Overexpression of mouse IsK protein fused to green fluorescent protein induces apoptosis of human astroglioma cells.

Intracellular K(+) plays an important role in controlling ion homeostasis for maintaining cell volume and inhibiting activity of pro-apoptotic enzymes. Cytoplasmic K(+) concentration is regulated by K(+) uptake via Na(+) -K(+) -ATPase and K(+) efflux through K(+) channels in the plasma membrane. The IsK (KCNE1) protein is known to co-assemble with KCNQ1 (KvLQT1) protein to form a K(+) channel underlying the slowly activating delayed rectifier K(+) outward current which delays voltage activation. In order to further study the activity and cellular localization of IsK protein, we constructed a C-terminal fusion of IsK with EGFP (enhanced green fluorescent protein). Expression of the fusion protein appeared as clusters located in the plasma membrane and induced degeneration of both transiently or stably transfected cells.

Is the WKL1 gene associated with schizophrenia?

A missense mutation Leu309Met in the WKL1 (MLC1, KIAA0027) gene, mapped to 22q13.3, was reported to co-segregate with periodic catatonic schizophrenia (SCZ) in a single large German pedigree with seven affected individuals (Meyer et al. [2001: Mol Psychiatry 6:302-306]). This report raised the following questions that were dealt with in the present study: does the Leu309Met mutation have a role in SCZ, or only in catatonic SCZ? Does the mutation Leu309Met in the WKL1 gene, encoding a putative membrane protein, non-selective cation channel, have any effect on the channel activity? Is the WKL1 gene, which is expressed exclusively in brain, expressed differently in SCZ brains compared to controls? These questions were answered by screening the Leu309Met mutation in 117 Israeli Jewish patients with SCZ (55 Ashkenazi and 62 non-Ashkenazi Jews) and 176 matched controls. In search of differences in the level of WKL1 gene expression, postmortem dorsalateral prefrontal cortex of 16 schizophrenic patients and 15 controls was checked. We also measured the putative channel activity of normal WKL1 subcloned in pcDNA3 to determine the effect of the reported Leu309Met mutation. Our results argue against the involvement of WKL1 in SCZ susceptibility.

Ca2+-activated K+ channels in human leukemic Jurkat T cells. Molecular cloning, biochemical and functional characterization.

Previous studies have demonstrated the presence of apamin-sensitive, small-conductance Ca(2+)-activated K(+) currents in human leukemic Jurkat T cells. Using a combined cDNA and reverse transcriptase-polymerase chain reaction cloning strategy, we have isolated from Jurkat T cells a 2.5-kilobase cDNA, hSK2, encoding the human isoform of SK2 channels. Northern blot analysis reveals the presence of a 2.5-kilobase hSK2 transcript in Jurkat T cells. While present in various human tissues, including brain, heart, skeletal muscle, kidney, and liver, no hSK2 mRNA could be detected in resting and activated normal human T cells. The hSK2 gene is encoded by 8 exons and could be assigned to chromosome 5 (q21.2-q22.1). The protein encoded by hSK2 is 579 amino acids long and exhibits 97% identity with its rat counterpart rSK2. When expressed in Chinese hamster ovary cells, hSK2 produces Ca(2+)-activated K(+) currents with a unitary conductance of 9.5 pS and a K(0.5) for calcium of 0.7 microm; hSK2 currents are inhibited by apamin, scyllatoxin, and d-tubocurarine. Overexpression of the Src family tyrosine kinase p56(lck) in Jurkat cells, up-regulates SK2 currents by 3-fold. While IKCa channels are transcriptionally induced upon activation of normal human T cells, our results show that in Jurkat cells SK2 channels are constitutively expressed and down-regulated following mitogenic stimulation.

Hypomyelination and increased activity of voltage-gated K(+) channels in mice lacking protein tyrosine phosphatase epsilon.

