Molecular determinants for sodium-dependent activation of G protein-gated K+ channels

G protein-gated inwardly rectifying K+ channels (GIRKs) are activated by a direct interaction with Gbetagamma subunits and also by raised internal [Na+]. Both processes require the presence of phosphatidylinositol bisphosphate (PIP2). Here we show that the proximal C-terminal region of GIRK2 mediates the Na+-dependent activation of both the GIRK2 homomeric channels and the GIRK1/GIRK2 heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na+-dependent activation of both the homomeric and heteromeric channels. Neutralizing a nearby negative charge, E234S had no effect. The reverse mutation in GIRK1, N217D, was sufficient to restore Na+-dependent activation to the GIRK1N217D/GIRK2D226N heteromeric channels. The D226N mutation did not alter either the single channel properties or the ability of these channels to be activated via the m2-muscarinic receptor. PIP2 dramatically increased the open probability of GIRK1/GIRK2 channels in the absence of Na+ or Gbetagamma but did not preclude further activation by Na+, suggesting that Na+ is not acting simply to promote PIP2 binding to GIRKs. We conclude that aspartate 226 in GIRK2 plays a crucial role in Na+-dependent gating of GIRK1/GIRK2 channels.     

Redox-dependent gating of G protein-coupled inwardly rectifying K+ channels

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 Gbetagamma 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 DTT-dependent 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.     

Rapid desensitization of G protein-gated inwardly rectifying K(+) currents is determined by G protein cycle

Activation of G protein-gated inwardly rectifying K⁺ (GIRK) channels, found in the brain, heart, and endocrine tissue, leads to membrane hyperpolarization that generates neuronal inhibitory postsynaptic potentials, slows the heart rate, and inhibits hormone release. During stimulation of G_i/o-coupled receptors and subsequent channel activation, it has been observed that the current desensitizes. In this study we examined mechanisms underlying fast desensitization of cloned heteromeric neuronal Kir3.1+3.2A and atrial Kir3.1+3.4 channels and also homomeric Kir3.0 currents in response to stimulation of several G_i/o G protein-coupled receptors (GPCRs) expressed in HEK-293 cells (adenosine A₁, adrenergic _2A, dopamine D_2S, M₄ muscarinic, and GABA_B1b/2 receptors). We found that all agonist-induced currents displayed a similar degree of desensitization except the adenosine A₁ receptor, which exhibits an additional desensitizing component. Using the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPS), we found that this is due to a receptor-dependent, G protein-independent process. Using Ca²⁺ imaging we showed that desensitization is unlikely to be accounted for solely by phospholipase C activation and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis. We examined the contribution of the G protein cycle and found the following. First, agonist concentration is strongly correlated with degree of desensitization. Second, competitive inhibition of GDP/GTP exchange by using nonhydrolyzable guanosine 5'-O-(2-thiodiphosphate) (GDPS) has two effects, a slowing of channel activation and an attenuation of the fast desensitization phenomenon. Finally, using specific G subunits we showed that ternary complexes with fast activation rates display more prominent desensitization than those with slower activation kinetics. Together our data suggest that fast desensitization of GIRK currents is accounted for by the fundamental properties of the G protein cycle. G protein-coupled receptor; potassium channel; inward rectifier; kinetics     

Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.     

Properties of inwardly rectifying K+ channels in ventricular myocytes

Summary The voltage- and time-dependent properties of whole-cell, multi-channel (outside-out), and single channel inwardly-rectifying K⁺ currents were studied using adult and neonatal rat, and embryonic chick ventricular myocytes. Inward rectification of the current-voltage relationship was found in the whole-cell and single channel measurements. The steady-state single channel probability of opening decreased with hyperpolarization from E_K, as did the mean open time, thereby explaining the time-dependent inactivation of the macroscopic current. Myocytes dialysed with a Mg⁺⁺-free K⁺ solution (to remove the property of inward rectification) displayed a quasi-linear current-voltage relationship. The outward K⁺ currents flowing through the modified inward rectifier channels were able to be blocked by the local anesthetic and anti-arrhythmic agent, lidocaine.     

Properties of inwardly rectifying K+ channels in ventricular myocytes

The voltage- and time-dependent properties of whole-cell, multi-channel (outside-out), and single channel inwardly-rectifying K+ currents were studied using adult and neonatal rat, and embryonic chick ventricular myocytes. Inward rectification of the current-voltage relationship was found in the whole-cell and single channel measurements. The steady-state single channel probability of opening decreased with hyperpolarization from EK, as did the mean open time, thereby explaining the time-dependent inactivation of the macroscopic current. Myocytes dialysed with a Mg++-free K+ solution (to remove the property of inward rectification) displayed a quasi-linear current-voltage relationship. The outward K+ currents flowing through the modified inward rectifier channels were able to be blocked by the local anesthetic and anti-arrhythmic agent, lidocaine.     

Agonist-induced localization of Gq-coupled receptors and G protein-gated inwardly rectifying K+ (GIRK) channels to caveolae determines receptor specificity of phosphatidylinositol 4,5-bisphosphate signaling

