Localization and mobility of the delayed-rectifer K+ channel Kv2.1 in adult cardiomyocytes

The delayed-rectifier voltage-gated K(+) channel (Kv) 2.1 underlies the cardiac slow K(+) current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard immunolocalization approaches do not detect cardiac Kv2.1; therefore, little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of green fluorescent protein (GFP)-Kv2.1 and yellow fluorescent protein-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, whereas Kv1.4 was mobile (tau = 252 +/- 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes (tau = 479 +/- 178 s), although its mobility is slower than Kv1.4 (tau(1) = 18.9 +/- 2.3 s; tau(2) = 305 +/- 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.     

Styrax blocks inward and outward current of Kir2.1 channel

Kir2.1 plays key roles in setting rest membrane potential and modulation of cell excitability. Mutations of Kir2.1, such as D172N or E299V, inducing gain-of-function, can cause type3 short QT syndrome (SQT3) due to the enlarged outward currents. So far, there is no clinical drug target to block the currents of Kir2.1. Here, we identified a novel blocker of Kir2.1, styrax, which is a kind of natural compound selected from traditional Chinese medicine. Our data show that styrax can abolish the inward and outward currents of Kir2.1. The IC₅₀ of styrax for WT, D172N and E299V are 0.0113 ± 0.00075, 0.0204 ± 0.0048 and 0.0122 ± 0.0012 (in volume), respectively. The results indicate that styrax can serve as a novel blocker for Kir2.1.     

Endogenous KCNE subunits govern Kv2.1 K+ channel activation kinetics in Xenopus oocyte studies

Kv2.1 is a voltage-gated potassium (Kv) channel that generates delayed rectifier currents in mammalian heart and brain. The biophysical properties of Kv2.1 and other ion channels have been characterized by functional expression in heterologous systems, and most commonly in Xenopus laevis oocytes. A number of previous oocyte-based studies of mammalian potassium channels have revealed expression-level-dependent changes in channel properties, leading to the suggestion that endogenous oocyte factors regulate channel gating. Here, we show that endogenous oocyte potassium channel KCNE ancillary subunits xMinK and xMiRP2 slow the activation of oocyte-expressed mammalian Kv2.1 channels two-to-fourfold. This produces a sigmoidal relationship between Kv2.1 current density and activation rate in oocyte-based two-electrode voltage clamp studies. The effect of endogenous xMiRP2 and xMinK on Kv2.1 activation is diluted at high Kv2.1 expression levels, or by RNAi knockdown of either endogenous subunit. RNAi knockdown of both xMiRP2 and xMinK eliminates the correlation between Kv2.1 expression level and activation kinetics. The data demonstrate a molecular basis for expression-level-dependent changes in Kv channel gating observed in heterologous expression studies.     

Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells

Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K(+) current of the OHC (I(K)) is also sensitive to Cl(-), although, in contrast to prestin, extracellularly. I(K) is inhibited by reducing extracellular Cl(-) levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K(+) (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl(-). To elucidate the molecular basis of this Cl(-)-sensitive I(K), we looked at potential molecular candidates based on Cl(-) sensitivity and/or similarities in kinetics. For I(K), we identified three different Ca(2+)-independent components of I(K) based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current (Ik(1)), and a slowly inactivating current (Ik(2)). Extracellular Cl(-) differentially affects these components. Because the inactivation time constants of Ik(1) and Ik(2) are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl(-) inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl(-)-sensitive K(+) channels, but found no extracellular Cl(-) sensitivity. The Cl(-) sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl(-) sensitivity.     

Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes.

The molecular structure of oxygen-sensitive delayed-rectifier K+ channels which are involved in hypoxic pulmonary artery (PA) vasoconstriction has yet to be elucidated. To address this problem, we identified the Shab K+ channel Kv2.1 and a novel Shab-like subunit Kv9.3, in rat PA myocytes. Kv9.3 encodes an electrically silent subunit which associates with Kv2.1 and modulates its biophysical properties. The Kv2.1/9.3 heteromultimer, unlike Kv2.1, opens in the voltage range of the resting membrane potential of PA myocytes. Moreover, we demonstrate that the activity of Kv2.1/Kv9.3 is tightly controlled by internal ATP and is reversibly inhibited by hypoxia. In conclusion, we propose that metabolic regulation of the Kv2.1/Kv9.3 heteromultimer may play an important role in hypoxic PA vasoconstriction and in the possible development of PA hypertension.     

