IRK1 inward rectifier K(+) channels exhibit no intrinsic rectification

In intact cells the depolarization-induced outward IRK1 currents undergo profound relaxation so that the steady-state macroscopic I-V curve exhibits strong inward rectification. A modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of Mg(2+) and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. Furthermore, IRK1 exhibits significant extracellular K(+)-sensitive relaxation of its inward current, a feature that has been likened to the C-type inactivation observed in the voltage-activated Shaker K(+) channels. We found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in HEPES and ethylenediamine in EDTA. Therefore, inherently, IRK1 channels are essentially ohmic at the macroscopic level, and the voltage jump-induced current relaxations do not reflect IRK1 gating but the unusually high affinity of its pore for cations. Furthermore, our study helps define the optimal experimental conditions for studying IRK1.     

Gating dependence of inner pore access in inward rectifier K(+) channels

Cation channel gating may occur either at or below the inner vestibule entrance or at the selectivity filter. To differentiate these possibilities in inward rectifier (Kir) channels, we examined cysteine accessibility in the ATP-gated Kir6.2 channel. MTSEA and MTSET both block channels and modify M2 cysteines with identical voltage dependence. If entry is restricted to open channels, modification rates will slow in ATP-closed channels, but because the reagent can be trapped in the pore following brief openings, this may not be apparent until open probability is extremely low (<0.01). When these conditions are met, modification does slow significantly, indicating gated access and highlighting an important caveat for interpretation of MTS-accessibility measurements: reagent "trapping" in nominally "closed" channels may obscure gated access.     

Importance of the G protein gamma subunit in activating G protein-coupled inward rectifier K(+) channels

Arachidonic acid (AA) is generated via Rac-mediated phospholipase A2 (PLA2) activation in response to growth factors and cytokines and is implicated in cell growth and gene expression. In this study, we show that AA activates the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in a time- and dose-dependent manner. Indomethacin and nordihydroguaiaretic acid, potent inhibitors of cyclooxygenase and lipoxygenase, respectively, did not exert inhibitory effects on AA-induced SAPK/JNK activation, thereby indicating that AA itself could activate SAPK/JNK. As Rac mediates SAPK/JNK activation in response to a variety of stressful stimuli, we examined whether the activation of SAPK/JNK by AA is mediated by Rac1. We observed that AA-induced SAPK/JNK activation was significantly inhibited in Rat2-Rac1N17 dominant-negative mutant cells. Furthermore, treatment of AA induced membrane ruffling and production of hydrogen peroxide, which could be prevented by Rac1N17. These results suggest that AA acts as an upstream signal molecule of Rac, whose activation leads to SAPK/JNK activation, membrane ruffling and hydrogen peroxide production.     

Interactions of cations with the cytoplasmic pores of inward rectifier K(+) channels in the closed state

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.     

Revisiting inward rectification: K ions permeate through Kir2.1 channels during high-affinity block by spermidine

Outward currents through Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells, and such currents are subjected to voltage-dependent block by intracellular Mg(2+) and polyamines that bind to both high- and low-affinity sites on the channels. Under physiological conditions, high-affinity block is saturated and yet outward Kir2.1 currents can still occur, implying that high-affinity polyamine block cannot completely eliminate outward Kir2.1 currents. However, the underlying molecular mechanism remains unknown. Here, we show that high-affinity spermidine block, rather than completely occluding the single-channel pore, induces a subconducting state in which conductance is 20% that of the fully open channel. In a D172N mutant lacking the high-affinity polyamine-binding site, spermidine does not induce such a substate. However, the kinetics for the transitions between the substate and zero-current state in wild-type channels is the same as that of low-affinity block in the D172N mutant, supporting the notion that these are identical molecular events. Thus, the residual outward current after high-affinity spermidine block is susceptible to low-affinity block, which determines the final amplitude of the outward current. This study provides a detailed insight into the mechanism underlying the emergence of outward Kir2.1 currents regulated by inward rectification attributed to high- and low-affinity polyamine blocks.     

CO(2) inhibits specific inward rectifier K(+) channels by decreases in intra- and extracellular pH

Hypercapnia has been shown to affect cellular excitability by modulating K(+) channels. To understand the mechanisms for this modulation, four cloned K(+) channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO(2) for 4-6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pH(i), pH(o)) dropped during CO(2) exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pH(i) produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pH(i) level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO(2) without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K(+) channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO(2), and (3) these effects are independent of cytosol-soluble factors.     

