K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.     

ATP-sensitive K(+)-channel run-down is Mg2+ dependent

ATP-sensitive K(+)-channel currents were recorded from isolated membrane patches and voltage-clamped CRI-G1 insulin-secreting cells. Internal Mg2+ ions inhibited ATP-K+ channels by a voltage-dependent block of the channel current and decrease of open-state probability. The run-down of ATP-K+ channel activity was also shown to be [Mg2+]i dependent, being almost abolished in Mg2(+)-free conditions. Substitution of Mn2+ for Mg2+ did not prevent run-down, nor did the presence of phosphate-donating nucleotides, a protease or phosphatase inhibitor or replacement of Cl- by gluconate.     

ABA regulation of K(+)-permeable channels in maize subsidiary cells

An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K(+) ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K(+) uptake and K(+) release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K(+) channels. ABA caused a pronounced rise in time-dependent outward-rectifying K(+) currents (K(out)) at alkaline pH and decreased inward-rectifying K(+) currents (K(in)) in a Ca(2+)-dependent manner. Our results show that the ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K(+) transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K(+) channels.     

Mechanism of estradiol-induced block of voltage-gated K+ currents in rat medial preoptic neurons

The present study was conducted to characterize possible rapid effects of 17-β-estradiol on voltage-gated K(+) channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-β-estradiol affects the K(+) channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-β-Estradiol rapidly (within seconds) and reversibly reduced the K(+) currents, showing an EC(50) value of 9.7 μM. The effect was slightly voltage dependent, but independent of external Ca(2+), and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K(+) currents, membrane-impermeant forms of estradiol did not reduce the K(+) currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the K(V)-2-channel blocker r-stromatoxin-1. The time course of K(+) current in 17-β-estradiol, with a time-dependent inhibition and a slight dependence on external K(+), suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-β-estradiol binds to open K(+) channels. It was concluded that 17-β-estradiol rapidly reduces voltage-gated K(+) currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.     

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.     

Activation of ATP-sensitive K+-channels of cardiomyocytes by endogenous cardiopeptides]

The "patch-clamp" method was applied to rat ventricular cardiomyocytes to study the effect of endogenous cardiopeptides (a component of a cardiologic medicine "cardialin") on single K(+)-channel currents. It was noted that ATP-sensitive K(+)-channel activity increased only in the presence of 10(-8)-10(-6) mg.ml-1 peptide solution in the patch-clamp micropipette. To check up the suggestion that G-protein takes part in a direct reaction of K(+)-channel by external ligand we studied the effect of GTP-gamma-S (0.1 mM) in the presence of Mg2+ (1 mM) on the K(+)-channel in "run-down" state in the "inside-out" configuration. It was demonstrated experimentally that reactivation of the ATP-sensitive K(+)-channel develops in the presence of GTP-analogue.     

Effects of endothelin-1 on K(+) currents from rat ventricular myocytes.

It has been suggested that the positive inotropic effect of the vasoactive peptide hormone, endothelin-1 (ET-1), involves inhibition of cardiac K(+) currents. In order to identify the K(+) currents modulated by ET-1, the outward K(+) currents of isolated rat ventricular myocytes were investigated using whole-cell patch-clamp recording techniques. Outward currents were elicited by depolarisation to +40 mV for 200 ms from the holding potential of -60 mV. Currents activated rapidly, reaching a peak (I(pk)) of 1310 +/- 115 pA and subsequently inactivating to an outward current level of 1063 +/- 122 pA at the end of the voltage-pulse (I(late)) (n = 11). ET-1 (20 nM) reduced I(pk) by 247.6 +/- 60.7 pA (n = 11, P < 0.01) and reduced I(late) by 323.2 +/- 43.9 pA (P < 0.001). The effects of ET-1 were abolished in the presence of the nonselective ET receptor antagonist, PD 142893 (10 microM, n = 5). Outward currents were considerably reduced and the effects of ET-1 were not observed when K(+) was replaced with Cs(+) in the experimental solutions; this indicates that ET-1 modulated K(+)-selective currents. A double-pulse protocol was used to investigate the inactivation of the currents. The voltage-dependent inactivation of the currents from potentials positive to -80 mV was fitted by a Boltzmann equation revealing the existence of an inactivating transient outward component (I(to)) and a noninactivating steady-state component (I(ss)). ET-1 markedly inhibited I(ss) by 43.0 +/- 3.8% (P < 0.001, n = 7) and shifted the voltage-dependent inactivation of I(to) by +3.3 +/- 1.2 mV (P < 0.05). Although ET-1 had little effect on the onset of inactivation of the currents elicited from a conditioning potential of -70 mV, the time-independent noninactivating component of the currents was markedly inhibited. In conclusion, the predominant effect of ET-1 was to inhibit a noninactivating steady-state background K(+) current (I(ss)). These results are consistent with the hypothesis that I(ss) inhibition contributes to the inotropic effects of ET-1.     

Recombinant hTASK1 is an O(2)-sensitive K(+) channel

Hypoxic inhibition of background K(+) channels is crucial to O(2) sensing by chemoreceptor tissues, but direct demonstration of O(2) sensitivity by any member of this K(+) channel family is lacking. HEK293 cells were transfected with a pcDNA3.1-hTASK1 construct; expression of hTASK1 was verified using RT-PCR and immunocytochemistry. Whole-cell K(+) currents of cells stably expressing hTASK-1 were, as anticipated, extremely sensitive to extracellular pH, within the physiological range (IC(50) approximately 7.0). All cells expressing this signature pH sensitivity were acutely modulated by pO(2); reduction of pO(2) from 150 to <40 mmHg (at pH 7.4) caused rapid and reversible suppression of pH-sensitive K(+) currents. Furthermore, these two regulatory signals clearly acted at the same channel, since the magnitude of the O(2)-sensitive current was dependent on the extracellular pH. These data represent the first direct verification that hTASK1 is O(2)-sensitive and reinforce the idea that this K(+) channel is key to O(2) sensing in chemoreceptors.     

Effects of choline on Na(+)- and K(+)-interactions with the Na+/K(+)-ATPase.

Choline chloride, 100 mM, stimulates Na+/K(+)-ATPase activity of a purified dog kidney enzyme preparation when Na+ is suboptimal (9 mM Na+ and 10 mM K+) and inhibits when K+ is suboptimal (90 mM Na+ and 1 mM K+), but has a negligible effect at optimal concentrations of both (90 mM Na+ and 10 mM K+). Stimulation occurs at low Na+ to K+ ratios, but not at those same ratios when the actual Na+ concentration is high (90 mM). Stimulation decreases or disappears when incubation pH or temperature is increased or when Li+ is substituted for K+ or Rb+. Choline+ also reduces the Km for MgATP at the low ratio of Na+ to K+ but not at the optimal ratio. In the absence of K+, however, choline+ does not stimulate at low Na+ concentrations: either in the Na(+)-ATPase reaction or in the E1 to E2P conformational transition. Together, these observations indicate that choline+ accelerates the rate-limiting step in the Na+/K(+)-ATPase reaction cycle, K(+)-deocclusion; consequently, optimal Na+ concentrations reflect Na+ accelerating that step also. Thus, the observed K0.5 for Na+ includes high-affinity activation of enzyme phosphorylation and low-affinity acceleration of K(+)-deocclusion. Inhibition of Na+/K(+)-ATPase and K(+)-nitrophenylphosphatase reactions by choline+ increases as the K(+)-concentration is decreased; the competition between choline+ and K+ may represent a similar antagonism between conformations selected by choline+ and by K+.     

Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system

A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B.     

DPP6 establishes the A-type K(+) current gradient critical for the regulation of dendritic excitability in CA1 hippocampal neurons

Subthreshold-activating A-type K(+) currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K(+) current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability.     

Genetic dissection of Ca2(+)-dependent ion channel function in Paramecium.

The ciliated protozoan, Paramecium, broadcasts the activity of its individual ion channel classes through its swimming behaviour. This fact has made it possible to isolate mutants with defective ion currents, simply by selecting individuals with abnormal swimming patterns. At least four of Paramecium's ion currents are activated by rising intracellular calcium concentration, including two K+ currents and a Na+ current. A variety of cell lines with defects in these Ca2(+)-dependent currents have been isolated: in several cases, the defects have been traced to mutations in the structural gene for calmodulin. Sequence analysis of calmodulins from these and other Ca2(+)-dependent ion-current mutants may enable a detailed mapping of putative channel interaction domains on the surface of the calmodulin molecule.     

TASK-5, a new member of the tandem-pore K(+) channel family.

TASKs are members of the recently identified K(+) channel family (KCNKx). Four TASKs (TASK1-4) identified so far form functional K(+) channels and encode background K(+) channels in various cell types. Recently, another member (TASK-5) was identified in the human genome. We cloned it and studied its tissue expression and functional properties. TASK-5 shares 51% amino acid identity with TASK-1 and TASK-3. Northern blot analysis showed that TASK-4 mRNA was expressed primarily in the adrenal gland and pancreas. Single nucleotide polymorphism (SNP) was found at amino acid position 95 that normally forms part of the K(+) channel selectivity filter. Neither form of TASK-5 showed channel activity when transfected in COS-7 cells. Exchange of C-termini of TASK-3 and TASK-5 failed to generate whole-cell currents. Thus, TASK-5 is a new member of the tandem-pore K(+) channel family but does not produce a functional plasma membrane K(+) current by itself.     

K(+) versus Na(+) ions in a K channel selectivity filter: a simulation study

Molecular dynamics simulations of a bacterial potassium channel (KcsA) embedded in a phospholipid bilayer reveal significant differences in interactions of the selectivity filter with K(+) compared with Na(+) ions. K(+) ions and water molecules within the filter undergo concerted single-file motion in which they translocate between adjacent sites within the filter on a nanosecond timescale. In contrast, Na(+) ions remain bound to sites within the filter and do not exhibit translocation on a nanosecond timescale. Furthermore, entry of a K(+) ion into the filter from the extracellular mouth is observed, whereas this does not occur for a Na(+) ion. Whereas K(+) ions prefer to sit within a cage of eight oxygen atoms of the filter, Na(+) ions prefer to interact with a ring of four oxygen atoms plus two water molecules. These differences in interactions in the selectivity filter may contribute to the selectivity of KcsA for K(+) ions (in addition to the differences in dehydration energy between K(+) and Na(+)) and the block of KcsA by internal Na(+) ions. In our simulations the selectivity filter exhibits significant flexibility in response to changes in ion/protein interactions, with a somewhat greater distortion induced by Na(+) than by K(+) ions.     

多不饱和脂肪酸对成年雪貂心肌钾通道的作用

本研究是在成年雪貂的心肌上研究多不饱和脂肪酸（PUFA）对电压门控钾通道的效应。我们观察到,n-3 PUFA能抑制短时性外向钾电流（Ito)和延迟整流钾电流（IK),而对内向整流钾电流（IK1）则没有明显影响。二十二碳六烯酸（DHA）对Ito和Ik能产生浓度依赖性的抑制作用,其IC50分别为7.5和20μmol/L,但不影响IK1。二十碳五烯酸（EPA）对这三种钾通道的作用与DHA相似。花生四烯酸（5或10μmol/L)先引起IK的抑制,然后引起IK,AA的激活;用环氧合酶抑制剂消炎痛可以阻断花生四烯酸激活IK,AA的作用。不具有抗心律失常作用的单不饱和脂肪酸和饱和脂肪酸都不明显影响这些钾通道的活性。上述实验结果证明,n-3 PUFA能抑制心肌细胞的Ito和IK,但和我们以前报道的PUFA对心肌钠电流和钙电流的作用相比,其对Ito和IK抑制作用的效能较低。n-3 PUFA的抗心律失常效应可能与它们抑制心肌钠、钙、钾通道的作用有关。     

Voltage dependence of slow inactivation in Shaker potassium channels results from changes in relative K(+) and Na(+) permeabilities

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.     

Two mechanisms of K(+)-dependent potentiation in Kv2.1 potassium channels

Elevation of external [K(+)] potentiates outward K(+) current through several voltage-gated K(+) channels. This increase in current magnitude is paradoxical in that it occurs despite a significant decrease in driving force. We have investigated the mechanisms involved in K(+)-dependent current potentiation in the Kv2.1 K(+) channel. With holding potentials of -120 to -150 mV, which completely removed channels from the voltage-sensitive inactivated state, elevation of external [K(+)] up to 10 mM produced a concentration-dependent increase in outward current magnitude. In the absence of inactivation, currents were maximally potentiated by 38%. At more positive holding potentials, which produced steady-state inactivation, K(+)-dependent potentiation was enhanced. The additional K(+)-dependent potentiation (above 38%) at more positive holding potentials was precisely equal to a K(+)-dependent reduction in steady-state inactivation. Mutation of two lysine residues in the outer vestibule of Kv2.1 (K356 and K382), to smaller, uncharged residues (glycine and valine, respectively), completely abolished K(+)-dependent potentiation that was not associated with inactivation. These mutations did not influence steady-state inactivation or the K(+)-dependent potentiation due to reduction in steady-state inactivation. These results demonstrate that K(+)-dependent potentiation can be completely accounted for by two independent mechanisms: one that involved the outer vestibule lysines and one that involved K(+)-dependent removal of channels from the inactivated state. Previous studies demonstrated that the outer vestibule of Kv2.1 can be in at least two conformations, depending on the occupancy of the selectivity filter by K(+) (Immke, D., M. Wood, L. Kiss, and S. J. Korn. 1999. J. Gen. Physiol. 113:819-836; Immke, D., and S. J. Korn. 2000. J. Gen. Physiol. 115:509-518). This change in conformation was functionally defined by a change in TEA sensitivity. Similar to the K(+)-dependent change in TEA sensitivity, the lysine-dependent potentiation depended primarily (>90%) on Lys-356 and was enhanced by lowering initial K(+) occupancy of the pore. Furthermore, the K(+)-dependent changes in current magnitude and TEA sensitivity were highly correlated. These results suggest that the previously described K(+)-dependent change in outer vestibule conformation underlies the lysine-sensitive, K(+)-dependent potentiation mechanism.