The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate possible expression of Kv1.3 channels in other types of tissue, such as epithelia, binding experiments, immunoprecipitation studies and immunohistochemical studies were performed. The double-mutated, radiolabeled peptidyl ligand, ¹²⁵I-HgTX₁-A19Y/Y37F, which selectively binds Kv1.1, Kv1.2, Kv1.3 and Kv1.6 channels, was used to perform binding studies in epithelia isolated from rabbit kidney and colon. The equilibrium dissociation constant for this ligand was found to be in the sub-picomolar range and the maximal receptor concentration (in fmol/mg protein) 1.68 for colon and 0.61-0.75 for kidney epithelium. To determine the subtype of Kv1 channels, immunoprecipitation studies with ¹²⁵I-HgTX₁-A19Y/Y37F labeled epithelial membranes were performed with specific antibodies against Kv1.1, Kv1.2, Kv1.3, Kv1.4 or Kv1.6 subunits. These studies demonstrated that Kv1.3 subunits constituted more than 50% of the entire Kv1 subunit population. The precise localization of Kv1.3 subunits in epithelia was determined by immunohistochemical studies.     

Actinomycin D-induced apoptosis involves the potassium channel Kv1.3

Several cytostatic agents are known to induce apoptosis in T-leukemic cells. Although a variety of studies show the central role of apoptosis in cytostatic drug-induced cell death, many molecular details require definition. Here, we demonstrate that cells genetically deficient for the potassium channel Kv1.3 are resistant to apoptosis initiated by the cytostatic drug actinomycin D. Retransfection of Kv1.3 restores sensitivity of the cells to actinomycin D. Cells lacking Kv1.3 fail to respond to actinomycin D with DNA fragmentation, release of cytochrome c, and loss of mitochondrial membrane potential (Delta Psi(m)), while cells functionally expressing Kv1.3 rapidly undergo those changes indicative for apoptosis. The data indicate a central role of the ion channel Kv1.3 in actinomycin D-triggered apoptosis.     

Ceramide inhibits the potassium channel Kv1.3 by the formation of membrane platforms

Previous studies suggested a central role of sphingomyelin- and cholesterol-enriched membrane rafts in the initiation of signaling via many receptors. Here, we investigated the role of membrane rafts for the function of the voltage-gated potassium channel Kv1.3. We demonstrate that Kv1.3 localizes in the cell membrane to pre-existing small, sphingolipid- and cholesterol-enriched membrane rafts. Transformation of these small rafts to large ceramide-enriched membrane platforms was achieved by stimulation of the endogenous acid sphingomyelinase, addition of exogenous sphingomyelinase or treatment of the cells with C(16)-ceramide and resulted in clustering of Kv1.3 within ceramide-enriched membrane platforms and inhibition of the channel's activity. Likewise, disruption of pre-existing small rafts inhibited Kv1.3 activity. This indicates that intact small membrane rafts are required for Kv1.3 activity and an alteration of the lipid environment of rafts inhibits Kv1.3. These data, thus, may suggest a novel concept for the regulation of ion channels by the cell membrane composition.     

Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers

Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.     

Expression of Kv1.3 potassium channels regulates density of cortical interneurons

The Kv1.3 protein is a member of the large family of voltage‐dependent K⁺ subunits (Kv channels), which assemble to form tetrameric membrane‐spanning channels that provide a selective pore for the conductance of K⁺ across the cell membrane. Kv1.3 differs from most other Kv channels in that deletion of Kv1.3 gene produces very striking changes in development and structure of the olfactory bulb, where Kv1.3 is expressed at high levels, resulting in a lower threshold for detection of odors, an increased number of synaptic glomeruli and alterations in the levels of a variety of neuronal signaling molecules. Because Kv1.3 is also expressed in the cerebral cortex, we have now examined the effects of deletion of the Kv1.3 gene on the expression of interneuron populations of the cerebral cortex. Using unbiased stereology we found an increase in the number of parvalbumin (PV) cells in whole cerebral cortex of Kv1.3^?/? mice relative to that in wild‐type mice, and a decrease in the number of calbindin (CB), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and somatostatin (SOM) interneurons. These changes are accompanied by a decrease in the cortical volume such that the cell density of PV interneurons is significantly increased and that of SOM neurons is decreased in Kv1.3^?/? animals. Our studies suggest that, as in the olfactory bulb, Kv1.3 plays a unique role in neuronal differentiation and/or survival of interneuron populations and that expression of Kv1.3 is required for normal cortical function. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:841–855, 2013     

A C-terminal PDZ binding domain modulates the function and localization of Kv1.3 channels

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms.     

Contribution of voltage-gated potassium channels to the regulation of apoptosis

Recent evidence points to the crucial involvement of voltage-gated potassium channels (Kv) in apoptotic volume decrease and in the regulation of apoptosis in several systems. We have recently described the presence of a Kv channel, Kv1.3, in the mitochondria of lymphocytes. Expression of the channel correlated with increased sensitivity to apoptotic stimuli. Mitochondrial Kv1.3 contributes to the apoptotic cascade in T lymphocytes by interacting with pro-apoptotic Bax resulting in alteration of mitochondrial functional parameters and ultimately, in cytochrome c release. The present review summarizes the current understanding of the function of Kv channels in apoptosis in several cell types as well as the role of mitochondrial Kv1.3 in the regulation of cell death in lymphocytes.     

Molecular mechanisms for Kv1.3 potassium channel current inhibition by CD3/CD28 stimulation in Jurkat T cells

In T lymphocyte, activation of Kv1.3 channel, the major voltage-dependent K⁺ channel, is an essential step for cell proliferation in immune responses. Here, effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current were examined in three types of human T lymphocyte derived cell lines, Jurkat E6-1, p56lck-kinase deficient mutant JCaM.1, and CD45-phosphatase deficient mutant J45.01. Kv1.3 current was partly reduced by CD3 stimulation and more strongly by addition of anti-CD28 antibody in E6-1. In JCaM.1, Kv1.3 current responses to anti-CD28/CD3 antibodies were similar to those in E6-1. In J45.01, CD3 stimulation partly inhibited Kv1.3 current, but the additive reduction by CD28 stimulation was not significant. The inhibition of tyrosine phosphatase in E6-1 abolished the additional inhibition by anti-CD28 antibody in a similar manner as in J45.01. In conclusion, the stimulation of CD28 in addition to CD3 strongly inhibits Kv1.3 current and this additive inhibition is mediated by CD45 activation.     

Identification of a new specific Kv1.3 channel blocker, Ctri9577, from the scorpion Chaerilus tricostatus

Scorpion toxins are valuable resources for discovering new ion channel modulators and drug candidates. Potassium channel Kv1.3 is an important pharmacological target of T cell-mediated autoimmune diseases, which are encouraging the screening and design of the specific peptide blockers for Kv1.3 channel. Ctri9577, the first neurotoxin gene of Chaerilidae family was cloned from the venom of the scorpion Chaerilus tricostatus through the constructing its cDNA library. The sequence analysis showed that the mature peptide of Ctri9577 contained 39 amino acid residues including six conserved cysteines, whose low sequence similarity indicated that it was a new member of α-KTx15 subfamily. By using expression and purification technology, the recombinant peptide was obtained. Subsequently, the electrophysiological experiments indicated that the Ctri9577 peptide selectively inhibited Kv1.3 channel current with an IC(50) of 0.49±0.45 nM without effectively blocking potassium channels Kv1.1, Kv1.2, hERG and SK3. All these findings not only enrich the knowledge of toxins from the Chaerilidae family, but also present a novel potential drug candidate targeting Kv1.3 channels for the therapy of autoimmune diseases.     

The role of the voltage-gated potassium channel,Kv2.1 in prostate cancer cell migration

Voltage-gated potassium (Kv) channels are involved in many important cellular functions and play pivotal roles in cancer progression. The expression level of Kv2.1 was observed to be higher in the highly metastatic prostate cancer cells (PC-3), specifically in their membrane, than in immortalized prostate cells (WPMY-1 cells) and comparatively less metastatic prostate cancer cells (LNCaP and DU145 cells). However, Kv2.1 expression was significantly decreased when the cells were treated with anti-oxidants, such as N-acetylcysteine or ascorbic acid, implying that the highly expressed Kv2.1 could detect reactive oxygen species (ROS) in malignant prostate cancer cells. In addition, the blockade of Kv2.1 with stromatoxin-1 or siRNA targeting Kv2.1 significantly inhibited the migration of malignant prostate cancer cells. Our results suggested that Kv2.1 plays an important role as a ROS sensor and that it is a promising therapeutic molecular target in metastasis of prostate cancer.     

AMIGO is an auxiliary subunit of the Kv2.1 potassium channel

Kv2.1 is a potassium channel α-subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (I(K)) in several neuronal types and a regulator of excitability during high-frequency firing. Here we identify AMIGO (amphoterin-induced gene and ORF), a neuronal adhesion protein with leucine-rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus-induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage-dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal I(K) at negative membrane voltages. In conclusion, our data indicate AMIGO as a function-modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.     

Modeling the binding of three toxins to the voltage-gated potassium channel (Kv1.3)

The conduction properties of the voltage-gated potassium channel Kv1.3 and its modes of interaction with several polypeptide venoms are examined using Brownian dynamics simulations and molecular dynamics calculations. Employing an open-state homology model of Kv1.3, we first determine current-voltage and current-concentration curves and ascertain that simulated results accord with experimental measurements. We then investigate, using a molecular docking method and molecular dynamics simulations, the complexes formed between the Kv1.3 channel and several Kv-specific polypeptide toxins that are known to interfere with the conducting mechanisms of several classes of voltage-gated K⁺ channels. The depths of potential of mean force encountered by charybdotoxin, α-KTx3.7 (also known as OSK1) and ShK are, respectively, −19, −27, and −25 kT. The dissociation constants calculated from the profiles of potential of mean force correspond closely to the experimentally determined values. We pinpoint the residues in the toxins and the channel that are critical for the formation of the stable venom-channel complexes.     