G protein-gated inwardly rectifying K(+) (GIRK) channels are parasympathetic effectors in cardiac myocytes that act as points of integration of signals from diverse pathways. Neurotransmitters and hormones acting on the Gq protein regulate GIRK channels by phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion. In previous studies, we found that endothelin-1, but not bradykinin, inhibited GIRK channels, even though both of them hydrolyze PIP(2) in cardiac myocytes, showing receptor specificity. The present study assessed whether the spatial organization of the PIP(2) signal into caveolar microdomains underlies the specificity of PIP(2)-mediated signaling. Using biochemical analysis, we examined the localization of GIRK and Gq protein-coupled receptors (GqPCRs) in mouse atrial myocytes. Agonist stimulation induced a transient co-localization of GIRK channels with endothelin receptors in the caveolae, excluding bradykinin receptors. Such redistribution was eliminated by caveolar disruption with methyl-β-cyclodextrin (MβCD). Patch clamp studies showed that the specific response of GIRK channels to GqPCR agonists was abolished by MβCD, indicating the functional significance of the caveolae-dependent spatial organization. To assess whether low PIP(2) mobility is essential for PIP(2)-mediated signaling, we blocked the cytoskeletal restriction of PIP(2) diffusion by latrunculin B. This abolished the GIRK channel regulation by GqPCRs without affecting their targeting to caveolae. These data suggest that without the hindered diffusion of PIP(2) from microdomains, PIP(2) loses its signaling efficacy. Taken together, these data suggest that specific targeting combined with restricted diffusion of PIP(2) allows the PIP(2) signal to be compartmentalized to the targets localized closely to the GqPCRs, enabling cells to discriminate between identical PIP(2) signaling that is triggered by different receptors.     

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.     

Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina

Kir5.1 is an inwardly rectifying K⁺ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD₆₅) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K⁺-buffering action of Müller cells. inwardly rectifying potassium channel; heteromerization; glial Müller cells; amacrine cells; potassium siphoning     

Expression of inwardly rectifying K+ channels in the carotid body of rat

The inwardly rectifying K+ channels, Kir1.1, Kir2.3, Kir4.1-Kir5.1, and Kir4.2-Kir5.1, are candidate chemosensory molecules for CO2/H+. Here, we determined the mRNA expression and immunohistochemical localization of these channels in the carotid body (CB) and petrosal ganglion (PG) of the rat. RT-PCR analysis revealed mRNA expression of Kir4.1 and Kir5.1 in CB, and Kir1.1, Kir4.1, and Kir5.1 in PG. Immunohistochemistry identified the glomus cells in CB to express both Kir4.1 and Kir5.1 protein, while the nerve fibers in CB were immunoreactive for Kir1.1, Kir4.1, and Kir5.1. In the PG, immunoreactivity for Kir1.1, Kir4.1, and Kir5.1 was observed in some ganglion cells. Our findings suggest that Kir channels in the peripheral chemoreceptors play a role in sensing hypercapnic acidosis and maintaining the resting membrane potentials.     

Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co-injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co-expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4-5 or 4-5-4-5, but not when they were connected 4-4-5-5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4-5-4-5. Further, the relative order of subunits within the channel contributes to their functional properties.     

Ion selectivity filter regulates local anesthetic inhibition of G-protein-gated inwardly rectifying K+ channels

The weaver mutation (G156S) in G-protein-gated inwardly rectifying K+ (GIRK) channels alters ion selectivity and reveals sensitivity to inhibition by a charged local anesthetic, QX-314, applied extracellularly. In this paper, disrupting the ion selectivity in another GIRK channel, chimera I1G1(M), generates a GIRK channel that is also inhibited by extracellular local anesthetics. I1G1(M) is a chimera of IRK1 (G-protein-insensitive) and GIRK1 and contains the hydrophobic domains (M1-pore-loop-M2) of GIRK1 (G1(M)) with the N- and C-terminal domains of IRK1 (I1). The local anesthetic binding site in I1G1(M) is indistinguishable from that in GIRK2(wv) channels. Whereas chimera I1G1(M) loses K+ selectivity, although there are no mutations in the pore-loop complex, chimera I1G2(M), which contains the hydrophobic domain from GIRK2, exhibits normal K+ selectivity. Mutation of two amino acids that are unique in the pore-loop complex of GIRK1 (F137S and A143T) restores K+ selectivity and eliminates the inhibition by extracellular local anesthetics, suggesting that the pore-loop complex prevents QX-314 from reaching the intrapore site. Alanine mutations in the extracellular half of the M2 transmembrane domain alter QX-314 inhibition, indicating the M2 forms part of the intrapore binding site. Finally, the inhibition of G-protein-activated currents by intracellular QX-314 appears to be different from that observed in nonselective GIRK channels. The results suggest that inward rectifiers contain an intrapore-binding site for local anesthetic that is normally inaccessible from extracellular charged local anesthetics.     

An inwardly rectifying K+ channel is required for patterning

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFβ/BMP signaling. We use Drosophila melanogaster to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, we generated flies in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse.     

Dynamic integration of alpha-adrenergic and cholinergic signals in the atria: role of G protein-regulated inwardly rectifying K+ channels

Numerous heptahelical receptors use activation of heterotrimeric G proteins to convey a multitude of extracellular signals to appropriate effector molecules in the cell. Both high specificity and correct integration of these signals are required for reliable cell function. Yet the molecular machineries that allow each cell to merge information flowing across different receptors are not well understood. Here we demonstrate that G protein-regulated inwardly rectifying K(+) (GIRK) channels can operate as dynamic integrators of alpha-adrenergic and cholinergic signals in atrial myocytes. Acting at the last step of the cholinergic signaling cascade, these channels are activated by direct interactions with betagamma subunits of the inhibitory G proteins (G betagamma), and efficiently translate M(2) muscarinic acetylcholine receptor (M2R) activation into membrane hyperpolarization. The parallel activation of alpha-adrenergic receptors imposed a distinctive "signature" on the function of M2R-activated GIRK1/4 channels, affecting both the probability of G betagamma binding to the channel and its desensitization. This modulation of channel function was correlated with a parallel depletion of G beta and protein phosphatase 1 from the oligomeric GIRK1 complexes. Such plasticity of the immediate GIRK signaling environment suggests that multireceptor integration involves large protein networks undergoing dynamic changes upon receptor activation.