Molecular determinants for activation of G-protein-coupled inward rectifier K+ (GIRK) channels by extracellular acidosis

Synaptic cleft acidification occurs following vesicle release. Such a pH change may affect synaptic transmissions in which G-protein-coupled inward rectifier K(+) (GIRK) channels play a role. To elucidate the effect of extracellular pH (pH(o)) on GIRK channels, we performed experiments on heteromeric GIRK1/GIRK4 channels expressed in Xenopus oocytes. A decrease in pH(o) to 6.2 augmented GIRK1/GIRK4 currents by approximately 30%. The channel activation was reversible and dependent on pH(o) levels. This effect was produced by selective augmentation of single channel conductance without change in the open-state probability. To determine which subunit was involved, we took advantage of homomeric expression of GIRK1 and GIRK4 by introducing a single mutation. We found that homomeric GIRK1-F137S and GIRK4-S143T channels were activated at pH(o) 6.2 by approximately 20 and approximately 70%, respectively. Such activation was eliminated when a histidine residue in the M1-H5 linker was mutated to a non-titratable glutamine, i.e. H116Q in GIRK1 and H120Q in GIRK4. Both of these histidines were required for pH sensing of the heteromeric channels, because the mutation of one of them diminished but not abolished the pH(o) sensitivity. The pH(o) sensitivity of the heteromeric channels was completely lost when both were mutated. Thus, these results suggest that the GIRK-mediated synaptic transmission is determined by both neurotransmitter and protons with the transmitter accounting for only 70% of the effect on postsynaptic cell and protons released together with the transmitter contributing to the other 30%.     

Regulation of inward rectifier K+ channels by shift of intracellular pH dependence

The mechanistic link between mitochondrial metabolism and inward rectifier K+ channel activity was investigated by studying the effects of a mitochondrial inhibitor, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on inward rectifiers of the Kir2 subfamily expressed in Xenopus oocytes, using two-electrode voltage-clamp, patch-clamp, and intracellular pH recording. FCCP inhibited Kir2.2 and Kir2.3 currents and decreased intracellular pH, but the pH change was too small to account for the inhibitory effect by itself. However, pre-incubation of oocytes with imidazole prevented both the pH decrease and the inhibition of Kir2.2 and Kir2.3 currents by FCCP. The pH dependence of Kir2.2 was shifted to higher pH in membrane patches from FCCP-treated oocytes compared to control oocytes. Therefore, the inhibition of Kir2.2 by FCCP may involve a combination of intracellular acidification and a shift in the intracellular pH dependence of these channels. To investigate the sensitivity of heteromeric channels to FCCP, we studied its effect on currents expressed by heteromeric tandem dimer constructs. While Kir2.1 homomeric channels were insensitive to FCCP, both Kir2.1-Kir2.2 and Kir2.1-Kir2.3 heterotetrameric channels were inhibited. These data support the notion that mitochondrial dysfunction causes inhibition of heteromeric inward rectifier K+ channels. The reduction of inward rectifier K+ channel activity observed in heart failure and ischemia may result from the mitochondrial dysfunction that occurs in these conditions.     

Regulation of the inward rectifying properties of G-protein-activated inwardly rectifying K+ (GIRK) channels by Gbeta gamma subunits

Gbetagamma subunits are known to bind to and activate G-protein-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 Gbetagamma subunit mediated regulation of the inwardly rectifying properties of these channels. I(KACh) activated by submaximal concentrations of acetylcholine exhibited a approximately 2.5-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 Gbetagamma subunits. Analysis of the outward current block underlying inward rectification demonstrated that the fraction of instantaneously blocked channels was reduced when Gbetagamma was over-expressed. The Gbetagamma 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 Gbetagamma 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.     

Hyposmotic challenge inhibits inward rectifying K+ channels in cerebral arterial smooth muscle cells

