The G Protein α Subunit Has a Key Role in Determining the Specificity of Coupling to, but Not the Activation of, G Protein-gated Inwardly Rectifying K+ Channels

Abstract

In neuronal and atrial tissue, G protein-gated inwardly rectifying K ؉ channels (Kir3.x family) are responsible for mediating inhibitory postsynaptic potentials and slowing the heart rate. They are activated by G␤␥ dimers released in response to the stimulation of receptors coupled to inhibitory G proteins of the G i/o family but not receptors coupled to the stimulatory G protein G s . We have used biochemical, electrophysiological, and molecular biology techniques to examine this specificity of channel activation. In this study we have succeeded in reconstituting such specificity in an heterologous expression system stably expressing a cloned counterpart of the neuronal channel (Kir3.1 and Kir3.2A heteromultimers). The use of pertussis toxinresistant G protein ␣ subunits and chimeras between G i1 and G s indicate a central role for the G protein ␣ subunits in determining receptor specificity of coupling to, but not activation of, G protein-gated inwardly rectifying K ؉ channels.G protein-gated, inwardly rectifying K ϩ channels were first identified in atrial myocytes where they were shown to be activated by acetylcholine at muscarinic m 2 receptors (1), and stimulation of this current is responsible for slowing of the heart rate in response to vagal nerve stimulation. It was subsequently shown that this activation was sensitive to pertussis toxin (PTx), 1 implicating the family of G i/o proteins (2-4). It is now apparent that G protein-gated inwardly rectifying K ϩ currents are present in many neuronal cell types, and they are involved in the postsynaptic inhibitory effects of stimulating G i/o -coupled receptors such as GABA B and adenosine A 1 (5).Cloning efforts from a number of laboratories have revealed the molecular counterparts of these currents (6 -10). The channel is a heteromultimer of members of the inwardly rectifying Kir3.x family of potassium channels. Co-expression of Kir3.1 with Kir3.2, Kir3.3, or Kir3.4 results in currents that show many of the basic characteristics of the native channels in neurons and atria (11-13). Homomultimers of splice variants of Kir3.2 may also form in certain neuronal regions (14).Channel activation of these currents in native tissues and of the cloned counterparts in heterologous expression systems was shown to be membrane-delimited (4, 15), involving a direct interaction with the G protein ␤␥ dimer (16). This finding was initially controversial, but there are now numerous lines of evidence that point to the involvement of the ␤␥ subunit as the direct channel activator (17). Recent studies have focused on domains and residues involved in G protein ␤␥ dimer binding (18 -24) and the cell biology of channel biogenesis (25-27).The question remains as to how receptor specificity is achieved and what are the key determinants involved in this. In native tissues only G i/o -coupled receptors, and not G s -coupled receptors, activate these channels, and yet both should liberate free G protein ␤␥ upon receptor stimulation. Receptor selectivity does not lie at the level of the G...

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“…No such differences are known for the classical PTX-sensitive inhibitory G a subunits, G i a1-3 (Taussig and Gilman 1995;Sunahara et al 1996). These regulatory patterns indicate that the functional significance of heterogeneity at the level of inhibitory G proteins lies not exclusively in conferring specificity to receptor/G coupling (Kleuss et al 1991;Leaney et al 2000). Instead, they open the opportunity for the existence of multiple inhibitory AC pathways within a single cell.…”

“…Identification of the signal transduction pathway mediating tolerance/dependence is hampered by the ability of ORs to activate multiple inhibitory G proteins and intracellular effector systems (Gudermann et al 1992). Several experimental approaches have been employed to elucidate the specificity of signal transduction cascades in situ, such as application of site-directed anti-G protein antibodies (Simonds et al 1989), antisense strategies (Kleuss et al 1991), and over-expression of genetically modified PTXresistant inhibitory G a subunits (Leaney et al 2000). However, each of these approaches failed to define specific inhibitory AC signalling pathways most likely because of a pronounced functional redundancy between inhibitory G a subunits (Tang et al 1991;Sunahara et al 1996).…”

Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K + channels

“…Using the whole-cell configuration of the patch-clamp technique we studied receptor-mediated G protein-gated Kir channel currents in a HEK293 stable cell line robustly expressing the Kir3.1ϩ3.2A channel complex (31). To apply agonists we used a rapid and localized drug-perfusion system that enabled us to apply and remove agonist in under 0.25 s. We used five different dual-receptor and channel stable lines to investigate receptormediated Kir3.…”

“…Cell-culture methods and the generation of stable cell lines were as described (30,31). In addition to the stable lines wae have described previously [Kir3.1ϩ3.2A channel plus either the A 1 adenosine receptor (HKIR3.1͞3.2͞A1) or the D 2S dopaminergic receptor (HKIR3.1͞3.2͞D2)], we used a further three-dualreceptor ϩ channel stable lines that were denoted as ␣ 2A adrenergic receptor (HKIR3.1͞3.2͞␣2), GABA-B 1b/2 receptor (HKIR3.1͞3.2͞ GGB), and M 4 -muscarinic receptor (HKIR3.1͞3.2͞M4).…”

Potassium channels in the sinoatrial node and their role in heart rate control

“…Anionic phospholipids particularly phosphatidylinositol- [4,5]-bisphosphate and sodium are known to be key modulators of the gating and a number of site-directed mutagenesis studies together with structural work have suggested models predicting how this might occur [38–40]. However the inhibitory G-protein α subunit also participates in determining the selectivity of activation but not directly in activation itself [41]. There are strands of evidence pointing to models in which the inhibitory heterotrimeric G-protein is complexed with the channel and receptor prior to activation [42–46].…”