The Dynamics of Formation and Action of the Ternary Complex Revealed in Living Cells Using a G-protein-gated K+ Channel as a Biosensor

Abstract

Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1؉3.2A) that is activated by the direct binding of G␤␥ subunit to the channel complex to report, in real-time, using the patch clamp technique the activity of the "ternary complex" of agonist/receptor/G-protein. This analysis is further facilitated by the use of pertussis toxin-resistant fluorescent and non-fluorescent G␣ i/o subunits and a series of HEK293 cell lines stably expressing both channel and receptors (including the adenosine A 1 receptor, the adrenergic ␣ 2A receptor, the dopamine D 2S receptor, the M4 muscarinic receptor, and the dimeric GABA-B 1b/2 receptor). We systematically analyzed the contribution of the various inputs to the observed kinetic response of channel activation. Our studies indicate that the combination of agonist, GPCR, and G-protein isoform uniquely specify the behavior of these channels and thus support the importance of the whole ternary complex at a kinetic level.The activation of G-protein-coupled receptors (GPCRs) 1 by extracellular ligands is an important mechanism involved in a multitude of physiological responses and is of central importance in drug development and therapeutics (1). The activated receptor couples to G-proteins of various subtypes that then activates effector pathways either directly or indirectly. This combination of agonist, receptor, and G-protein is referred to as the "ternary complex" and is thought to be the key essential determinant of the magnitude of the downstream response (2, 3). The most recent formulations propose a cubic ternary complex model with a large number of equilibrium constants between various states governing efficacy (2, 4, 5). The important species is the activated receptor/agonist/G-protein complex. It is proposed that for any combination of these three elements the particular active conformation (or conformational space) is unique and can thus have distinctive signaling consequences (2, 4 -6). An agonist binds more favorably to the active receptor species and thus at equilibrium favors its' formation. Recently this model has been extended to also incorporate the kinetics of G-protein activation and deactivation and indicate that a kinetic model, as opposed to an equilibrium model, may potentially have quite different properties (7).Generally these phenomena have been studied by the use of biochemical assays, using cell homogenates or fractions, or by measuring the behavior of a physiological response many steps downstream from the G-protein cycle. It is apparent that there is a gap in our understanding about how these signaling pathways behave dynamically in intact cells. This is important, because, in reality, the release of hormones and neurotransmitters varies over the second time scale. Agonist binding to receptor is generally agreed to be diffusion-limited and much faster th...

Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

“…We originally investigated such issues to explain what factors contribute to the rapid activation of GIRK channels by Gi͞o coupled receptors. In addition, we had previously found that the activation rate through a receptor-G protein fusion was identical to that of the normal receptor and increasing G protein concentration did not affect signaling kinetics (12). GPCR G protein precoupling can contribute to fast activation rates and can explain our previous experimental observations.…”

“…Patch clamping was carried out as described (12,30). Cell capacitance was Ϸ15 pF, and series resistance (Ͻ10 M⍀) was at least 75% compensated by using the amplifier.…”

“…Activation of native and cloned GIRK channels has been shown to involve a direct, membrane-delimited interaction with the G␤␥ subunit (9,10). One critical point is that the activation occurs rapidly in both native and heterologous settings: complete channel activation can occur within 1 s of the addition of agonist (11)(12)(13). Such fast rates of signaling suggest that the components diffuse only small distances, if at all.…”

Rapid Activation of Inwardly Rectifying Potassium Channels by Immobile G-Protein-Coupled Receptors

“…Previous studies have addressed the role of receptor-Gprotein collision in GIRK activation by fusing GPCRs and G-proteins and by varying the abundance of these proteins to either eliminate or manipulate the frequency of receptor-Gprotein collision. One of these studies concluded that receptor-G-protein collision was rate-limiting (Vorobiov et al, 2000), whereas the other reached the opposite conclusion (Benians et al, 2003). In both of these studies, the GIRK activation rate was limited by the heterologous expression system, and it has since been shown that fusing receptor and G-protein does not eliminate intermolecular collision coupling (Molinari et al, 2003).…”

Gβγ-activated Inwardly Rectifying K+ (GIRK) Channel Activation Kinetics via Gαi and Gαo-coupled Receptors Are Determined by Gα-specific Interdomain Interactions That Affect GDP Release Rates

“…In addition to the G protein activation steps, signal termination with GTP hydrolysis by the G␣ subunit and G␤␥ reassociation also impacts the kinetics and amplitude of agonist-activated GIRK currents. The ternary complex consisting of agonist, GPCR, and G protein influences the time course of agonist-elicited GIRK channel currents (3), supporting the notion that isoform composition of different GPCR-G␣ i/o ␤␥ protein-RGS protein-GIRK channel signaling complexes have different kinetic properties that affect their functional output (4). We recently reported notable differences in the gating properties of GIRK channels activated by muscarinic m2 receptors coupled specifically to PTX-insensitive G␣ i isoforms (G␣ i1 , G␣ i2 , or G␣ i3 ) versus G␣ o isoforms (G␣ oA or G␣ oB ) in the Xenopus oocyte system (4).…”