Nathan Dascal - US grants

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).

DESCRIPTION (provided by applicant): G-protein activated K+ channels (GIRK; Kir3) play an important role in the cardiac and brain function, mediating the inhibitory action of many neurotransmitters. GIRK channels are activated by direct binding G-Beta and G-Alpha subunits of G-proteins also directly interact with GIRK and play an important role in determining the specificity of signaling. Over the last years, we have presented evidence that Galpha subunits affect GIRK gating directly and indirectly. However, much of the phenomenology, the physiological impact, and the mechanisms of interaction of Galpha with GIRK remains unclear. These issues are in the focus of the proposed study. Our long-term goal is to understand the molecular mechanisms and the physiological significance of GIRK modulation by Galpha subunits, and to utilize GIRK to study the general problem of specificity and G protein effector interactions in G protein pathways. The specific aims are: (1) Further study of the roles and mechanisms of actions of Ga in GIRK gating. By monitoring protein-protein interactions in vitro and by using physiological assays in Xenopus oocytes and HEK cells, mutagenesis, immunocytochemistry, and fluorescence energy transfer (FRET), we will: i) verify the existence of GcdGIRK interactions and their impact on GIRK activity; ii) clarify which form(s) of Gm is physiologically important; iii) map the sites of interaction between Galpha and GIRK, and study the role of these sites in channel gating and regulation; iv) investigate how GIRK gating is affected by Galpha (2) Understanding of the role of Galpha-GIRK interactions in determining specificity of signaling in this pathway. The physical interactions between the GIRK channel and various Galpha subunits will be monitored, and correlated with the effects of coexpression of these Galpha subunits, on the function of GIRK channels. (3). Study of the physiological correlates of modulation of GIRK by Ga. The roles of GIRK modulation by Galpha in cultured atrial myocytes and neurons will be addressed using electrophysiological methods, by co-expression of proteins, addition of purified proteins and peptides to excised patches, and by knock-down of endogenous G protein subunits. Colocalization and complex formation between Galpha and GIRK will be explored in atrium and brain using biochemical and immunohistochemical methods.