This project addresses a general problem of synaptic physiology, the role played by both heterotrimeric GTP-binding proteins (G-proteins) and phosphorylation cascades in transducing the slow electrical actions of transmitters. In the neuroblastoma-glioma cell line (NG108-15), a model neuron used in this project, the neuropeptide bradykinin (BK) has a dual action, activation of a K+ current, and inhibition of the voltage-dependent Ca2+ current, ICa V. These two effects of BK are mediated by distinct G-proteins, both insensitive to pertussis toxin (PTX): the G-protein Gq/11 mediates the activation of K+ current, while G13 mediates the inhibition of the ICa V. What is the mechanism of the action of these two G-proteins on the ion channels they modulate? At least part of the action of Gq/11 is via activation of phospholipase C (PI-PLC) and release of IP3, which in turn raises intracellular Ca2+ and activates Ca2+ -dependent K+ channels. The mechanism of action of G13 is not known. Preliminary observations from the PI's laboratory indicate that the ion channel regulatory actions of both Gq/11 and G13 are transduced by phosphorylation cascade(s). Furthermore, there are indications that these cascades require activation of mitogen-activated protein kinases (MAPKs), a family of kinases which transduce the effects of hormones, growth factors and transmitters. Thus, this project aims to determine the organization of the phosphorylation pathways activated by Gq/11 and G13, to probe the coupling between G-proteins and their cascades, and to investigate the relationship between the phosphorylation pathways and the PI- PLC/IP3/Ca2+ pathway.
These aims will be achieved by combining patch- clamp recording of the electrical responses to BK with intracellular perfusion of specific reagents, including constitutively active or inactive kinases, G-proteins and enzyme inhibitors. While BK is an important mediator of pain and inflammation, in general G-protein- coupled receptors transduce the actions of numerous neurotransmitters, hormones and drugs. Thus this project is relevant for understanding and treating diseases of both the central nervous system and the peripheral tissues.
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