Regulation of the Inward Rectifying Properties of G-protein-activated Inwardly Rectifying K+ (GIRK) Channels by Gβγ Subunits

Abstract

G␤␥ subunits are known to bind to and activate Gprotein-activated inwardly rectifying K ؉ channels (GIRK) by regulating their open probability and bursting behavior. Studying G-protein regulation of either native GIRK (I KACh ) channels in feline atrial myocytes or heterologously expressed GIRK1/4 channels in Chinese hamster ovary cells and HEK 293 cells uncovered a novel G␤␥ subunit mediated regulation of the inwardly rectifying properties of these channels. I KACh activated by submaximal concentrations of acetylcholine exhibited a ϳ2.5-fold stronger inward rectification than I KACh activated by saturating concentrations of acetylcholine. Similarly, the inward rectification of currents through GIRK1/4 channels expressed in HEK cells was substantially weakened upon maximal stimulation with co-expressed G␤␥ subunits. Analysis of the outward current block underlying inward rectification demonstrated that the fraction of instantaneously blocked channels was reduced when G␤␥ was over-expressed. The G␤␥ induced weakening of inward rectification was associated with reduced potencies for Ba 2؉ and Cs ؉ to block channels from the extracellular side. Based on these results we propose that saturation of the channel with G␤␥ leads to a conformational change within the pore of the channel that reduced the potency of extracellular cations to block the pore and increased the fraction of channels inert to a pore block in outward direction.G-protein-activated inwardly rectifying K ϩ channels (GIRKs) 1 are expressed in many areas of the brain and in supraventricular myocytes of the heart (1, 2). Activation of G-protein-coupled receptors that couple to G i -proteins such as the M 2 muscarinic acetylcholine receptors (M 2 -mAChRs) lead to a dissociation of heterotrimeric G-proteins into activated ␣ subunits and ␤␥ dimers. G␤␥ subunits are known to bind to GIRK channels and increase the open probability of these channels (3, 4). Cardiac I KACh channels are formed by heteromultimers of GIRK1 and GIRK4 subunits (4). The binding site of G␤␥ subunits to GIRK channels was mapped primarily to the C terminus of GIRK1 and GIRK4 (4 -8). Cross-linking studies have demonstrated that the heterotetrameric channel can bind up to 4 G␤␥ subunits (9). However, despite much experimental effort the mechanism by which G␤␥ activates these channels is not well understood.GIRK channels belong to the family of strong inwardly rectifying K ϩ channels, which are characterized by their strong inwardly rectifying current-voltage relationships. The inward rectification has been linked to the presence of intracellular Mg 2ϩ and polyamines (10 -12). These positively charged cytoplasmic ions are thought to block outward K ϩ currents by blocking the pore of channels from the inside (10 -13); however, for a related inwardly rectifying channel Kir2.1 this hypothesis has recently been questioned (14). Inward rectification of K ϩ channels is not only voltage-dependent but also dependent on the extracellular K ϩ concentration (11). The inward rectification of these K ...

Fluorescence/Bioluminescence Resonance Energy Transfer Techniques to Study G-Protein-Coupled Receptor Activation and Signaling

“…Functional and kinetic studies comparing the activation of G i -proteins and GIRK channels by FRET and by patch clamping also support the notion of a tight kinetic and presumably also spatial coupling of G-proteins and their effector channels Lohse et al, 2008b). This is further supported by the notion that GIRK channel regulation involves both G␣ subunits and, primarily, the G␤␥ complex (Hommers et al, 2003;Berlin et al, 2010). This was additionally shown by a combined approach using FRET and total internal reflection microscopy that demonstrated a preformed complex of G-protein and GIRK channel; this preformed complex would allow a precise temporal control of activation and add to the selective activation of the channel (Riven et al, 2006).…”

Membrane and synaptic properties of pyramidal neurons in the anterior olfactory nucleus

“…7D) solution and reduced the input resistance with a potassium-based (n ϭ 3; 2 ϭ 6; P Ͻ 0.05; control, 69.7 Ϯ 14.6 M⍀; baclofen, 48.1 Ϯ 6.0 M⍀; CGP55845, 78.4 Ϯ 9.7 M⍀) but not with a cesium-based pipette solution (n ϭ 3; 2 ϭ 4.7; P ϭ 0.10; control, 232 Ϯ 53 M⍀; baclofen, 195 Ϯ 42 M⍀; CGP55845, 261 Ϯ 46 M⍀). The small effect of GABA B receptors in cesium may reflect a small drift in EPSC amplitude as well as the presence of postsynaptic cesium-permeable GIRK channels (Hommers et al 2003).…”

Gi protein activation in intact cells involves subunit rearrangement rather than dissociation

“…GIRK currents activated by fluorescent G protein subunits were measured as described (23), except that, in the present study, an Axopatch 200B and CLAMPEX software (Axon Instruments, Foster City, CA) was used. Experimental conditions including external and internal solutions were the same as described (24).…”

“…G␤␥-subunits when coexpressed without G␣-subunits are known to constitutively activate GIRK currents (24,30). To test for functional interaction of fluorescent G␤␥-subunits, HEK cells were transiently transfected with ␣ 2A -AR GIRK1͞4 channel subunits and with or without G␤␥-subunits composed of either WT G␤ 1 ␥ 2 or G␤ 1 plus CFP-N-G␥ 2 or CFP-N-G␤ 1 plus G␥ 2 .…”