Effects of intracellular magnesium on Kv1.5 and Kv2.1 potassium channels

We characterized the effects of intracellular Mg²⁺ (Mg²⁺_i) on potassium currents mediated by the Kv1.5 and Kv2.1 channels expressed in Xenopus oocytes. Increase in Mg²⁺_i caused a voltage-dependent block of the current amplitude, apparent acceleration of the current kinetics (explained by a corresponding shift in the steady-state activation) and leftward shifts in activation and inactivation dependencies for both channels. The voltage-dependent block was more potent for Kv2.1 [dissociation constant at 0 mV, K_d(0), was ~70 mM and the electric distance of the Mg²⁺ binding site, , was 0.2] than for the Kv1.5 channel [K_d(0)~40 mM and =0.1]. Similar shifts in the voltage-dependent parameters for both channels were described by the Gouy-Chapman formalism with the negative charge density of 1 e^–/100 Ų. Additionally, Mg²⁺_i selectively reduced a non-inactivating current and increased the accumulation of inactivation of the Kv1.5, but not the Kv2.1 channel. A potential functional role of the differential effects of Mg²⁺_i on the Kv channels is discussed.     

PKC regulation of ion channels: The involvement of PIP2

Ion channels are integral membrane proteins whose gating has been increasingly shown to depend on the presence of the low-abundance membrane phospholipid, phosphatidylinositol (4,5) bisphosphate. The expression and function of ion channels is tightly regulated via protein phosphorylation by specific kinases, including various PKC isoforms. Several channels have further been shown to be regulated by PKC through altered surface expression, probability of channel opening, shifts in voltage dependence of their activation, or changes in inactivation or desensitization. In this review, we survey the impact of phosphorylation of various ion channels by PKC isoforms and examine the dependence of phosphorylated ion channels on phosphatidylinositol (4,5) bisphosphate as a mechanistic endpoint to control channel gating.     

Differential inhibition of transient outward currents of Kv1.4 and Kv4.3 by endothelin

The effects of endothelin on the transient outward K(+) currents were compared between Kv1.4 and Kv4.3 channels in Xenopus oocytes expression system. Both transient outward K(+) currents were decreased by stimulation of endothelin receptor ET(A) coexpressed with the K(+) channels. Transient outward current of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, while that of Kv4.3 was decreased by about 60%. By mutagenesis experiments we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in I(to) by ET-1. In Kv4.3 a PKC phosphorylation site was identified which is in part responsible for the decrease in I(to). Differences in the suppression of I(to) could be ascribed to the difference in intracellular signaling including the number of phosphorylation sites. These findings might give clues for the understanding of molecular mechanism of ventricular arrhythmias in heart failure, in which endothelin is involved in the pathogenesis.     

Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.     

Mutant LGI1 inhibits seizure-induced trafficking of Kv4.2 potassium channels

Activity-dependent redistribution of ion channels mediates neuronal circuit plasticity and homeostasis, and could provide pro-epileptic or compensatory anti-epileptic responses to a seizure. Thalamocortical neurons transmit sensory information to the cerebral cortex and through reciprocal corticothalamic connections are intensely activated during a seizure. Therefore, we assessed whether a seizure alters ion channel surface expression and consequent neurophysiologic function of thalamocortical neurons. We report a seizure triggers a rapid (<2h) decrease of excitatory postsynaptic current (EPSC)-like current-induced phasic firing associated with increased transient A-type K(+) current. Seizures also rapidly redistributed the A-type K(+) channel subunit Kv4.2 to the neuronal surface implicating a molecular substrate for the increased K(+) current. Glutamate applied in vitro mimicked the effect, suggesting a direct effect of glutamatergic transmission. Importantly, leucine-rich glioma-inactivated-1 (LGI1), a secreted synaptic protein mutated to cause human partial epilepsy, regulated this seizure-induced circuit response. Human epilepsy-associated dominant-negative-truncated mutant LGI1 inhibited the seizure-induced suppression of phasic firing, increase of A-type K(+) current, and recruitment of Kv4.2 surface expression (in vivo and in vitro). The results identify a response of thalamocortical neurons to seizures involving Kv4.2 surface recruitment associated with dampened phasic firing. The results also identify impaired seizure-induced increases of A-type K(+) current as an additional defect produced by the autosomal dominant lateral temporal lobe epilepsy gene mutant that might contribute to the seizure disorder.     

Kv4.2通道电流与大鼠海马神经元瞬间外向钾电流特性的比较

本研究比较了转染的Kv4.2钾电流与原代培养大鼠海马神经元上瞬间外向钾电流(IA)动力学特征。实验采用瞬时转染,细胞培养和全细胞膜片钳记录等方法。结果表明:转染的Kv4.2通道电流和海马神经元上IA均具有明显的A型电流特征。海马神经元IA的半数最大激活电位和斜率因子分别为-10.0±3.3 mV和13.9±2.6 mV;半数最大失活电位和斜率因子分别为-93.0±11.4 mV和-9.0±1.5 mV;失活后再激活恢复时间常数(T)为27.9±14.1 ms。Kv4.2的半数最大激活电位和斜率因子分别为-9.7±4.1 mV和15.8±5.7 mV;半数最大失活电位和斜率因子分别为-59.4±12.2 mV和8.0±3.1 mV;Kv4.2的灭活后再激活的恢复时间常数τ为172.8±10.0 ms。结果提示:Kv4.2通道电流可能是海马神经元上的IA电流的主要成分,但不是唯一成分。     

Modulation of Native TREK-1 and Kv1.4 K<Superscript>+</Superscript> Channels by Polyunsaturated Fatty Acids and Lysophospholipids

The modulation of TREK-1 leak and Kv1.4 voltage-gated K⁺ channels by fatty acids and lysophospholipids was studied in bovine adrenal zona fasciculata (AZF) cells. In whole-cell patch-clamp recordings, arachidonic acid (AA) (1–20 µM) dramatically and reversibly increased the activity of bTREK-1, while inhibiting bKv1.4 current by mechanisms that occurred with distinctly different kinetics. bTREK-1 was also activated by the polyunsaturated cis fatty acid linoleic acid but not by the trans polyunsaturated fatty acid linolelaidic acid or saturated fatty acids. Eicosatetraynoic acid (ETYA), which blocks formation of active AA metabolites, failed to inhibit AA activation of bTREK-1, indicating that AA acts directly. Compared to activation of bTREK-1, inhibition of bKv1.4 by AA was rapid and accompanied by a pronounced acceleration of inactivation kinetics. Cis polyunsaturated fatty acids were much more effective than trans or saturated fatty acids at inhibiting bKv1.4. ETYA also effectively inhibited bKv1.4, but less potently than AA. bTREK-1 current was markedly increased by lysophospholipids including lysophosphatidyl choline (LPC) and lysophosphatidyl inositol (LPI). At concentrations from 1–5 µM, LPC produced a rapid, transient increase in bTREK-1 that peaked within one minute and then rapidly desensitized. The transient lysophospholipid-induced increases in bTREK-1 did not require the presence of ATP or GTP in the pipette solution. These results indicate that the activity of native leak and voltage-gated K⁺ channels are directly modulated in reciprocal fashion by AA and other cis unsaturated fatty acids. They also show that lysophospholipids enhance bTREK-1, but with a strikingly different temporal pattern. The modulation of native K⁺ channels by these agents differs from their effects on the same channels expressed in heterologous cells, highlighting the critical importance of auxiliary subunits and signaling. Finally, these results reveal that AZF cells express thousands of bTREK-1 K⁺ channels that lie dormant until activated by metabolites including phospholipase A₂ (PLA₂)-generated fatty acids and lysophospholipids. These metabolites may alter the electrical and secretory properties of AZF cells by modulating bTREK-1 and bKv1.4 K⁺ channels.     

Neuronal transmission stimulates the phosphorylation of Kv1.4 channel at Ser229 through protein kinase A1

Phosphorylation of voltage-gated K+ channels (Kv) is involved in regulation of neuronal excitability, synaptic plasticity and neuronal survival. Among Kv channels expressed in the CNS, Kv1.4 is located in the soma, dendrite and axon terminus of neurones in most regions of the brain. Here, we show that Ser229 found within the highly conserved T1 domain of Kv1.4 in cultured rat cortical neurones is phosphorylated by protein kinase A (PKA), as demonstrated by in vitro protein kinase assay and Western blotting with a polyclonal antibody specific against phosphorylated Ser229. Glutamate, high concentrations of K+ or K+ channel blockers known to increase neurotransmission all stimulated the phosphorylation of Kv1.4 at Ser229 via N-methyl-D-aspartate (NMDA), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor, whereas tetradotoxin (TTX), known to block neuronal transmission, and depletion of extracellular Ca2+ inhibited phosphorylation induced by tetraethylammonium (TEA), a non-selective K+ channel blocker. Mutation of Ser229 to Ala229 enhanced the current density. Taken together, elevation of the neuronal transmission stimulates the phosphorylation of Kv1.4 at Ser229 via the Ca2+ influx through NMDA receptor. Thus, it is possible that neuronal transmission regulates neuronal excitability partially through the phosphorylation of Kv1.4S229.     

Contribution of the functional dyad of animal toxins acting on voltage-gated Kv1-type channels.

The 'functional dyad', a well-defined pair of amino acid residues (basic and hydrophobic residues), is a key molecular determinant present in most animal toxins acting on voltage-gated Kv1 channels. It is increasingly used as a working concept to explain how toxins are able to recognize and block their specific ion channel targets. However, other crucial toxin determinants are emerging and the actual role of this 'functional dyad' ought to be clarified, which is the object of the present mini-review.     

Sex differences in and hormonal regulation of Kv1 potassium channel gene expression in the electric organ: molecular control of a social signal

Electric fish communicate with electric organ (EO) discharges (EODs) that are sexually dimorphic, hormone-sensitive, and often individually distinct. The cells of the EO (electrocytes) of the weakly electric fish Sternopygus possess delayed rectifying K+ currents that systematically vary in their activation and deactivation kinetics, and this precise variation in K+ current kinetics helps shape sex and individual differences in the EOD. Because members of the Kv1 subfamily produce delayed rectifier currents, we cloned a number of genes in the Kv1 subfamily from the EO of Sternopygus. Using our sequences and those from genome databases, we found that in teleost fish Kv1.1 and Kv1.2 exist as duplicate pairs (Kv1.1a&b, Kv1.2a&b) whereas Kv1.3 does not. Using real-time quantitative RT-PCR, we found that Kv1.1a and Kv1.2a, but not Kv1.2b, expression in the EO is higher in high EOD frequency females (which have fast EO K+ currents) than in low EOD frequency males (which have slow EO K+ currents). Systemic treatment with dihydrotestosterone decreased Kv1.1a and Kv1.2a, but not Kv1.2b, expression in the EO, whereas treatment with human chorionic gonadotropin (hCG) increased Kv1.2a but not Kv1.1a or Kv1.2b expression in the EO. Thus, systematic variation in the ratios of Kv1 channels expressed in the EO is correlated with individual differences in and sexual dimorphism of a communication signal.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Volume stabilization in human erythrocytes: combined effects of Ca-dependent potassium channels and adenylate metabolism

A mathematical model describing the possible role of Ca²⁺-dependent K⁺ channels and adenylate metabolism in volume stabilization of human erythrocytes was developed. The model predicts that the red blood cell volume can be stabilized either dynamically or stationary over a broad range of cell membrane permeabilities to cations. The dynamic stabilization results from the operation of Ca²⁺-dependent potassium channels. The erythrocyte volume changes less than 10% if the membrane permeability changes abruptly to a value in the range from half to sevenfold higher than the normal one. The stationary stabilization is achieved via controlling the adenylate metabolism. The stationary value of cell volume changes less than 10% when the membrane permeability varies from half the normal value to 15-fold higher than the normal value.     

Regulation of CRAC channels by protein interactions and post-translational modification

Store-operated Ca²⁺ entry (SOCE) is a widespread mechanism to elevate the intracellular Ca²⁺ concentrations and stimulate downstream signaling pathways affecting proliferation, secretion, differentiation and death in different cell types. In immune cells, immune receptor stimulation induces intracellular Ca²⁺ store depletion that subsequently activates Ca²⁺-release-activated-Ca²⁺ (CRAC) channels, a prototype of store-operated Ca²⁺ (SOC) channels. CRAC channel opening leads to activation of diverse downstream signaling pathways affecting proliferation, differentiation, cytokine production and cell death. Recent identification of STIM1 as the endoplasmic reticulum Ca²⁺ sensor and Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tools to dissect the mechanism of activation and regulation of CRAC channels. In this review, we discuss the recent advances in understanding the associating partners and posttranslational modifications of Orai1 and STIM1 proteins that regulate diverse aspects of CRAC channel function.     