The pore of plant K(+) channels is involved in voltage and pH sensing: domain-swapping between different K(+) channel alpha-subunits

Plant K(+) uptake channel types differ with respect to their voltage, Ca(2)+, and pH dependence. Here, we constructed recombinant chimeric channels between KST1, a member of the inward-rectifying, acid-activated KAT1 family, and AKT3, a member of the weakly voltage-dependent, proton-blocked AKT2/3 family. The homologous pore regions of AKT3 (amino acids 216 to 287) and KST1 (amino acids 217 to 289) have been exchanged to generate the two chimeric channels AKT3/(p)KST1 and KST1/(p)AKT3. In contrast to AKT3 wild-type channels, AKT3/(p)KST1 revealed a strong inward rectification reminiscent of that of KST1. Correspondingly, the substitution of the KST1 by the AKT3 pore led to less pronounced rectification properties of KST1/(p)AKT3 compared with wild-type KST1. Besides the voltage dependence, the interaction between the chimera and extracellular H(+) and Ca(2)+ resembled the properties of the inserted rather than the respective wild-type pore. Whereas AKT3/(p)KST1 was acid activated and Ca(2)+ insensitive, extracellular protons and Ca(2)+ inhibited KST1/(p)AKT3. The regulation of the chimeric channels by cytoplasmic protons followed the respective wild-type backbone of the chimeric channels, indicating that the intracellular pH sensor is located outside the P domain. We thus conclude that essential elements for external pH and Ca(2)+ regulation and for the rectification of voltage-dependent K(+) uptake channels are located within the channel pore.     

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.     

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.     

Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.     

Oncogenic potential of EAG K(+) channels.

We have investigated the possible implication of the cell cycle-regulated K(+) channel ether à go-go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune-depressed mice. These data provide evidence for the oncogenic potential of EAG.     

Role of inwardly rectifying K(+) channels in K(+)-induced cerebral vasodilatation in vivo

We tested whether activation of inwardly rectifying K(+) (Kir) channels, Na(+)-K(+)-ATPase, or nitric oxide synthase (NOS) play a role in K(+)-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K(+)] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 +/- 1, 27 +/- 4, 35 +/- 4, 43 +/- 12, and 47 +/- 16%, respectively). Responses to K(+) were inhibited by approximately 50% by the Kir channel inhibitor BaCl(2) (30 and 100 microM). In contrast, neither ouabain (1-100 microM, a Na(+)-K(+)-ATPase inhibitor) nor N(G)-nitro-L-arginine (30 microM, a NOS inhibitor) had any effect on K(+)-induced vasodilatation. These concentrations of K(+) also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 microM BaCl(2). RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K(+)-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na(+)-K(+)-ATPase, or NOS.     

Molecular basis of ion selectivity, block, and rectification of the inward rectifier Kir3.1/Kir3.4 K(+) channel

The glycine-tyrosine-glycine (GYG) sequence in the p-loop of K+ channel subunits lines a narrow pore through which K+ ions pass in single file intercalated by water molecules. Mutation of the motif can give rise to non-selective channels, but it is clear that other structural features are also required for selectivity because, for instance, a recently identified class of cyclic nucleotide-gated pacemaker channels has the GYG motif but are poorly K+ selective. We show that mutation of charged glutamate and arginine residues behind the selectivity filter in the Kir3.1/Kir3.4 K+ channel reduces or abolishes K+ selectivity, comparable with previously reported effects in the Kir2.1 K+ channel. It has been suggested that a salt bridge exists between the glutamate-arginine residue pair. Molecular modeling indicates that the salt bridge does exist, and that it acts as a "bowstring" to maintain the rigid bow-like structure of the selectivity filter and restrict selectivity to K+. The modeling shows that relaxation of the bowstring by mutation of the residue pair leads to enhanced flexibility of the p-loop, allowing permeation of other cations, including polyamines. In experiments, mutation of the residue pair can also abolish polyamine-induced inward rectification. The latter effect occurs because polyamines now permeate rather than block the channel, to the remarkable extent that large polyamine currents can be measured.     

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic cells where targets ATP-sensitive K⁺ (K_ATP) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K_ATP channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic KATP channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K_ATP channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K_ATP channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.     

Internal and external K+ help gate the inward rectifier.

