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 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.     

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.     

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.     

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.     

Ligand-induced closure of inward rectifier Kir6.2 channels traps spermine in the pore

Small organic amines block open voltage-gated K+ channels and can be trapped by subsequent closure. Such studies provide strong evidence for voltage gating occurring at the intracellular end of the channel. We engineered the necessary properties (long block times with unblock kinetics comparable to, or slower than, the kinetics of gating) into spermine-blocked, ATP-gated (N160D,L157C) mutant KATP channels, in order to test the possibility of "blocker trapping" in ligand-gated Kir channels. Spermine block of these channels is very strongly voltage dependent, such that, at positive voltages, the off-rate of spermine is very low. A brief pulse to negative voltages rapidly relieves the block, but no such relief is observed in ATP-closed channels. The results are well fit by a simple kinetic model that assumes no spermine exit from closed channels. The results incontrovertibly demonstrate that spermine is trapped in channels that are closed by ATP, and implicate the M2 helix bundle crossing, or somewhere lower, as the probable location of the gate.     

Internal aluminum block of plant inward K(+) channels

Aluminum (Al) inhibits inward K(+) channels (K(in)) in both root hair and guard cells, which accounts for at least part of the Al toxicity in plants. To understand the mechanism of Al-induced K(in) inhibition, we performed patch clamp analyses on K(in) in guard cells and on KAT1 channels expressed in Xenopus oocytes. Our results show that Al inhibits plant K(in) by blocking the channels at the cytoplasmic side of the plasma membrane. In guard cells, single-channel recording revealed that Al inhibition of K(in) occurred only upon internal exposure. Using both "giant patch" recording and single-channel analyses, we found that Al reduced KAT1 open probability and changed its activation kinetics through an internal membrane-delimited mechanism. We also provide evidence that a Ca(2)+ channel-like pathway that is sensitive to antagonists verapamil and La(3)+ mediates Al entry across the plasma membrane. We conclude that Al enters plant cells through a Ca(2)+ channel-like pathway and inhibits K(+) uptake by internally blocking K(in).     

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.     

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.     

[K+] dependence of polyamine-induced rectification in inward rectifier potassium channels (IRK1, Kir2.1)

The effects of permeant (K+) ions on polyamine (PA)-induced rectification of cloned strong inwardly rectifying channels (IRK1, Kir2.1) expressed in Xenopus oocytes were examined using patch-clamp techniques. The kinetics of PA-induced rectification depend strongly on external, but not internal, K+ concentration. Increasing external [K+] speeds up "activation" kinetics and shifts rectification to more positive membrane potentials. The shift of rectification is directly proportional to the shift in the K+ reversal potential (EK) with slope factors +0.62, +0.81, and +0.91 for 1 mM putrescine (Put), 100 microM spermidine and 20 microM spermine (Spm), respectively. The time constant of current activation, resulting from unblock of Spm, also shifts directly in proportion to EK with slope factor +1.1. Increasing internal [K+] slows down activation kinetics and has a much weaker relieving effect on block by PA: Spm-induced rectification and time constant of activation (Spm unblock) shift directly in proportion to the corresponding change in EK with slope factors -0.15 and +0.31, respectively, for 20 microM Spm. The speed up of activation kinetics caused by increase of external [K+] cannot be reversed by equal increase of internal [K+]. The data are consistent with the hypothesis that the conduction pathway of strong inward rectifiers is a long and narrow pore with multiple binding sites for PA and K+.     

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.     

[K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1).

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.     

Gating the pore of potassium leak channels

A key feature of potassium channel function is the ability to switch between conducting and non-conducting states by undergoing conformational changes in response to cellular or extracellular signals. Such switching is facilitated by the mechanical coupling of gating domain movements to pore opening and closing. Two-pore domain potassium channels (K_2P) conduct leak or background potassium-selective currents that are mostly time- and voltage-independent. These channels play a significant role in setting the cell resting membrane potential and, therefore modulate cell responsiveness and excitability. Thus, K_2P channels are key players in numerous physiological processes and were recently shown to also be involved in human pathologies. It is well established that K_2P channel conductance, open probability and cell surface expression are significantly modulated by various physical and chemical stimuli. However, in understanding how such signals are translated into conformational changes that open or close the channels gate, there remain more open questions than answers. A growing line of evidence suggests that the outer pore area assumes a critical role in gating K_2P channels, in a manner reminiscent of C-type inactivation of voltage-gated potassium channels. In some K_2P channels, this gating mechanism is facilitated in response to external pH levels. Recently, it was suggested that K_2P channels also possess a lower activation gate that is positively coupled to the outer pore gate. The purpose of this review is to present an up-to-date summary of research describing the conformational changes and gating events that take place at the K_2P channel ion-conducting pathway during the channel regulation.     

The interaction of Na(+) and K(+) in the pore of cyclic nucleotide-gated channels

The permeability ratio between K(+) and Na(+) ions in cyclic nucleotide-gated channels is close to 1, and the single channel conductance has almost the same value in the presence of K(+) or Na(+). Therefore, K(+) and Na(+) ions are thought to permeate with identical properties. In the alpha-subunit from bovine rods there is a loop of three prolines at positions 365 to 367. When proline 365 is mutated to a threonine, a cysteine, or an alanine, mutant channels exhibit a complex interaction between K(+) and Na(+) ions. Indeed K(+), Rb(+) and Cs(+) ions do not carry any significant macroscopic current through mutant channels P365T, P365C and P365A and block the current carried by Na(+) ions. Moreover in mutant P365T the presence of K(+) in the intracellular (or extracellular) medium caused the appearance of a large transient inward (or outward) current carried by Na(+) when the voltage command was quickly stepped to large negative (or positive) membrane voltages. This transient current is caused by a transient potentiation, i.e., an increase of the open probability. The permeation of organic cations through these mutant channels is almost identical to that through the wild type (w.t.) channel. Also in the w.t. channel a similar but smaller transient current is observed, associated to a slowing down of the channel gating evident when intracellular Na(+) is replaced with K(+). As a consequence, a rather simple mechanism can explain the complex behavior here described: when a K(+) ion is occupying the pore there is a profound blockage of the channel and a potentiation of gating immediately after the K(+) ion is driven out. Potentiation occurs because K(+) ions slow down the rate constant K(off) controlling channel closure. These results indicate that K(+) and Na(+) ions do not permeate through CNG channels in the same way and that K(+) ions influence the channel gating.     

Mutational analysis of the Shab-encoded delayed rectifier K(+) channels in Drosophila.

K(+) currents in Drosophila muscles have been resolved into two voltage-activated currents (I(A) and I(K)) and two Ca(2+)-activated currents (I(CF) and I(CS)). Mutations that affect I(A) (Shaker) and I(CF) (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I(K)) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K(+) channel gene, we show that this gene encodes the delayed rectifier K(+) channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K(+) current (I(K)). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivo physiological role for the Shab-encoded K(+) channels in Drosophila. The availability of mutations that affect I(K) opens up possibilities for studying I(K) and its role in larval muscle excitability.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

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.