Regulation of neuronal ion channels via P2Y receptors

Within the last 15 years, at least 8 different G protein-coupled P2Y receptors have been characterized. These mediate slow metabotropic effects of nucleotides in neurons as well as non-neural cells, as opposed to the fast ionotropic effects which are mediated by P2X receptors. One class of effector systems regulated by various G protein-coupled receptors are voltage-gated and ligand-gated ion channels. This review summarizes the current knowledge about the modulation of such neuronal ion channels via P2Y receptors. The regulated proteins include voltage-gated Ca²⁺ and K⁺ channels, as well as N-methyl-d-aspartate, vanilloid, and P2X receptors, and the regulating entities include most of the known P2Y receptor subtypes. The functional consequences of the modulation of ion channels by nucleotides acting at pre- or postsynaptic P2Y receptors are changes in the strength of synaptic transmission. Accordingly, ATP and related nucleotides may act not only as fast transmitters (via P2X receptors) in the nervous system, but also as neuromodulators (via P2Y receptors). Hence, nucleotides are as universal transmitters as, for instance, acetylcholine, glutamate, or -aminobutyric acid.     

香烟提取物活化大鼠支气管平滑肌细胞蛋白激酶C亚型及下调钾通道BKCa、Kv1.5表达

大电导的钙活化钾通道（large—conductance calcium—activated potassium channel,BKCa）和电压依赖性钾通道Kv1．5在气道高反应性的发生机制中具有重要作用。已知吸烟可致气道高反应,但钾通道的变化在其发病中的作用尚需进一步阐明。本文旨在研究香烟提取物（cigarette smoke extract,CSE）对培养的大鼠支气管平滑肌细胞（bronchial smooth muscle cells,BSMCs）钾通道BKCa和Kv1．5表达的直接作用,以及蛋白激酶C（protein kinaseC,PKC）在其中的作用。实验采用原代培养大鼠BSMCs,用5％CSE刺激,免疫印迹检测PKC亚型的表达和转位,半定量RT—PCR、免疫印迹实验检测BKCa和Kv1．5的mRNA和蛋白表达,然后用PKC抑制剂BIM和G6e6983与CSE共作用,检测其对BKCa和Kv1．5的mRNA和蛋白表达的影响。结果显示,5％CSE使PKCε、η、θ发生明显的膜转位,并使BKCa。和Kv1．5的蛋白和mRNA表达明显降低;选择性PKC抑制剂BIM或G6e6983与CSE共同作用,均可使BKCa和Kv1．5的蛋白和mRNA表达部分恢复。上述结果提示,CSE可引起BSMCs的BKCa和Kv1．5表达下调,PKCε、η、θ参与其信号转导。     

Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.     

Kinetics of xylem loading,membrane potential maintenance,and sensitivity of K+‐permeable channels to reactive oxygen species: physiological traits that differentiate salinity tolerance between pea and barley

Salt sensitive (pea) and salt tolerant (barley) species were used to understand the physiological basis of differential salinity tolerance in crops. Pea plants were much more efficient in restoring otherwise depolarized membrane potential thereby effectively decreasing K⁺ efflux through depolarization‐activated outward rectifying potassium channels. At the same time, pea root apex was 10‐fold more sensitive to physiologically relevant H₂O₂ concentration and accumulated larger amounts of H₂O₂ under saline conditions. This resulted in a rapid loss of cell viability in the pea root apex. Barley plants rapidly loaded Na⁺ into the xylem; this increase was only transient, and xylem and leaf Na⁺ concentration remained at a steady level for weeks. On the contrary, pea plants restricted xylem Na⁺ loading during the first few days of treatment but failed to prevent shoot Na⁺ elevation in the long term. It is concluded that superior salinity tolerance of barley plants compared with pea is conferred by at least three different mechanisms: (1) efficient control of xylem Na⁺ loading; (2) efficient control of H₂O₂ accumulation and reduced sensitivity of non‐selective cation channels to H₂O₂ in the root apex; and (3) higher energy saving efficiency, with less ATP spent to maintain membrane potential under saline conditions.