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Emerging role(s) of G‐protein α‐subunits in the gating of GIRKs

In the early days following the discovery of heterotrimeric G-Proteins, the GTP associated Gα subunits were regarded as the active principle in signalling to G-protein effectors. Gβ/γ subunits were seen as inactive, being required as ‘docking-stations’ for α-subunits when signalling was over. The first direct Gβ/γ effector to be discovered was a G-protein activated potassium channel from atrium (GIRK; Logothetis et al. 1987). Subsequently, certain isoforms of phospholipase Cβ (PLCβ) and other G-protein effectors were also found to be triggered directly by the Gβ/γ dimer. Finally the view that Gβ/γ and the Gα subunits are in fact equal partners with respect to signalling became generally accepted (for review see Birnbaumer, 2007). Moreover, with the uncovering of G-protein effector isoforms, it became clear that some effector isoforms are regulated by Gαs, others by Gβ/γs, in a differential and eventually divergent way, sometimes both entities acting cooperatively on the same effector isoform (see e.g. Tang & Gilman, 1991). 
 
With respect to GIRKs, Gβ/γ dimers of pertussis toxin (PTX) sensitive G-proteins were long believed to act as the exclusive mediators of G-protein coupled receptor (GPCR) activation. Only sparse evidence for a role of G-protein α-subunits existed: in 1995, direct binding of Gβ/γ to both the carboxy- and the amino-terminal cytosolic parts (C-T and N-T, respectively) of the GIRK1 subunit was observed (Huang et al. 1995). Interestingly, also the inactive G-protein trimer, including the Gα subunit, was found to directly interact with the GIRK1 N-T in the same study. First functional effects of G-protein α-subunits on GIRK channel activation by Gβ/γ were described in 1996 (Schreibmayer et al. 1996). Despite these early findings the exact role(s) of G-protein α-subunits as mediators of GIRK gating remained obscure. 
 
In a seminal series of papers, starting in 2002 and coming to a preliminary end with Rubinstein et al. (2009) in a recent issue of The Journal of Physiology, scientists around Prof. Dascal from Tel Aviv University have established the GIRK1 subunit as target for the inactive, GDP bound, form of Gα. The entire story started with the seminal observation, that the basal, agonist-independent K+ current through GIRK1/GIRK2 heterooligomeric channels grows disproportionally with the level of expression in Xenopus oocytes. Thus, at high levels of expression of GIRK1/GIRK2 the basal current is abnormally high, indicating a limiting endogenous compound. Surprisingly, Gαi3 was identified as being this limiting compound, since Gαi3 overexpression sufficed to restore low basal current. Moreover, Gαi3 was found to directly interact with and bind to the GIRK1 N-T, confirming the previous data of Huang et al. (1995) and Peleg et al. (2002). In two subsequent papers (Ivanina et al. 2003, 2004), the binding sites for both Gβ/γ and Gαi1/Gαi3 on the GIRK1 and GIRK2 C-T and N-T were mapped with biochemical methods. In addition to confirming and extending data of several other groups, a low-affinity Gβ/γ-binding site was detected in the unique distal C-T of GIRK1. When compared to Gαi1, Gαi3 binding to GIRK1 and to GIRK2 was stronger. Accordingly, Gαi3 better supported activation by Gβ/γ when compared to Gαi1 in excised membrane patches as well as in whole oocytes. The correlation between the specificity of the two Gαs and their binding strength to GIRK subunits indicated that direct binding of Gαi plays a role in channel regulation. Subsequently, Rishal et al. (2005) discovered, that the high basal activity upon overexpression of GIRK1/GIRK2 was the result of co-trafficking of the Gβ/γ subunit with the GIRK1/GIRK2 heterooligomer, which ‘hauls’ Gβ/γ to the plasma membrane. Co-trafficking of Gα subunits was also observed, but to a lesser extent. Preassociation of Gβ/γ with GIRKs with signalling complexes was independently confirmed by others using BRET and FRET (Rebois et al. 2006; Riven et al. 2006). The knowledge on the regulation of GIRK channels by G-protein α-subunits was meanwhile extended independently by another group, when Koike-Tani et al. (2005) observed direct inhibition of GIRK1/GIRK2 heterooligomeric channels by Gαq. Rubinstein et al. (2007) refined and extended the experimental paradigms and further distinguished the regulation by Gαi3 from the effect of a simple Gβ/γ scavenger. 
 
Notably, Rubinstein et al. (2009) show that the regulation of GIRK1/GIRK2 by Gαi3 is not a peculiar property of the Xenopus laevis oocyte expression system, but also occurs in mammalian (HEK 293) cells. Furthermore, by using constitutively active and inactive mutants of Gαi3, it was demonstrated that Gαi3GDP, but not Gαi3GTP is required to regulate basal activity. Moreover, this regulation is isoform specific with respect to GIRK subunits: only GIRK1 binds to the Gαi3βγ heterotrimer, while GIRK2 does not. Accordingly, functional effects are conferred exclusively by the GIRK1 subunit. The responsible structural determinant is located in the unique proximal C-T of GIRK1. Rubinstein et al. propose a model where GIRK1/2 has two sites, an ‘activation’ site for Gβ/γ which is occupied by Gβ/γ when Gα is GTP-bound, and an ‘anchoring’ site where the ineffective Gαβγ heterotrimer is bound to GIRK, preventing Gβ/γ from activating the channel. 
 
New perspectives come forth and reveal important questions to be answered in the future. Does this regulation via GIRK associated Gα subunits participate in the specificity of G-protein isoform mediated signalling? Are also GIRK3 and GIRK4 subunits affected? Does this regulation of basal current by Gαi3GDP occur in native cells? In atrial cells, for example, the agonist independent, basal activity of IK+ACh is extremely low, to an extent that has never been observed in heterologous systems (see e.g. Schreibmayer et al. 1996). It will be interesting to see whether regulation by an endogenous Gα subunit is responsible.