Recovery from slow inactivation of Shab K+ channels

We have recently examined slow inactivation of Shab channels. Here we extend our characterization of Shab slow inactivation by presenting the properties of recovery from inactivation. The observations support our proposal that Shab reaches the same inactivated state either from open or closed states and suggest that closed and open state inactivation share the same mechanism. Regarding the latter, we also show that external K⁺ and TEA slow down recovery from inactivation in agreement with the hypothesis that the mechanism of Shab inactivation qualitatively differs from C-type inactivation.     

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.     

Phosphorylation affects voltage gating of the delayed rectifier K+ channel by electrostatic interactions

The delayed rectifier K+ channel of the squid axon undergoes a series of modifications in its kinetic and conductive parameters when it is phosphorylated as the result of shifts in its voltage-dependent parameters. These effects can be interpreted as due to electrostatic interaction between the voltage sensor of the channel and the transferred phosphate from ATP. Using different concentrations of intracellular Mg2+, we determined the density of surface charges seen by the K+ channel voltage sensor before and after phosphorylation. Values for the surface charge density in the cytoplasmic side of the membrane were between 1/350 and 1/250 e-/A2 in the absence of ATP and between 1/160 and 1/155 e-/A2 under phosphorylating conditions. Incorporation of a surface potential into a kinetic model for the delayed rectifier channel can predict quantitatively phosphorylation-like changes in K+ currents. These results provide evidence for the importance of electrostatic interactions as one of the mechanisms by which phosphorylation modulates the behavior of voltage-dependent channels.     

Subcellular localization of the delayed rectifier K(+) channels KCNQ1 and ERG1 in the rat heart

In the heart, several K(+) channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K(+) currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K(+) channels, KCNQ1 and ether-a-go-go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter. Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular localization pattern, KCNQ1 is more widely distributed within the cardiac cells. The localization of these K(+) channels to the transverse tubular system close to the Ca(2+) channels renders them with maximal repolarizing effect.     

Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels

K(+) channels encoded by the human ether-à-go-go-related gene (HERG) are distinguished from most other voltage-gated K(+) channels by an unusually slow deactivation process that enables cardiac I(Kr), the corresponding current in ventricular cells, to contribute to the repolarization of the action potential. When the first 16 amino acids are deleted from the amino terminus of HERG, the deactivation rate is much faster (Wang, J., M.C. Trudeau, A.M. Zappia, and G.A. Robertson. 1998. J. Gen. Physiol. 112:637-647). In this study, we determined whether the first 16 amino acids comprise a functional domain capable of slowing deactivation. We also tested whether this "deactivation subdomain" slows deactivation directly by affecting channel open times or indirectly by a blocking mechanism. Using inside-out macropatches excised from Xenopus oocytes, we found that a peptide corresponding to the first 16 amino acids of HERG is sufficient to reconstitute slow deactivation to channels lacking the amino terminus. The peptide acts as a soluble domain in a rapid and readily reversible manner, reflecting a more dynamic regulation of deactivation than the slow modification observed in a previous study with a larger amino-terminal peptide fragment (Morais Cabral, J.H., A. Lee, S.L. Cohen, B.T. Chait, M. Li, and R. Mackinnon. 1998. Cell. 95:649-655). The slowing of deactivation by the peptide occurs in a dose-dependent manner, with a Hill coefficient that implies the cooperative action of at least three peptides per channel. Unlike internal TEA, which slows deactivation indirectly by blocking the channels, the peptide does not reduce current amplitude. Nor does the amino terminus interfere with the blocking effect of TEA, indicating that the amino terminus binding site is spatially distinct from the TEA binding site. Analysis of the single channel activity in cell-attached patches shows that the amino terminus significantly increases channel mean open time with no alteration of the mean closed time or the addition of nonconducting states expected from a pore block mechanism.We propose that the four amino-terminal deactivation subdomains of the tetrameric channel interact with binding sites uncovered by channel opening to specifically stabilize the open state and thus slow channel closing.     

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.     

Changes in the inactivation of rat Kv1.4 K(+) channels induced by varying the number of inactivation particles

N-type inactivation of rat Kv1.4 channels with one, two, or four inactivation balls was investigated using homogeneous populations of channels expressed in Xenopus oocytes. Tandem dimeric and tetrameric constructs of Kv1.4 were made. Channels encoded by tandem cDNAs Kv1. 4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) have two or only one tethered inactivation ball, respectively, whereas Kv1.4 itself encodes channels having four inactivation balls. The time constants for inactivation of macroscopic currents were increased significantly as the number of inactivation balls was decreased, whereas the time constants for recovery from inactivation were not modified. The ratios of the rate constants of inactivation (k(inact)) of Kv1.4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) channels to that of the Kv1.4 channel were 0.65 and 0.4, respectively, whereas the ratios of the rate constant of recovery (k(rec)) of these channels to that of Kv1.4 were almost unity. The rate constants k(inact) for channels having two and four inactivation balls are smaller than those that would be expected if inactivation balls on each channel are independent, suggesting some interaction occurs between inactivation balls. Furthermore, noninactivating current became apparent as the number of inactivation balls on a channel was decreased.     

NADPH binding to beta-subunit regulates inactivation of voltage-gated K(+) channels

Ancillary beta-subunits regulate the voltage-dependence and the kinetics of Kv currents. The Kvbeta proteins bind pyridine nucleotides with high affinity but the role of cofactor binding in regulating Kv currents remains unclear. We found that recombinant rat Kvbeta 1.3 binds NADPH (K(d)=1.8+/-0.02 microM) and NADP(+) (K(d)=5.5+/-0.9 microM). Site-specific modifications at Tyr-307 and Arg-316 decreased NADPH binding; whereas, K(d) NADPH was unaffected by the R241L mutation. COS-7 cells transfected with Kv1.5 cDNA displayed non-inactivating currents. Co-transfection with Kvbeta1.3 accelerated Kv activation and inactivation and induced a hyperpolarizing shift in voltage-dependence of activation. Kvbeta-mediated inactivation of Kv currents was prevented by the Y307F and R316E mutations but not by the R241L substitution. Additionally, the R316E mutation weakened Kvalpha-beta interaction. Inactivation of Kv currents by Kvbeta:R316E was restored when excess NADPH was included in the patch pipette. These observations suggest that NADPH binding is essential for optimal interaction between Kvalpha and beta subunits and for Kvbeta-induced inactivation of Kv currents.     

Acceleration of P/C-type inactivation in voltage-gated K(+) channels by methionine oxidation

Oxidation of amino acid residues causes noticeable changes in gating of many ion channels. We found that P/C-type inactivation of Shaker potassium channels expressed in Xenopus oocytes is irreversibly accelerated by patch excision and that this effect was mimicked by application of the oxidant H(2)O(2), which is normally produced in cells by the dismutase action on the superoxide anion. The inactivation time course was also accelerated by high concentration of O(2). Substitution of a methionine residue located in the P-segment of the channel with a leucine largely eliminated the channel's sensitivity to patch excision, H(2)O(2), and high O(2). The results demonstrate that oxidation of methionine is an important regulator of P/C-type inactivation and that it may play a role in mediating the cellular responses to hypoxia/hyperoxia.     

温度对下丘脑神经元膜上延迟整流性K^＋通道活动密度的影响

目的：探讨温度对下丘脑神经元延迟整流性K^＋单离子通道（Ik）活动密度的影响。方法：采用膜片钳细胞贴附式技术观察和记录32℃、37℃和39℃时下丘脑神经元Ik的活动。结果：温度升高,记录到封 膜片上Ik活动的机率增大,由32℃时的34.6％升至39℃的51.05,而且出现通道活动;Ik通道活动密度明显增高（P＜0.05）,32℃、37℃和39℃时膜上的通道活动密度分别为0.71、1.22 和1.52channels/μm^2。结论：温度升高,下丘脑神经元上Ik通道更多地处于活动状态,这可能利于丘脑神经元对体温活动的调节。     

Inhibition of delayed rectifier potassium channels and induction of arrhythmia: a novel effect of celecoxib and the mechanism underlying it

Selective inhibitors of cyclooxygenase-2 (COX-2), such as rofecoxib (Vioxx), celecoxib (Celebrex), and valdecoxib (Bextra), have been developed for treating arthritis and other musculoskeletal complaints. Selective inhibition of COX-2 over COX-1 results in preferential decrease in prostacyclin production over thromboxane A2 production, thus leading to less gastric effects than those seen with nonselective COX inhibitors such as acetylsalicylic acid (aspirin). Here we show a novel effect of celecoxib via a mechanism that is independent of COX-2 inhibition. The drug inhibited the delayed rectifier (Kv2) potassium channels from Drosophila, rats, and humans and led to pronounced arrhythmia in Drosophila heart and arrhythmic beating of rat heart cells in culture. These effects occurred despite the genomic absence of cyclooxygenases in Drosophila and the failure of acetylsalicylic acid, a potent inhibitor of both COX-1 and COX-2, to inhibit rat Kv2.1 channels. A genetically null mutant of Drosophila Shab (Kv2) channels reproduced the cardiac effect of celecoxib, and the drug was unable to further enhance the effect of the mutation. These observations reveal an unanticipated effect of celecoxib on Drosophila hearts and on heart cells from rats, implicating the inhibition of Kv2 channels as the mechanism underlying this effect.     

GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K(+)-selective, K(+)-sensing ion channel

Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K(+) channel. GORK represents a new member of the plant Shaker K(+) channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K(+) currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage- and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells.     

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.     

K(+)-dependent composite gating of the yeast K(+) channel, Tok1

TOK1 encodes an outwardly rectifying K(+) channel in the plasma membrane of the budding yeast Saccharomyces cerevisiae. It is capable of dwelling in two kinetically distinct impermeable states, a near-instantaneously activating R state and a set of related delayed activating C states (formerly called C(2) and C(1), respectively). Dwell in the R state is dependent on membrane potential and both internal and external K(+) in a manner consistent with the K(+) electrochemical potential being its determinant, where dwell in the C states is dependent on voltage and only external K(+). Whereas activation from the C states showed high temperature dependencies, typical of gating transitions in other Shaker-like channels, activation from the R state had a temperature dependence nearly as low as that of simple ionic diffusion. These findings lead us to conclude that although the C states reflect the activity of an internally oriented channel gate, the R state results from an intrinsic gating property of the channel filter region.     

Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K(+) channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K(+) channel inhibitors. Unlike most other K(+) channel-blocking toxins, HaTx1 adopts an "inhibitor cystine knot" motif and is composed of two beta-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca(2+) and Na(+) channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels.     

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.     

Saxitoxin is a gating modifier of HERG K+ channels

Potassium (K+) channels mediate numerous electrical events in excitable cells, including cellular membrane potential repolarization. The hERG K+ channel plays an important role in myocardial repolarization, and inhibition of these K+ channels is associated with long QT syndromes that can cause fatal cardiac arrhythmias. In this study, we identify saxitoxin (STX) as a hERG channel modifier and investigate the mechanism using heterologous expression of the recombinant channel in HEK293 cells. In the presence of STX, channels opened slower during strong depolarizations, and they closed much faster upon repolarization, suggesting that toxin-bound channels can still open but are modified, and that STX does not simply block the ion conduction pore. STX decreased hERG K+ currents by stabilizing closed channel states visualized as shifts in the voltage dependence of channel opening to more depolarized membrane potentials. The concentration dependence for steady-state modification as well as the kinetics of onset and recovery indicate that multiple STX molecules bind to the channel. Rapid application of STX revealed an apparent "agonist-like" effect in which K+ currents were transiently increased. The mechanism of this effect was found to be an effect on the channel voltage-inactivation relationship. Because the kinetics of inactivation are rapid relative to activation for this channel, the increase in K+ current appeared quickly and could be subverted by a decrease in K+ currents due to the shift in the voltage-activation relationship at some membrane potentials. The results are consistent with a simple model in which STX binds to the hERG K+ channel at multiple sites and alters the energetics of channel gating by shifting both the voltage-inactivation and voltage-activation processes. The results suggest a novel extracellular mechanism for pharmacological manipulation of this channel through allosteric coupling to channel gating.     

Energetics of Shaker K channels block by inactivation peptides

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage- dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations.