouse models and electrophysiological approaches for these studies. Accomplishing the proposed work will strongly enhance our understanding on PTX-sensitive G proteins in neurotransmission and neuronal development and help devise new strategies to treat neurological disease.
Neurotransmitters and their receptors are essential components of neural development and neuronal communication which are involved in all aspects of brain function. Often the intracellular signals are transmitted through heterotrimeric G proteins, among which the Gi/o proteins are commonly thought to mediate inhibitory neurotransmission. Recent studies suggest that Gi/o proteins can also mediate excitatory neurotransmission through activation of TRPC4 and TRPC5, calcium permeable cation channels of the Transient Receptor Potential (TRP) protein family. We show that through TRPC4, Gi/o proteins regulate outgrowth and branching of dendritic trees of hippocampal neurons and neuronal excitability of lateral septal neurons. This proposed project will elucidate the mechanism and physiological significance of these novel Gi/o-TRPC4 mediated functions. The results will greatly enrich our understanding on brain development and brain function and inform new therapeutic strategies to treat neurological disorders.
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