Abstract
The G protein-activated Inwardly Rectifying K+-channel (GIRK) modulates heart rate and neuronal excitability. Following G-Protein Coupled Receptor (GPCR)-mediated activation of heterotrimeric G proteins (Gαβγ), opening of the channel is obtained by direct binding of Gβγ subunits. Interestingly, GIRKs are solely activated by Gβγ subunits released from Gαi/o-coupled GPCRs, despite the fact that all receptor types, for instance Gαq-coupled, are also able to provide Gβγ subunits. It is proposed that this specificity and fast kinetics of activation stem from pre-coupling (or pre-assembly) of proteins within this signaling cascade. However, many studies, including our own, point towards a diffusion-limited mechanism, namely collision coupling. Here, we set out to address this long-standing question by combining electrophysiology, imaging, and mathematical modeling. Muscarinic-2 receptors (M2R) and neuronal GIRK1/2 channels were coexpressed in Xenopus laevis oocytes, where we monitored protein surface expression, current amplitude, and activation kinetics. Densities of expressed M2R were assessed using a fluorescently labeled GIRK channel as a molecular ruler. We then incorporated our results, along with available kinetic data reported for the G-protein cycle and for GIRK1/2 activation, to generate a comprehensive mathematical model for the M2R-G-protein-GIRK1/2 signaling cascade. We find that, without assuming any irreversible interactions, our collision coupling kinetic model faithfully reproduces the rate of channel activation, the changes in agonist-evoked currents and the acceleration of channel activation by increased receptor densities.
Highlights
GIRK (G protein-activated Inwardly Rectifying K+-channel) channels play fundamental physiological roles, such as control of heart rate and neuronal excitability, and are highly implicated in disease such as addiction, depression, bipolar disorder, and cardiac arrhythmias (Dascal, 1997; Hibino et al, 2010; Luscher and Slesinger, 2010; Voigt et al, 2014; Mayfield et al, 2015; Rifkin et al, 2017)
All authors contributed to the article and approved the submitted version
Summary
GIRK (G protein-activated Inwardly Rectifying K+-channel) channels play fundamental physiological roles, such as control of heart rate and neuronal excitability, and are highly implicated in disease such as addiction, depression, bipolar disorder, and cardiac arrhythmias (Dascal, 1997; Hibino et al, 2010; Luscher and Slesinger, 2010; Voigt et al, 2014; Mayfield et al, 2015; Rifkin et al, 2017). Heterologous overexpression of proteins of the b-adrenergic-Gas cascade can lead to activation of GIRK via Gas-derived Gbg (Lim et al, 1995; Bender et al, 1998; Touhara and MacKinnon, 2018) These results show that the system can proceed, at least in some instances, via a collision coupling mechanism. We set out to understand the mode of coupling in the classical M2R-Gai/o-GIRK cascade, by combining electrophysiological, fluorescence, and biochemical measurements in Xenopus oocytes with kinetic modeling We studied this cascade by systematically varying and quantifying surface densities of proteins involved in it, and monitored outcomes on GIRK activation. We find that a collision coupling model faithfully reproduces both the fast activation kinetics of agonist-induced GIRK responses, and their dependence on GPCR surface density
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