G protein-coupled inwardly rectifying potassium channels, GIRK/Kir3.x, are gated by the Gbetagamma subunits of the G protein. The molecular mechanism of gating was investigated by employing a novel yeast-based random mutagenesis approach that selected for channel mutants that are active in the absence of Gbetagamma. Mutations in TM2 were found that mimicked the Gbetagamma-activated state. The activity of these channel mutants was independent of receptor stimulation and of the availability of heterologously expressed Gbetagamma subunits but depended on PtdIns(4,5)P(2). The results suggest that the TM2 region plays a key role in channel gating following Gbetagamma binding in a phospholipid-dependent manner. This mechanism of gating in inwardly rectifying K+ channels may be similar to the involvement of the homologous region in prokaryotic KcsA potassium channel and, thus, suggests evolutionary conservation of the gating structure.

G protein coupled inwardly rectifying K ؉ channels (GIRK͞Kir3.x) are mainly activated by a direct interaction with G␤␥ subunits, released upon the activation of inhibitory neurotransmitter receptors. Although G␤␥ binding domains on all four subunits have been found, the relative contribution of each of these binding sites to channel gating has not yet been defined. It is also not known whether GIRK channels open once all G␤␥ sites are occupied, or whether gating is a graded process. We used a tandem tetrameric approach to enable the selective elimination of specific G␤␥ binding domains in the tetrameric context. Here, we show that tandem tetramers are fully operational. Tetramers with only one wild-type channel subunit showed receptor-independent high constitutive activity. The presence of two or three wild-type subunits reconstituted receptor activation gradually. Furthermore, a tetramer with no GIRK1 G␤␥ binding domain displayed slower kinetics of activation. The slowdown in activation was found to be independent of regulator of G protein signaling or receptor coupling, but this slowdown could be reversed once only one G␤␥ binding domain of GIRK1 was added. These results suggest that partial activation can occur under low G␤␥ occupancy and that full activation can be accomplished by the interaction with three G␤␥ binding subunits. The G protein-gated K ϩ channels (GIRK͞Kir3.x) play an important role in various physiological actions. They have been extensively studied in the heart and the brain, where they are involved in the control of heart rate upon vagal stimulation (1) and in the generation of neurotransmitter-mediated slow inhibitory postsynaptic potentials (2). The gating of these channels is mainly mediated by the direct interaction of the G␤␥ subunits of the G protein (3-5), in concert with other factors such as phosphatidylinositol-4,5-bisphosphate (6-8) and Na ϩ ions (refs. 9 and 10; for review see ref. 11).The functional unit of the GIRK channels is a tetramer, usually composed of GIRK1 and either GIRK2 or GIRK4, in the brain and heart, respectively. The GIRK heterotetramers have been suggested to contain two subunits of each of the two gene products in alternating positions (12)(13)(14). A few cases are noted where the functional unit is a homotetramer of either GIRK2 (in the substantia nigra) or GIRK4 (in the atrium) (15, 16).The molecular elements involved in the binding of G␤␥ to the channel and the mechanism of channel activation are not well defined. However, evidence from many laboratories suggests that multiple binding domains, localized to both the N-and C-terminal cytosolic domains, are involved in this action (17-26). Huang et al. (19) identified a G␤␥ binding site within amino acids 318-374 of GIRK1, with a downstream region (amino acids 390-462) acting to enhance the binding of the first segment. They identified these regions in GIRK2-GIRK4 as G␤␥ binding sites as well. The proximal C-terminal binding site may be responsible for agonist-induced activation but does not affect basal acti...

G protein-coupled inwardly rectifying K؉ channels (GIRK) play a major role in inhibitory signaling in excitable and endocrine tissues. The gating mechanism of these channels is mediated by a direct interaction of the G␤␥ subunits of G protein, which are released upon inhibitory neurotransmitter receptor activation. This gating mechanism is further manifested by intracellular factors such as anionic phospholipids and Na ؉ and Mg 2؉ions. In addition to the essential role of these components for channel function, phosphorylation events can also modulate channel activity. In this study we explored the involvement of redox modulation on GIRK channel function. Extracellular application of the reducing agent dithiothreitol (DTT), but not reduced glutathione, activated GIRK channels without affecting their permeation or rectification properties. The DTTdependent activation was found to mimic receptor activation and to act directly on the channel in a membrane delimited fashion. A critical cysteine residue located in the N-terminal cytoplasmic domain was found to be essential for DTT-dependent activation in hetero-and homotetrameric contexts. Interestingly, when mutating this cysteine residue, DTT-dependent activation was abolished, but receptor-mediated channel activation was not affected. These results suggest that intracellular redox potential can play a major role in tuning GIRK channel activity in a receptor-independent manner. This sort of redox modulation can be part of an important cellular protective mechanism against ischemic or hypoxic insults.

G protein-coupled inwardly rectifying potassium channels (GIRK/Kir3) are important elements in controlling cellular excitability. In recent years, tremendous progress has been made toward understanding various components involved in channel activation, modulation, and signaling specificity. In this review, we summarize these recent findings and attempt to put them in context with recently available structural data.

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.