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.     

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.     

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.     

Backbone resonance assignments for the cytoplasmic regions of G protein-activated inwardly rectifying potassium channel 1 (GIRK1)

G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. A variety of ligands, including heterotrimeric G protein βγ subunits (Gβγ), bind to the cytoplasmic regions of GIRK and modulate its activity. We established the backbone resonance assignments of ²H/¹³C/¹⁵N-labeled cytoplasmic regions of mouse GIRK1, which form a tetramer with a molecular weight of 96 K.     

Critical determinants of the G protein gamma subunits in the Gbetagamma stimulation of G protein-activated inwardly rectifying potassium (GIRK) channel activity

The betagamma subunits of G proteins modulate inwardly rectifying potassium (GIRK) channels through direct interactions. Although GIRK currents are stimulated by mammalian Gbetagamma subunits, we show that they were inhibited by the yeast Gbetagamma (Ste4/Ste18) subunits. A chimera between the yeast and the mammalian Gbeta1 subunits (ymbeta) stimulated or inhibited GIRK currents, depending on whether it was co-expressed with mammalian or yeast Ggamma subunits, respectively. This result underscores the critical functional influence of the Ggamma subunits on the effectiveness of the Gbetagamma complex. A series of chimeras between Ggamma2 and the yeast Ggamma revealed that the C-terminal half of the Ggamma2 subunit is required for channel activation by the Gbetagamma complex. Point mutations of Ggamma2 to the corresponding yeast Ggamma residues identified several amino acids that reduced significantly the ability of Gbetagamma to stimulate channel activity, an effect that was not due to improper association with Gbeta. Most of the identified critical Ggamma residues clustered together, forming an intricate network of interactions with the Gbeta subunit, defining an interaction surface of the Gbetagamma complex with GIRK channels. These results show for the first time a functional role for Ggamma in the effector role of Gbetagamma.     

Calcium-sensing receptor decreases cell surface expression of the inwardly rectifying K+ channel Kir4.1

The Ca(2+)-sensing receptor (CaR) regulates salt and water transport in the kidney as demonstrated by the association of gain of function CaR mutations with a Bartter syndrome-like, salt-wasting phenotype, but the precise mechanism for this effect is not fully established. We found previously that the CaR interacts with and inactivates an inwardly rectifying K(+) channel, Kir4.1, which is expressed in the distal nephron that contributes to the basolateral K(+) conductance, and in which loss of function mutations are associated with a complex phenotype that includes renal salt wasting. We now find that CaR inactivates Kir4.1 by reducing its cell surface expression. Mutant CaRs reduced Kir4.1 cell surface expression and current density in HEK-293 cells in proportion to their signaling activity. Mutant, activated Gα(q) reduced cell surface expression and current density of Kir4.1, and these effects were blocked by RGS4, a protein that blocks signaling via Gα(i) and Gα(q). Other α subunits had insignificant effects. Knockdown of caveolin-1 blocked the effect of Gα(q) on Kir4.1, whereas knockdown of the clathrin heavy chain had no effect. CaR had no comparable effect on the renal outer medullary K(+) channel, an apical membrane distal nephron K(+) channel that is internalized by clathrin-coated vesicles. Co-immunoprecipitation studies showed that the CaR and Kir4.1 physically associate with caveolin-1 in HEK cells and in kidney extracts. Thus, the CaR decreases cell surface expression of Kir4.1 channels via a mechanism that involves Gα(q) and caveolin. These results provide a novel molecular basis for the inhibition of renal NaCl transport by the CaR.     

Permeance of Cs+ and Rb+ through the inwardly rectifying K+ channel in guinea pig ventricular myocytes

Summary Inward currents carried by external Cs, Rb, NH₄ and K through theI _K1 channel were studied using a whole-cell voltage clamp technique. Cs, NH₄, and Rb currents could be recorded negative to –40 mV following depolarizing prepulses (0 mV and 200–1000 msec in duration). The current activation displayed an instantaneous component followed by a monoexponential increase () to a peak amplitude. Subsequent inactivation was fit by a single exponential, _i. With hyperpolarization, and _i decreasede-fold per 36 and 25 mV, respectively. In Ca-free external solutions (pipette [Mg]0.3mm), inactivation was absent, consistent with the hypothesis that inactivation represents time- and voltage-dependent block of Cs, NH₄, and Rb currents by external Ca. The inactivation and degree of steady-state block was greatest when Cs was the charge carrier, followed by NH₄, and then Rb. K currents, however, did not inactivate in the presence of Ca. Na and Li did not carry any significant current within the resolution of our recordings. Comparison ofpeak inward current ratios (I _x/I_K) as an index of permeability revealed a higher permeance of Cs (0.15), NH₄ (0.30), and Rb (0.51) relative to K (1.0) than that obtained by comparing thesteady-state current ratios (CsNH₄RbK0.010.060.211.0). At any given potential, was smaller the more permeant the cation. In the absence of depolarizing prepulses, the amplitude of was reduced. Divalent-free solutions did not significantly affect activatio in the presence of 0.3mm pipette [Mg]. When pipette [Mg] was buffered to 50 m, however, removal of external Ca and Mg lead to a four- to fivefold increase in Cs currents and loss of both time-dependent activation and inactivation (reversible upon repletion of external Ca).These results suggest that (i) permeability ratios forI _K1 should account for differences in the degree to which monovalent currents are blocked by extracellular Ca and (ii) extracellular or intracellular divalent cations contribute to the slow phase of activation which may represent either (a) the actual rate of Mg or Ca extrusion from the channel into the cell, a process which may be enhanced by repulsive interaction with the incoming permeant monovalent cation or (b) an intrinsic gating process that is strongly modulated by the permeant monovalent ion and divalent cations.     

Molecular determinants responsible for differential cellular distribution of G protein-gated inwardly rectifying K+ channels

Activation of the heteromeric G protein-gated inwardly rectifying K(+) channel (GIRK) GIRK1 and GIRK4 subunits gives rise to I(KACh), which controls excitability in atrial tissue. Although homomeric GIRK4 channels localize to the plasma membrane and display moderate function, GIRK1 channels fail to localize to the cell surface and do not exhibit significant function as homomers. Using oocytes to express GFP-tagged GIRK1 and GIRK4 and chimeras between these two proteins, we have identified two regions, one in the proximal C terminus and another in the distal N terminus that are critical for their subcellular localization. Replacement of both of these regions in GIRK1 with corresponding regions from GIRK4 was required for efficient expression of GIRK1 on the plasma membrane. Replacement of either region by itself was ineffective. The distal N terminus and proximal C terminus have been previously suggested to play important roles in ER-export and subunit co-assembly respectively in this family of channels. Our data indicate for the first time that both of these regions need to work in concert to mediate efficient targeting of these channels to the plasma membrane.     

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.     

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.     

Activation of the endothelin receptor inhibits the G protein-coupled inwardly rectifying potassium channel by a phospholipase A2-mediated mechanism

To develop a malleable system to model the well-described, physiological interactions between Gq/11 - coupled receptor and Gi/o-coupled receptor signaling, we coexpressed the endothelin A receptor, the mu-opioid receptor, and the G protein-coupled inwardly rectifying potassium channel (Kir 3) heteromultimers in Xenopus laevis oocytes. Activation of the Gi/o-coupled mu-opioid receptor strongly increased Kir 3 channel current, whereas activation of the Gq/11-coupled endothelin A receptor inhibited the Kir 3 response evoked by mu-opioid receptor activation. The magnitude of the inhibition of Kir 3 was channel subtype specific; heteromultimers composed of Kir 3.1 and Kir 3.2 or Kir 3.1 and Kir 3.4 were significantly more sensitive to the effects of endothelin-1 than heteromultimers composed of Kir 3.1 and Kir 3.5. The difference in sensitivity of the heteromultimers suggests that the endothelin-induced inhibition of the opioid- activated current was caused by an effect at the channel rather than at the opioid receptor. The endothelin-1-mediated inhibition was mimicked by arachidonic acid and blocked by the phospholipase A2 inhibitor arachidonoyl trifluoromethyl ketone. Consistent with a possible phospholipase A2-mediated mechanism, the endothelin-1 effect was blocked by calcium chelation with BAPTA-AM and was not affected by kinase inhibition by either staurosporine or genistein. The data suggest the hypothesis that Gq/11-coupled receptor activation may interfere with Gi/o-coupled receptor signaling by the activation of phospholipase A2 and subsequent inhibition of effector function by a direct effect of an eicosanoid on the channel.     

G protein beta2 subunit-derived peptides for inhibition and induction of G protein pathways. Examination of voltage-gated Ca2+ and G protein inwardly rectifying K+ channels

Voltage-gated Ca2+ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K+ channels (GIRK) are modulated via direct binding of G proteins. The modulation is mediated by G protein betagamma subunits. By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the modulatory domains of the G protein beta subunit on the recombinant P/Q-type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2+ currents of cultured hippocampal neurons. We found that Gbeta2 subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein modulation of the examined ion channels. In particular, the 25-amino acid peptide derived from the Gbeta2 N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the Gbeta2 C terminus induced modulation of voltage-gated Ca2+ channels and GIRK channels. Fluorescence resonance energy transfer (FRET) analysis of the live action of these peptides revealed that the 25-amino acid peptide diminished the FRET signal between G protein beta2gamma3 subunits, indicating a reorientation between G protein beta2gamma3 subunits in the presence of the peptide. In contrast, the 35-amino acid peptide increased the FRET signal between GIRK1,2 channel subunits, similarly to the Gbetagamma-mediated FRET increase observed for this GIRK subunit combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is structured. These results indicate that individual G protein beta subunit domains can act as independent, separate modulatory domains to either induce or inhibit G protein modulation for several effector proteins.     