This study sought to define whether inward rectifying K(+) (K(IR)) channels were modulated by vasoactive stimuli known to depolarize and constrict intact cerebral arteries. Using pressure myography and patch-clamp electrophysiology, initial experiments revealed a Ba(2+)-sensitive K(IR) current in cerebral arterial smooth muscle cells that was active over a physiological range of membrane potentials and whose inhibition led to arterial depolarization and constriction. Real-time PCR, Western blot, and immunohistochemical analyses established the expression of both K(IR)2.1 and K(IR)2.2 in cerebral arterial smooth muscle cells. Vasoconstrictor agonists known to depolarize and constrict rat cerebral arteries, including uridine triphosphate, U46619, and 5-HT, had no discernable effect on whole cell K(IR) activity. Control experiments confirmed that vasoconstrictor agonists could inhibit the voltage-dependent delayed rectifier K(+) (K(DR)) current. In contrast to these observations, a hyposmotic challenge that activates mechanosensitive ion channels elicited a rapid and sustained inhibition of the K(IR) but not the K(DR) current. The hyposmotic-induced inhibition of K(IR) was 1) mimicked by phorbol-12-myristate-13-acetate, a PKC agonist; and 2) inhibited by calphostin C, a PKC inhibitor. These findings suggest that, by modulating PKC, mechanical stimuli can regulate K(IR) activity and consequently the electrical and mechanical state of intact cerebral arteries. We propose that the mechanoregulation of K(IR) channels plays a role in the development of myogenic tone.     

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.     

Inhibition of inward rectifying tonoplast channels by a vacuolar factor: Physiological and kinetic implications

Summary Regulation of ion-channel activity must take place in order to regulate ion transport. In case of tonoplast ion channels, this is possible on both the cytoplasmic and the vacuolar side. Isolated vacuoles of youngVigna unguiculata seedlings show no or hardly any channel activity at tonoplast potentials >80 mV, in the vacuole-attached configuration. When the configuration is changed to an excised patch or whole vacuole, a fast (excised patch) or slow (whole vacuole) increase of inward rectifying channel activity is seen. This increase is accompanied by a shift in the voltage-dependent gating to less hyperpolarized potentials. In the whole vacuole configuration the level of inward current increases and also the activation kinetics changes. Induction of channel activity takes up to 20 min depending on the age of the plants used and the diameter of the vacuole. On the basis of the estimated diffusion velocities, it is hypothesized that a compound with a mol wt of 20,000 to 200,000 is present in vacuoles of young seedlings, which shifts the population of channels to a less voltage-sensitive state.Ecotrans publication no. 27.     

Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade

Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.     

Direct activation of cloned K(atp) channels by intracellular acidosis

ATP-sensitive K(+) (K(ATP)) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the K(ATP) changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted K(ATP) was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all K(ATP). The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous K(ATP) in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the K(ATP). The pH-dependent activation may enable the K(ATP) to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions.     

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.     

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.     

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.     

Dual-mode phospholipid regulation of human inward rectifying potassium channels

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using ⁸⁶Rb⁺ flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP₂) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP₂ increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP₂, and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP₂ and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.     

Inhibition of inwardly rectifying K(+) channels by cGMP in pulmonary vascular endothelial cells

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca(2+) concentration. Membrane-permeable analogs of guanosine 3',5'-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential (E(m)), which influences the electrochemical gradient for Ca(2+) influx, is regulated by K(+) channels, we investigated the effect of 8-bromo-cGMP on E(m) and inwardly rectifying K(+) (K(IR)) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to -50 mV that were abolished by application of 10 microM Ba(2+), consistent with K(IR) current. Ba(2+) also depolarized both cell types. 8-Bromo-cGMP (10(-3) M) depolarized BPAEC and BMVEC and inhibited K(IR) current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of K(IR) current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca(2+) entry.     

The polyamine binding site in inward rectifier K+ channels

Strongly inwardly rectifying potassium channels exhibit potent and steeply voltage-dependent block by intracellular polyamines. To locate the polyamine binding site, we have examined the effects of polyamine blockade on the rate of MTSEA modification of cysteine residues strategically substituted in the pore of a strongly rectifying Kir channel (Kir6.2[N160D]). Spermine only protected cysteines substituted at a deep location in the pore, between the "rectification controller" residue (N160D in Kir6.2, D172 in Kir2.1) and the selectivity filter, against MTSEA modification. In contrast, blockade with a longer synthetic polyamine (CGC-11179) also protected cysteines substituted at sites closer to the cytoplasmic entrance of the channel. Modification of a cysteine at the entrance to the inner cavity (169C) was unaffected by either spermine or CGC-11179, and spermine was clearly "locked" into the inner cavity (i.e., exhibited a dramatically slower exit rate) following modification of this residue. These data provide physical constraints on the spermine binding site, demonstrating that spermine stably binds at a deep site beyond the "rectification controller" residue, near the extracellular entrance to the channel.     

Gating of inward-rectifier K+ channels by intracellular pH.

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.     

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.     

Differential sensitivity of inward rectifier K+ channels to metabolic inhibitors

Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.     

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO(2)/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO(2) and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K(+) channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.     

Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.