The inwardly rectifying, G protein-activated K+ channels of the GIRK family are important in regulation of heartbeat and mediate the inhibitory effects of many neurotransmitters in the brain. Phenomenology and mechanisms of modulation of GIRK by neurotransmitters are poorly understood. Unresolved problems include: determinants of specificity of interaction between G proteins and the GIRK channels; mechanisms of gating, desensitization, and inhibitory modulation by neurotransmitters. The long term goal is to understand the molecular mechanisms and the physiological significance of the inhibitory GIRK modulation by G-alpha subunits and by G protein- coupled neurotransmitters.
The specific aims are: 1. To understand the molecular mechanisms of membrane-delimited interaction between GIRK subunits and the G-alpha proteins, by examining the effects of purified G proteins and agents affecting phosphorylation in excised patches of Xenopus oocyte membrane, and by monitoring protein-protein interactions by coimmunoprecipitation and overlay methodologies. 2. To study modulation of GIRK, via protein phosphorylation, by neurotransmitters that activate G-q, and the process of GIRK desensitization, by examining effects of protein kinase inhibitors and purified protein kinases in Xenopus oocytes, and by mutating putative phosphorylation sites in target GIRK subunit(s). 3. To evaluate the physiological significance of modulations of GIRK by G-alpha subunits. The existence of the modulations described in the oocytes, and the identity of their molecular mechanisms, will be confirmed in primary cultures of cardiac and nerve cells by testing the direct effects of G-alpha subunits and relevant protein kinase(s), and by eliminating the protein components of signaling pathways by antisense knockout. 4. To investigate how G-ail, G-as and protein phosphorylation interfere with the process of channel gating. Details of gating process will be explored by examining interactions of parts of channel involved in gating with the rest of the channel, and with agents that modulate gating (G-alpha proteins and protein kinases).
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