GPCR Activation of the G Protein-Gated Inward Rectifier Potassium Channel
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy
Heart rate is tightly regulated by the combined effects of the sympathetic and parasympathetic branches of the autonomic nervous system. These two branches control heart rate by stimulating different G protein-coupled receptors (GPCRs), which in turn activate ion channels that modify the electrical properties of cardiac pacemaker cells. Sympathetic stimulation accelerates heart rate through activation of beta-adrenergic receptors (βARs), which open excitatory ion channels through the stimulatory G protein (Gαs) pathway. Parasympathetic stimulation slows heart rate through activation of the muscarinic acetylcholine receptor M2 (M2Rs), which inhibits the effect of sympathetic stimulation through the inhibitory G protein (Gαi) pathway. M2Rs also release G protein beta-gamma subunits (Gβγ), which slow heart rate by activating G protein-gated inwardrectifier potassium (GIRK) channels. Interestingly, βARs also release the very same free Gβγ, but GIRK is not activated. The molecular mechanism underlying this specificity is poorly characterized. What is the molecular basis behind signaling specificity? It has been proposed that GIRK channels form a macromolecular supercomplex with Gαi-coupled receptors and G proteins, allowing released Gβγ to bind to and activate GIRK by proximity. However the evidence for the existence of the complex remains controversial. In the first part of my thesis, I challenge the supercomplex hypothesis by providing three experimental sets against the theory. First, GIRK co-localization with GPCRs shows no preference for M2Rs over β2ARs. Second, β2ARs do not activate GIRK channels even when they are co-localized. Third, neither Gαi1 nor G protein heterotrimers functionally interact with purified GIRK1/4 channels in the planar lipid bilayer system. I conclude that protein co-localization is not the underlying mechanism to explain why GIRK channels are specifically activated by Gαi-coupled receptors. I then set out to determine the molecular basis behind signaling specificity. Using electrophysiological technologies and bioluminescent resonance electron transfer (BRET) assays, I show that M2Rs catalyze release of Gβγ subunits at higher rates than β2ARs, generating higher Gβγ concentrations that activate GIRK and regulate other targets of Gβγ. The higher rate of Gβγ release is attributable to a faster GPCR-G protein association rate in M2Rs compared to β2ARs. I conclude that the activity of GIRK channels is simply determined by the efficiency of Gβγ release from GPCRs. Physiologically, only Gαi-coupled receptors can provide sufficient concentration of Gβγ to activate GIRK channels. In the second part of my thesis, I present my work on the functional characterization of Gβγ and Na+ regulation of two cardiac GIRK channels, GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. It is known that cardiac GIRK channels are composed of GIRK1/4 heterotetramers and GIRK4 homo-tetramers. However little is known about the functional difference between the two channels. Using purified proteins and the planar lipid bilayer system, I find that Na+ binding increases Gβγ affinity in GIRK4 homo-tetramers and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound.