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.

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.

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.

ISK, a slowly activating voltage-sensitive K+ channel. Characterization of multiple cDNAs and gene organization in the mouse.

mISK is a protein consisting of 129 amino acids with a single putative transmembrane domain. The injection of mISK cRNA into Xenopus oocytes directs the expression of a voltage-gated K+ current. A heart mRNA blot probed with mISK DNA revealed at least two transcripts. The messenger diversity of mISK was investigated by cloning and characterization of multiple cDNAs of one genomic clone, and by performing primer extension experiments. All cDNAs characterized have the same protein-coding sequence, and heterogeneity of the transcripts arises from alternative splicing, and multiple sites of transcription start and polyadenylation. ISK is encoded by a single gene in the mouse genome. The gene organization reveals the existence of an exon containing the whole protein-coding sequence and of two alternative exons corresponding to the 5' untranslated sequences. We failed to detect the presence of another exon capable of extending the protein-coding sequence. The diversity of mISK messengers is not associated with a diversity of the mISK protein.

Developmental expression of voltage-sensitive K+ channels in mouse skeletal muscle and C2C12 cells.

The developmental expression of voltage-sensitive K+ channels was analyzed by Northern blot in mouse skeletal muscle. Of nine Shaker-like genes studied, eight are expressed in this mammalian muscle. Their expression is differentially regulated during development. The mouse cell line C2C12 has been used to study expression of voltage-sensitive K+ channels during in vitro myotube differentiation. Different voltage-sensitive K+ channel messages are also expressed in these cells which display a pattern of expression depending upon the differentiation stage. The message for the very peculiar K+ channel of IsK type could only be detected by polymerase chain reaction on skeletal muscle mRNA.

Regulation of a major cloned voltage-gated K+ channel from human T lymphocytes.

When expressed into Xenopus oocytes, HLK3 K+ channel (Kv1-3) induced a slowly inactivating voltage-dependent K+ current. We have studied the modulation of this K+ current by co-expressing a cloned 5-HT2 receptor together with HLK3 K+ channel protein. Application of 5-HT caused a long-lasting inhibition of the voltage-gated K+ current. This inhibitory modulation was mimicked by intracellular injection of inositol triphosphate or Ca2+, as well as by incubation with phorbol esters or diacylglycerol analogs. Oocytes pretreatment with staurosporine and EGTA fully prevented 5-HT inhibitory action. Elevation of cAMP and cGMP levels into oocytes did not produce any detectable effect on the current recorded in the absence or the presence of 5-HT. These data suggest that the second messengers generated by phospholipase C activation may be important modulators of HLK3 K+ channels in the immune and the central nervous systems.

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.

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.

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.

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.