Recent investigations have demonstrated substantial reductions in internal [K+] in cardiac Purkinje fibers during myocardial ischemia (Dresdner, K.P., R.P. Kline, and A.L. Wit. 1987, Circ. Res. 60: 122-132). We investigated the possible role these changes in internal K+ might play in abnormal electrical activity by studying the effects of both internal and external [K+] on the gating of the inward rectifier iK1 in isolated Purkinje myocytes with the whole-cell patch-clamp technique. Increasing external [K+] had similar effects on the inward rectifier in the Purkinje myocyte as it does in other preparations: increasing peak conductance and shifting the activation curve in parallel with the potassium reversal potential. A reduction in pipette [K+] from 145 to 25 mM, however, had several dramatic previously unreported effects. It decreased the rate of activation of iK1 at a given voltage by several-fold, reversed the voltage dependence of recovery from deactivation, so that the deactivation rate decreased with depolarization, and caused a positive shift in the midpoint of the activation curve of iK1 that was severalfold smaller than the associated shift of reversal potential. These changes suggest an important role of internal K+ in gating iK1 and may contribute to changes in the electrical properties of the myocardium that occur during ischemia.     

Dynamic sensitivity of ATP-sensitive K(+) channels to ATP

ATP and MgADP regulate K(ATP) channel activity and hence potentially couple cellular metabolism to membrane electrical activity in various cell types. Using recombinant K(ATP) channels that lack sensitivity to MgADP, expressed in COSm6 cells, we demonstrate that similar on-cell activity can be observed with widely varying apparent submembrane [ATP] ([ATP](sub)). Metabolic inhibition leads to a biphasic change in the channel activity; activity first increases, presumably in response to a fast decrease in [ATP](sub), and then declines. The secondary decrease in channel activity reflects a marked increase in ATP sensitivity and is correlated with a fall in polyphosphoinositides (PPIs), including phosphatidylinositol 4,5-bisphosphate, probed using equilibrium labeling of cells with [(3)H]myo-inositol. Both ATP sensitivity and PPIs rapidly recover following removal of metabolic inhibition, and in both cases recovery is blocked by wortmannin. These data are consistent with metabolism having a dual effect on K(ATP) channel activity: rapid activation of channels because of relief of ATP inhibition and much slower reduction of channel activity mediated by a fall in PPIs. These two mechanisms constitute a feedback system that will tend to render K(ATP) channel activity transiently responsive to a change in [ATP](sub) over a wide range of steady state concentrations.     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.     

Interaction mechanisms between polyamines and IRK1 inward rectifier K+ channels

Rectification of macroscopic current through inward-rectifier K+ (Kir) channels reflects strong voltage dependence of channel block by intracellular cations such as polyamines. The voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field, which accompanies the binding-unbinding of a blocker. Residues D172, E224, and E299 in IRK1 are critical for high-affinity binding of blockers. D172 appears to be located somewhat internal to the narrow K+ selectivity filter, whereas E224 and E299 form a ring at a more intracellular site. Using a series of alkyl-bis-amines of varying length as calibration, we investigated how the acidic residues in IRK1 interact with amine groups in the natural polyamines (putrescine, spermidine, and spermine) that cause rectification in cells. To block the pore, the leading amine of bis-amines of increasing length penetrates ever deeper into the pore toward D172, while the trailing amine in every bis-amine binds near a more intracellular site and interacts with E224 and E299. The leading amine in nonamethylene-bis-amine (bis-C9) makes the closest approach to D172, displacing the maximal number of K+ ions and exhibiting the strongest voltage dependence. Cells do not synthesize bis-amines longer than putrescine (bis-C4) but generate the polyamines spermidine and spermine by attaching an amino-propyl group to one or both ends of putrescine. Voltage dependence of channel block by the tetra-amine spermine is comparable to that of block by the bis-amines bis-C9 (shorter) or bis-C12 (equally long), but spermine binds to IRK1 with much higher affinity than either bis-amine does. Thus, counterintuitively, the multiple amines in spermine primarily confer the high affinity but not the strong voltage dependence of channel block. Tetravalent spermine achieves a stronger interaction with the pore by effectively behaving like a pair of tethered divalent cations, two amine groups in its leading half interacting primarily with D172, whereas the other two in the trailing half interact primarily with E224 and E299. Thus, nature has optimized not only the blocker but also, in a complementary manner, the channel for producing rapid, high-affinity, and strongly voltage-dependent channel block, giving rise to exceedingly sharp rectification.