Protein tyrosine phosphatase epsilon (PTP epsilon) is strongly expressed in the nervous system; however, little is known about its physiological role. We report that mice lacking PTP epsilon exhibit hypomyelination of sciatic nerve axons at an early post-natal age. This occurs together with increased activity of delayed- rectifier, voltage-gated potassium (Kv) channels and with hyperphosphorylation of Kv1.5 and Kv2.1 Kv channel alpha-subunits in sciatic nerve tissue and in primary Schwann cells. PTP epsilon markedly reduces Kv1.5 or Kv2.1 current amplitudes in Xenopus oocytes. Kv2.1 associates with a substrate-trapping mutant of PTP epsilon, and PTP epsilon profoundly reduces Src- or Fyn-stimulated Kv2.1 currents and tyrosine phosphorylation in transfected HEK 293 cells. In all, PTP epsilon antagonizes activation of Kv channels by tyrosine kinases in vivo, and affects Schwann cell function during a critical period of Schwann cell growth and myelination.

Extracellular K(+) and opening of voltage-gated potassium channels activate T cell integrin function: physical and functional association between Kv1.3 channels and beta1 integrins.

Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.

A recessive C-terminal Jervell and Lange-Nielsen mutation of the KCNQ1 channel impairs subunit assembly.

The LQT1 locus (KCNQ1) has been correlated with the most common form of inherited long QT (LQT) syndrome. LQT patients suffer from syncopal episodes and high risk of sudden death. The KCNQ1 gene encodes KvLQT1 alpha-subunits, which together with auxiliary IsK (KCNE1, minK) subunits form IK(s) K(+) channels. Mutant KvLQT1 subunits may be associated either with an autosomal dominant form of inherited LQT, Romano-Ward syndrome, or an autosomal recessive form, Jervell and Lange-Nielsen syndrome (JLNS). We have identified a small domain between residues 589 and 620 in the KvLQT1 C-terminus, which may function as an assembly domain for KvLQT1 subunits. KvLQT1 C-termini do not assemble and KvLQT1 subunits do not express functional K(+) channels without this domain. We showed that a JLN deletion-insertion mutation at KvLQT1 residue 544 eliminates important parts of the C-terminal assembly domain. Therefore, JLN mutants may be defective in KvLQT1 subunit assembly. The results provide a molecular basis for the clinical observation that heterozygous JLN carriers show slight cardiac dysfunctions and that the severe JLNS phenotype is characterized by the absence of KvLQT1 channel.

Molecular impact of MinK on the enantiospecific block of I(Ks) by chromanols.

Slowly activating I:(Ks) (KCNQ1/MinK) channels were expressed in Xenopous: oocytes and their sensitivity to chromanols was compared to homomeric KCNQ1 channels. To elucidate the contribution of the ss-subunit MinK on chromanol block, a formerly described chromanol HMR 1556 and its enantiomer S5557 were tested for enantio-specificity in blocking I:(Ks) and KCNQ1 as shown for the single enantiomers of chromanol 293B. Both enantiomers blocked homomeric KCNQ1 channels to a lesser extent than heteromeric I:(Ks) channels. Furthermore, we expressed both WT and mutant MinK subunits to examine the involvement of particular MinK protein regions in channel block by chromanols. Through a broad variety of MinK deletion and point mutants, we could not identify amino acids or regions where sensitivity was abolished or strikingly diminished (>2.5 fold). This could indicate that MinK does not directly take part in chromanol binding but acts allosterically to facilitate drug binding to the principal subunit KCNQ1.

Tyrosine kinases modulate K+ channel gating in mouse Schwann cells.

1. The whole-cell configuration of the patch-clamp technique and immunoprecipitation experiments were used to investigate the effects of tyrosine kinases on voltage-dependent K+ channel gating in cultured mouse Schwann cells. 2. Genistein, a broad-spectrum tyrosine kinase inhibitor, markedly reduced the amplitude of a slowly inactivating delayed-rectifier current (IK) and, to a lesser extent, that of a transient K+ current (IA). Similar results were obtained on IK with another tyrosine kinase inhibitor, herbimycin A. Daidzein, the inactive analogue of genistein, was without effect. 3. Unlike herbimycin A, genistein produced additional effects on IA by profoundly affecting its gating properties. These changes consisted of slower activation kinetics with an increased time to peak, a positive shift in the voltage dependence of activation (by +30 mV), a decrease in the steepness of activation gating (9 mV per e-fold change) and an acceleration of channel deactivation. 4. The steepness of the steady-state inactivation was increased by genistein treatment, while the recovery from inactivation was not significantly altered. 5. The action of genistein on voltage-dependent K+ (Kv) currents was accompanied by a decrease in tyrosine phosphorylation of Kv1.4 as well as Kv1.5 and Kv2.1 encoding transient and slowly inactivating delayed-rectifier K+ channel alpha subunits, respectively. 6. In conclusion, the present study shows that tyrosine kinases markedly affect the amplitude of voltage-dependent K+ currents in Schwann cells and finely tune the gating properties of the transient K+ current component IA. These modulations may be functionally relevant in the control of K+ channel activity during Schwann cell development and peripheral myelinogenesis.

Stilbenes and fenamates rescue the loss of I(KS) channel function induced by an LQT5 mutation and other IsK mutants.

Genetic and physiological studies have established a link between potassium channel dysfunction and a number of neurological and muscular disorders. Many 'channelopathies' are accounted for by a dominant-lethal suppression of potassium channel function. In the cardiac I(KS) channel complex comprising the alpha and beta subunits, KvLQT1 and IsK, respectively, several mutations lead to a dominant-negative loss of channel function. These defects are responsible for a human cardiovascular disease called long QT (LQT) syndrome. Here we show that binding of I(KS) channel activators, such as stilbenes and fenamates, to an extracellular domain flanking the human IsK transmembrane segment, restores normal I(KS) channel gating in otherwise inactive IsK C-terminal mutants, including the naturally occurring LQT5 mutant, D76N. Our data support a model in which allosteric interactions exist between the extracellular and intracellular boundaries of the IsK transmembrane segment as well as between domains of the alpha and beta subunits. Disruption of this allosteric interplay impedes slow activation gating, decreases current amplitude and restores channel inactivation. Owing to allosteric interactions, stilbene and fenamate compounds can rescue the dominant-negative suppression of I(KS) produced by IsK mutations and thus, may have important therapeutic relevance for LQT syndrome.

Heteromultimeric delayed-rectifier K+ channels in schwann cells: developmental expression and role in cell proliferation.

Schwann cells (SCs) are responsible for myelination of nerve fibers in the peripheral nervous system. Voltage-dependent K+ currents, including inactivating A-type (KA), delayed-rectifier (KD), and inward-rectifier (KIR) K+ channels, constitute the main conductances found in SCs. Physiological studies have shown that KD channels may play an important role in SC proliferation and that they are downregulated in the soma as proliferation ceases and myelination proceeds. Recent studies have begun to address the molecular identity of K+ channels in SCs. Here, we show that a large repertoire of K+ channel alpha subunits of the Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2) families is expressed in mouse SCs and sciatic nerve. We characterized heteromultimeric channel complexes that consist of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve, most of the Kv1.2 channel subunits are involved in heteromultimeric association with Kv1.5. Despite the presence of Kv1. 1 and Kv1.2 alpha subunits, the K+ currents were unaffected by dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do not account substantially for SC KD currents. SC proliferation was found to be potently blocked by quinidine or 4-aminopyridine but not by DTX. Consistent with previous physiological studies, our data show that there is a marked downregulation of all KD channel alpha subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest that KD currents are accounted for by a complex combinatorial activity of distinct K+ channel complexes and confirm that KD channels are involved in SC proliferation.

A Ca2+/calmodulin-dependent protein kinase modulates Drosophila photoreceptor K+ currents: a role in shaping the photoreceptor potential.

Light activation of Drosophila photoreceptors leads to the generation of a depolarizing receptor potential via opening of transient receptor potential and transient receptor potential-like cationic channels. Counteracting the light-activated depolarizing current are two voltage-gated K+ conductances, IA and IK, that are expressed in these sensory neurons. Here we show that Drosophila photoreceptors IA and IK are regulated by calcium-calmodulin (Ca2+/calmodulin) via a Ca2+/calmodulin-dependent protein kinase (CaM kinase), with IK being far more sensitive than IA. Inhibition of Ca2+/calmodulin by N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide or trifluoperazine markedly reduced the K+ current amplitudes. Likewise, inhibition of CaM kinases by KN-93 potently depressed IK and accelerated its C-type inactivation kinetics. The effect of KN-93 was specific because its structurally related but functionally inactive analog KN-92 was totally ineffective. In Drosophila photoreceptor mutant ShKS133, which allows isolation of IK, we demonstrate by current-clamp recording that inhibition of IK by quinidine or tetraethylammonium increased the amplitude of the photoreceptor potential, depressed light adaptation, and slowed down the termination of the light response. Similar results were obtained when CaM kinases were blocked by KN-93. These findings place photoreceptor K+ channels as an additional target for Ca2+/calmodulin and suggest that IK is well suited to act in concert with other components of the signaling machinery to sharpen light response termination and fine tune photoreceptor sensitivity during light adaptation.