A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons

The discrete localization of ion channels is a critical determinant of neuronal excitability. We show here that the dendritic K+ channels Kv2.1 and Kv2.2 were differentially targeted in cultured hippocampal neurons. Kv2.1 was found in high-density clusters on the soma and proximal dendrites, while Kv2.2 was uniformly distributed throughout the soma and dendrites. Chimeras revealed a proximal restriction and clustering domain on the cytoplasmic tail of Kv2.1. Truncations and internal deletions revealed a 26-amino acid targeting signal within which four residues were critical for localization. This signal is not related to other known sequences for neuronal and epithelial membrane protein targeting and represents a novel cytoplasmic signal responsible for proximal restriction and clustering.     

Identification of a cytoplasmic domain important in the polarized expression and clustering of the Kv2.1 K+ channel

The voltage-sensitive K+ channel Kv2.1 has a polarized and clustered distribution in neurons. To investigate the basis for this localization, we expressed wild-type Kv2.1 and two COOH-terminal truncation mutants, delta C318 and delta C187, in polarized epithelial MDCK cells. These functional channel proteins had differing subcellular localization, in that while both wild-type Kv2.1 and delta C187 localized to the lateral membrane in high density clusters, delta C318 was expressed uniformly on both apical and lateral membranes. A chimeric protein containing the hemagglutinin protein from influenza virus and the region of Kv2.1 that differentiates the two truncation mutants (amino acids 536-666) was also expressed in MDCK cells, where it was found in high density clusters similar to those observed for Kv2.1. Polarized expression and clustering of Kv2.1 correlates with detergent solubility, suggesting that interaction with the detergent insoluble cytoskeleton may be necessary for proper localization of this channel.     

Control of outer vestibule dynamics and current magnitude in the Kv2.1 potassium channel

In Kv2.1 potassium channels, changes in external [K+] modulate current magnitude as a result of a K+-dependent interconversion between two outer vestibule conformations. Previous evidence indicated that outer vestibule conformation (and thus current magnitude) is regulated by the occupancy of a selectivity filter binding site by K+. In this paper, we used the change in current magnitude as an assay to study how the interconversion between outer vestibule conformations is controlled. With 100 mM internal K+, rapid elevation of external [K+] from 0 to 10 mM while channels were activated produced no change in current magnitude (outer vestibule conformation did not change). When channels were subsequently closed and reopened in the presence of elevated [K+], current magnitude was increased (outer vestibule conformation had changed). When channels were activated in the presence of low internal [K+], or when K+ flow into conducting channels was transiently interrupted by an internal channel blocker, increasing external [K+] during activation did increase current magnitude (channel conformation did change). These data indicate that, when channels are in the activated state under physiological conditions, the outer vestibule conformation remains fixed despite changes in external [K+]. In contrast, when channel occupancy is lowered, (by channel closing, an internal blocker or low internal [K+]), the outer vestibule can interconvert between the two conformations. We discuss evidence that the ability of the outer vestibule conformation to change is regulated by the occupancy of a nonselectivity filter site by K+. Independent of the outer vestibule-based potentiation mechanism, Kv2.1 was remarkably insensitive to K+-dependent processes that influence current magnitude (current magnitude changed by <7% at membrane potentials between -20 and 30 mV). Replacement of two outer vestibule lysines in Kv2.1 by smaller neutral amino acids made current magnitude dramatically more sensitive to the reduction in K+ driving force (current magnitude changed by as much as 40%). When combined, these outer vestibule properties (fixed conformation during activation and the presence of lysines) all but prevent variation in Kv2.1 current magnitude when [K+] changes during activation. Moreover, the insensitivity of Kv2.1 current magnitude to changes in K+ driving force promotes a more uniform modulation of current over a wide range of membrane potentials by the K+-dependent regulation of outer vestibule conformation.     

Single amino acid change in the fifth transmembrane segment of the TRP Ca2+ channel causes massive degeneration of photoreceptors

The trp gene encodes subunits of a highly Ca(2+)-permeable class of light-activated channels of Drosophila photoreceptors. The recently characterized mutation in this gene, Trp(P365), is semidominant and causes massive degeneration of photoreceptors by making the TRP channel constitutively active. We show that a single amino acid change, Phe-550 to Ile, near the beginning of the fifth transmembrane domain of TRP channel subunits is necessary to induce, and sufficient to closely mimic, the original mutant phenotypes of Trp(P365). Hypotheses are presented as to why the amino acid residues at position 550 and its immediate vicinity might be important in influencing the regulation of the TRP channel and why the substitution of Phe for Ile at this position, in particular, could result in constitutive activity of the channel.     

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.     

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K(+) current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.     

Light-induced changes in GTP and ATP in frog rod photoreceptors. Comparison with recovery of dark current and light sensitivity during dark adaptation

Light decreases GTP and ATP levels in purified suspensions of physiologically active frog rod outer segments still attached to their inner segment ellipsoids (OS-IS). (a) The GTP decrease is slower in OS-IS (t1/2 = 40 s) than in isolated outer segments (t1/2 = 7 s), which suggests there is more effective buffering in OS-IS. (b) The GTP decrease becomes detectable only at intensities greater than those required to saturate the photoresponse. As the intensity of a continuous light is increased over 4 log units, GTP levels decrease linearly with log intensity by as much as 60%. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity. (c) At levels of illumination bleaching greater than 0.003% of the rhodopsin, a decrease in ATP levels becomes detectable. (d) Following a flash, GTP levels fall and then rise with a recovery time dependent on the intensity of the flash. (e) After both 0.2 and 2% flash bleaches, the recovery of GTP levels parallels the recovery of light sensitivity, which is slower than the recovery of the dark current. This raises the possibility of a link between GTP levels and light sensitivity.     

Inhibition of Kv2.1 voltage-dependent K+ channels in pancreatic beta-cells enhances glucose-dependent insulin secretion

Voltage-dependent (Kv) outward K(+) currents repolarize beta-cell action potentials during a glucose stimulus to limit Ca(2+) entry and insulin secretion. Dominant-negative "knockout" of Kv2 family channels enhances glucose-stimulated insulin secretion. Here we show that a putative Kv2.1 antagonist (C-1) stimulates insulin secretion from MIN6 insulinoma cells in a glucose- and dose-dependent manner while blocking voltage-dependent outward K(+) currents. C-1-blocked recombinant Kv2.1-mediated currents more specifically than currents mediated by Kv1, -3, and -4 family channels (Kv1.4, 3.1, 4.2). Additionally, C-1 had little effect on currents recorded from MIN6 cells expressing a dominant-negative Kv2.1 alpha-subunit. The insulinotropic effect of acute Kv2.1 inhibition resulted from enhanced membrane depolarization and augmented intracellular Ca(2+) responses to glucose. Immunohistochemical staining of mouse pancreas sections showed that expression of Kv2.1 correlated highly with insulin-containing beta-cells, consistent with the ability of C-1 to block voltage-dependent outward K(+) currents in isolated mouse beta-cells. Antagonism of Kv2.1 in an ex vivo perfused mouse pancreas model enhanced first- and second-phase insulin secretion, whereas glucagon secretion was unaffected. The present study demonstrates that Kv2.1 is an important component of beta-cell stimulus-secretion coupling, and a compound that enhances, but does not initiate, beta-cell electrical activity by acting on Kv2.1 would be a useful antidiabetic agent.     

Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment

All-atom molecular dynamics simulations are used to better understand the dynamic environment experienced by the Kv1.2 channel in a lipid membrane. The structure of the channel is stable during the trajectories. The pore domain keeps a well-defined conformation, whereas the voltage-sensing domains undergo important lateral fluctuations, consistent with their modular nature. A channel-like region at the center of the S1-S4 helical bundle fills rapidly with water, reminiscent of the concept of high-dielectric aqueous crevices. The first two arginines along S4 (R294 and R297) adopt an interfacial position where they interact favorably with water and the lipid headgroups. The following two arginines (R300 and R303) interact predominantly with water and E226 in S2. Despite the absence of a structurally permanent gating pore formed by protein residues and surrounding the S4 helix, as traditionally pictured, the charged residues are located in a favorable environment and are not extensively exposed to the membrane nonpolar region. Continuum electrostatic computations indicate that the transmembrane potential sensed by the charged residues in the voltage sensor varies abruptly over the outer half of the membrane in the arginine-rich region of S4; thus, the voltage gradient or membrane electric field is "focused". Interactions of basic residues with the lipid headgroups at the intracellular membrane-solution interface reduce the membrane thickness near the channel, resulting in an increased transmembrane field.     

粒细胞巨噬细胞集落刺激因子诱导的巨噬细胞外向K+电流的特性

以粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ,１６ｎｇ／ｍｌ）长期（０．５－６ｄ）刺激小鼠腹腔渗了的巨噬细胞,采用全细胞膜片箝技术研究在ＧＭ－ＣＳＦ刺激过程中细胞膜电流的变化,观察到一种ＧＭ－ＣＳＦ诱导的瞬间失活的外向Ｋ电流,该电流在生理电压范围内可发生稳态失活,且当施加０．５ＨＺ的去极化脉冲刺激时其失活具有频率依赖性。该电流对胞外４－ＡＰ高度敏感,胞内Ｃａ＾２＋浓度「Ｃａ＾２＋」升主可抑制其幅度,     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.