G protein regulation of inwardly rectifying potassium channels

The work presented in this thesis focuses on mechanistic aspects related to the regulation of ion channel activity by heterotrimeric G proteins. Many ion channels in the heart and nervous system are subject to the modulatory actions of chemical transmitters. The binding of endogenously secreted ligands to distinct classes of cell surface receptors represents a major mechanism whereby membrane excitability is altered in response to external cues. Most hormones and neuroactive peptides bind to members of the large superfamily of G protein-coupled receptors (GPCRs) to adequately adjust the excitatory states of their target cells. In addition, metabotropic receptors for many small-molecule transmitters like acetylcholine, GABA and glutamate have been identified and characterized in many neuronal cell types.; G protein-gated inwardly rectifying potassium channels (GIRKs) are activated by the direct binding of G protein beta-gamma (Gbetagamma) subunits to the channel. These channels are widely distributed throughout the mammalian brain where they generate decreases in postsynaptic excitability to influence various aspects of information processing. The activity of GIRK channels is tightly coupled to the G protein regulatory cycle, which governs the overall duration and extent of G protein activation. This thesis examines one particular aspect of this cycle in closer detail to understand how it affects the response of GIRK channels to G protein-coupled receptor stimulation.; These studies reveal that RGS proteins, in particular RGS4, are able to negatively modulate GIRK channel activity when monitored in the excised-patch configuration. This inhibitory effect is likely to originate largely from increases in the rates of GTP hydrolysis by Galpha subunits, since pharmacological impairment of the hydrolysis reaction (by addition of GTPgammaS) prevents the inhibitory actions of RGS4. In contrast, RGS4 does not appear to directly affect single-channel conductance or channel mean open time. Despite its ability to negatively influence channel activity in excised inside-out patches, RGS4 appears to primarily enhance the rates of GIRK current activation and deactivation in response to receptor stimulation when studied in the whole-cell configuration, with no significant attenuation of maximal current amplitude. A kinetic model is presented and evaluated in terms of its ability to predict changes in GIRK current behavior that may occur in response to increasing the rates of GTP turnover. (Abstract shortened by UMI.)...