Expression of two inward rectifier potassium channels is essential for differentiation of primitive human hematopoietic progenitor cells.

A potassium inward rectifier (K(ir)) current was previously shown by us to be induced in primitive hematopoietic progenitor cells, stimulated with the combination of interleukin-3 (IL-3) and stem cell factor (SCF). Biophysical features of whole cell currents implicated the involvement of more than one K(ir) channel type. Employing IL-3 + SCF stimulated human cord blood CD34+38- cells, we isolated and characterized different components of this current. Reverse transcription-polymerase chain reaction (RT-PCR) subcloning identified the expression of a strongly rectifying K(ir) channel (K(ir) 4.3) as well as a weakly rectifying K(ir) channel (K(ir) 1.1) in these cells. Inhibition of the expression of each of the channels suppressed progenitor cell generation by IL-3 and SCF-stimulated CD34+38- cells in 7-day suspension cultures. The variable expression of two essential inward rectifying potassium channels early in the course of hematopoietic progenitor cell differentiation may play a potentially important role in potassium homeostasis in these cells.

Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells.

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+-channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed-rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+-channel alpha subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1- channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+-channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.

IKs, a slow and intriguing cardiac K+ channel and its associated long QT diseases.

Shaping of cardiac action potentials depends on a finely tuned orchestra of ion channels. Among them, K(+) channels probably form the most diverse family. They are responsible for inwardly rectifying (I(K1), I(KAch), I(KATP)), transient (I(to)), and sustained outward rectifying (I(Kur), I(Kr), I(Ks)) K(+) currents. The properties of these cardiac K(+) channels have recently been extensively reviewed. This article focuses on recent progress made toward understanding the molecular structure of the particular channel responsible for the slow outward K(+) current I(Ks) and its implication in the delayed ventricular repolarization that characterizes the congenital long QT syndrome.

Characterization of delayed rectifier Kv channels in oligodendrocytes and progenitor cells.

We examined the molecular identity of K+ channel genes underlying the delayed rectifier (IK) in differentiated cultured oligodendrocytes (OLGs) and oligodendrocyte progenitor (OP) cells. Using reverse transcription-PCR cloning, we found that OP cells and OLGs expressed multiple Kv transcripts, namely Kv1.2, Kv1.4, Kv.1.5, and Kv1.6. Immunocytochemical and Western blot analyses revealed that Kv1.5 and Kv1.6 as well as Kv1.2 and Kv1.4 channel proteins could be detected in these cells, but definitive evidence for functional K+ channel expression was obtained only for the Kv1.5 channel. In addition, mRNA and immunoreactive protein levels of both Kv1.5 and Kv1.6 channels were significantly lower in differentiated OLGs when compared with levels in OP cells. Proliferation of OP cells was inhibited by K+ channel blockers, but not by incubation with either Kv1.5 or Kv1.6 antisense oligonucleotides. We conclude that (1) IK in OP cells and OLGs is encoded partly by Kv1.5 subunits, possibly forming heteromultimeric channels with Kv1.6 or other Kv subunits; and (2) inhibition of Kv1.5 or Kv1.6 channel expression alone does not prevent mitogenesis. Concomitant inhibition of other Kv channels underlying IK may be necessary for OP cells to exit from cell cycle.

The role of the IsK protein in the specific pharmacological properties of the IKs channel complex.