Mapping the Gbetagamma-binding sites in GIRK1 and GIRK2 subunits of the G protein-activated K+ channel

G protein-activated K+ channels (Kir3 or GIRK) are activated by direct binding of Gbetagamma. The binding sites of Gbetagamma in the ubiquitous GIRK1 (Kir3.1) subunit have not been unequivocally charted, and in the neuronal GIRK2 (Kir3.2) subunit the binding of Gbetagamma has not been studied. We verified and extended the map of Gbetagamma-binding sites in GIRK1 by using two approaches: direct binding of Gbetagamma to fragments of GIRK subunits (pull down), and competition of these fragments with the Galphai1 subunit for binding to Gbetagamma. We also mapped the Gbetagamma-binding sites in GIRK2. In both subunits, the N terminus binds Gbetagamma. In the C terminus, the Gbetagamma-binding sites in the two subunits are not identical; GIRK1, but not GIRK2, has a previously unrecognized Gbetagamma-interacting segments in the first half of the C terminus. The main C-terminal Gbetagamma-binding segment found in both subunits is located approximately between amino acids 320 and 409 (by GIRK1 count). Mutation of C-terminal leucines 262 or 333 in GIRK1, recognized previously as crucial for Gbetagamma regulation of the channel, and of the corresponding leucines 273 and 344 in GIRK2 dramatically altered the properties of K+ currents via GIRK1/GIRK2 channels expressed in Xenopus oocytes but did not appreciably reduce the binding of Gbetagamma to the corresponding fusion proteins, indicating that these residues are mainly important for the regulation of Gbetagamma-induced changes in channel gating rather than Gbetagamma binding.     

Phosphatase is responsible for run down, and probably G protein- mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3- thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Calcium influx mediated by the inwardly rectifying K+ channel Kir4.1 (KCNJ10) at low external K+ concentration

COS-1 cells with heterologeous expression of the Kir4.1 (KCNJ10) channel subunit, possess functional Kir4.1 channels and become capable to generating cytosolic Ca2+ transients, upon lowering of the extracellular K+ concentration to 2 mM or below. These Ca2+ transients are blocked by external Ba2+ (100 microM). Acute brain stem slices from wild-type mice (second post-natal week), which were loaded with the fluorescent Ca2+ indicator Oregon Green BAPTA-1-AM, were exposed to 0.2 mM K+. Under these conditions astrocytes, but not neurons, responded with cytosolic Ca2+ elevations in wild-type mice. This astrocyte-specific response has previously been used to identify astroglial cells type [R. Dallwig, H. Vitten, J.W. Deitmer, A novel barium-sensitive calcium influx into rat astrocytes at low external potassium. Cell Calcium 28 (2000) 247-259]. In Kir4.1 knock-out (Kir4.1-/-) mice, the number of responding cells was dramatically reduced and the Ca2+ transients in responding cells were significantly smaller than in wild-type mice. Our results indicate that Kir4.1 channels are the molecular substrate for the observed Ca2+ influx in astrocytes under conditions of low external K+-concentration.     

Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart

G protein-activated inwardly rectifying potassium channels (Kir3, GIRK) provide an important mechanism for neurotransmitter regulation of membrane excitability. GIRK channels are tetramers containing various combinations of Kir3 subunits (Kir3.1--Kir3.4). We find that different combinations of Kir3 subunits exhibit a surprisingly complex spectrum of trafficking phenotypes. Kir3.2 and Kir3.4, but not Kir3.1, contain ER export signals that are important for plasma membrane expression of Kir3.1/Kir3.2 and Kir3.1/Kir3.4 heterotetramers, the GIRK channels found in the brain and the heart, respectively. Additional motifs in Kir3.2 and Kir3.4 control the trafficking between endosome and plasma membrane. In contrast, the Kir3.3 subunit potently inhibits plasma membrane expression by diverting the heterotetrameric channels to lysosomes. Such rich trafficking behaviors provide a mechanism for dynamic regulation of GIRK channel density in the plasma membrane.     

Quantitative analysis of outward rectifying K+ channel currents in guard cell protoplasts fromVicia faba

Summary A quantitative analysis of the time and voltage dependence of outward-rectifying K⁺ currents ( ) in guard cells fromVicia faba is described using the whole-cell patch-clamp technique. After step depolarizations from –75 mV to potentials positive to –40 mV, time-dependent outward currents were produced, which have recently been identified as K⁺ channel currents. This K⁺ current was characterized according to its time dependence and its steady-state activation. could be described in terms of a Hodgkin-Huxley type conductance. Activation of the current in time was sigmoid and was well fitted by raising the activation variable to the second power. Deactivating tail currents were single exponentials, which suggests that only one conductance underlies this slow outward K⁺ current. Rates of channel closing were strongly dependent on the membrane potential, while rates of channel opening showed only limited voltage dependence leading to a highly asymmetric voltage dependence for channel closing and opening. The presented analysis provides a quantitative basis for the understanding of channel gating and channel functions in plant cells.