IKs channels are composed of IsK and KvLQT1 subunits and underly the slowly activating, voltage-dependent IKs conductance in heart. Although it appears clear that the IsK protein affects both the biophysical properties and regulation of IKs channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K+ channels are inhibited by the IKs blockers 293B, azimilide and 17-beta-oestradiol. However, IKs channels induced by the coexpression of IsK and KvLQT1 subunits have a 6-100 fold higher affinity for these blockers. Moreover, the IKs activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady-state currents through heteropolymeric IKs channels by arresting them in an open state. In summary, the IsK protein modulates the effects of both blockers and activators of IKs channels. This finding is important for the action and specificity of these drugs as IsK protein expression in heart and other tissues is regulated during development and by hormones.

Molecular mechanism and functional significance of the MinK control of the KvLQT1 channel activity.

The very slowly activating delayed rectifier K+ channel IKs is essential for controlling the repolarization phase of cardiac action potentials and K+ homeostasis in the inner ear. The IKs channel is formed via the assembly of two transmembrane proteins, KvLQT1 and MinK. Mutations in KvLQT1 are associated with a long QT syndrome that causes syncope and sudden death and also with deafness. Here, we show a new mode of association between ion channel forming subunits in that the cytoplasmic C-terminal end of MinK interacts directly with the pore region of KvLQT1. This interaction reduces KvLQT1 channel conductance from 7.6 to 0.58 picosiemens. However, because MinK also reveals a large number of previously silent KvLQT1 channels (x 60), the overall effect is a large increase (x 4) in the macroscopic K+ current. Conformational changes associated with the KvLQT1/MinK association create very slow and complex activation kinetics without much alteration in the deactivation process. Changes induced by MinK have an essential regulatory role in the development of this K+ channel activity upon repetitive electrical stimulation with a particular interest in tachycardia.

The inhibitory effect of the antipsychotic drug haloperidol on HERG potassium channels expressed in Xenopus oocytes.

1. The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K+ channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity. 2. Haloperidol had only little effects on the delayed rectifier channels Kv1.1, Kv1.2, Kv1.5 and IsK, the A-type channel Kv1.4 and the inward rectifier channel Kir2.1 (inhibition < 6% at 3 microM haloperidol). 3. In contrast, haloperidol blocked HERG channels potently with an IC50 value of approximately 1 microM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC50 value of 2.6 microM. 4. Haloperidol block was use- and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested. 5. The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol affinity for HERG S631A was increased four fold at +40 mV compared to 0 mV. 6. In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side effects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

Chronic morphine administration enhances the expression of Kv1.5 and Kv1.6 voltage-gated K+ channels in rat spinal cord.

Prolonged opiate administration leads to the development of tolerance and dependence. These phenomena are accompanied by selective regulation of distant cellular proteins and mRNAs, including ionic channels. Acute opiate administration differentially affects voltage-dependent K+ currents. Whereas, opiate activation of K+ channels is well established opioid-induced inhibition of K+ conductance has also been studied. In this study, we focused on the effect of chronic morphine exposure on voltage-dependent Shaker-related Kv1.5 and Kv1.6 K+ channel gene expression and on Kv1.5 protein levels in the rat spinal cord. Several experimental approaches including in-situ hybridization, RNAse protection, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry were employed. We found that motor neurons are highly enriched in Kv1.5 and Kv1.6 mRNA and in Kv1.5 channel protein. Moreover, we found significant increases in the amount of mRNA encoding for these two K+ channels and in Kv1.5 channel protein in the spinal cord of morphine-treated rats, compared with controls. For example, quantitative in-situ hybridization, revealed a 2.1 +/- 0.15- and 2.3 +/- 0.5-fold increase in Kv1.5 and Kv1.6 channel mRNA levels, respectively. Similar results were obtained by semiquantitative RT-PCR analyses. Kv1.5 protein level was increased by 1.9-fold in the spinal cord or morphine-treated rats. Our results suggest that Kv1.5 and Kv1.6 Shaker K+ channels play an important role in regulating motor activity that increases in mRNA and protein levels of the spinal cord K+ channels after chronic morphine exposure could be viewed as a cellular adaptation which compensates for a persistent opioid-induced inhibition of K+ channel activity. These alterations may account, in part, for the cellular events leading to opiate tolerance and dependence.