Synthesis and biological evaluation of chalcones as inhibitors of the voltage-gated potassium channel Kv1.3

Chalcone derivatives of the natural product khellinone were synthesised and screened for bioactivity against the voltage-gated potassium channel Kv1.3. X-ray crystallography was employed to investigate relationships between the structure and function of a selection of the reported chalcones.     

Effects and mechanism of Chinese tarantula toxins on the Kv2.1 potassium channels

Three neurotoxins, Jingzhaotoxin-I, -III, and -V (JZTX-I, -III, and -V), isolated from the venom of the Chinese tarantula Chilobrachys Jingzhao, are 29-36-amino acid peptides. Electrophysiological recordings carried out in Xenopus laevis oocytes show that these toxins acted as gating modifier of voltage-dependent K+ channels. They slow the rate of Kv2.1 channel activation and increase the tail current deactivation, suggesting that toxin-bound channels can still open but are modified. JZTX-III selectively inhibits Kv2.1 channels, and JZTX-V exhibits a higher affinity to Kv4.2 channels than to Kv2.1 channels, whereas JZTX-I inhibits Kv2.1 and Kv4.1 channels with low affinity. Structure-function analysis indicates that electrostatic interactions can benefit for toxin affinity and the feature of electrostatic anisotropy may be correlated with the different affinity of the toxins for the Kv2.1 and Kv4.1 channels. Furthermore, phylogenetic analysis of these and other gating modifiers provides clues for the exploration of toxin-channel interaction.     

Activation and closed-state inactivation mechanisms of the human voltage-gated KV4 channel complexes

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Engineering a potent and specific blocker of voltage-gated potassium channel Kv1.3, a target for autoimmune diseases

A polypeptide toxin extracted from scorpion venom, OSK1, is modified such that its potency is drastically enhanced in blocking one class of voltage-gated potassium channels, Kv1.3, which is a pharmacological target for immunosuppressive therapy. The bound complex of Kv1.3 and OSK1 reveals that one lysine residue of the toxin is in the proximity of another lysine residue on the external vestibule of the channel, just outside of the selectivity filter. This unfavorable electrostatic interaction is eliminated by interchanging the positions of two amino acids in the toxin. The potentials of mean force of the wild-type and mutant OSK1 bound to Kv1.1-Kv1.3 channels are constructed using molecular dynamics, and the half-maximal inhibitory concentration (IC(50)) of each toxin-channel complex is computed. We show that the IC(50) values predicted for three toxins and three channels match closely with experiment. Kv1.3 is half-blocked by 0.2 pM mutant OSK1; it is >10000-fold more specific for this channel than for Kv1.1 and Kv1.2.     

Characterization of a new Kv1.3 channel-specific blocker, J123, from the scorpion Buthus martensii Karsch

The potassium channel Kv1.3 is an attractive pharmacological target for T-cell-mediated autoimmune diseases, and specific and selective peptidic blockers of Kv1.3 channels have served as valuable therapeutic leads for treating these diseases. Here, we found a new peptide toxin, J123, with 43 amino acids including six cysteine residues by screening the venomous cDNA library of scorpion Buthus martensii Karsch, which has been used as traditional medicine in China for more than 2000 years. The sequence analysis suggested that peptide J123 constituted a new member of the alpha-KTx toxins. The electrophysiological experiments further indicated that peptide J123 has a novel pharmacological profiles: it blocked Kv1.3 channel with high potency (IC(50)=0.79nM), and exhibited good selectivity on Kv1.3 over Kv1.1 (>1000-fold) and Kv1.2 (about 30-fold), respectively. Furthermore, peptide J123 had no activity on SKCa2 and SKCa3 channels at micromolar concentration level. Based on the pharmacological activities, the possible channel-interacting surface of peptide J123 was also predicted by molecular modeling and docking. All these data not only enrich the knowledge of the structure-function relationship of the new Kv1.3-speicific peptide but also present a potential drug candidate for selectively targeting Kv1.3 channels.     

A specific N-terminal residue in Kv1.5 is required for upregulation of the channel by SAP97

We have previously reported that SAP97 enhancement of hKv1.5 currents requires an intact Kv1.5 N-terminus and is independent of the PDZ-binding motif at the C-terminus of the channel [J. Eldstrom, W.S. Choi, D.F. Steele, D. Fedida, SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism, FEBS Lett. 547 (2003) 205-211]. Here, we report that an interaction between the two proteins can be detected under certain conditions but their interaction is irrelevant to the enhancement of channel expression. Instead, a threonine residue at position 15 in the hKv1.5 N-terminus is critically important. Mutation of this residue, which lies within a consensus site for phosphorylation by protein kinase C, to an alanine, completely abrogated the effect of SAP97 on channel expression. Although we were unable to detect phosphorylation of this residue, specific inhibition of kinase C by Calphostin C eliminated the increase in wild-type hKv1.5 currents associated with SAP97 overexpression suggesting a role for this kinase in the response.     

Characterization of the functional properties of the voltage-gated potassium channel Kv1.3 in human CD4+ T lymphocytes

Previous studies have shown that central memory T (T(CM)) cells predominantly use the calcium-dependent potassium channel KCa3.1 during acute activation, whereas effector memory T (T(EM)) cells use the voltage-gated potassium channel Kv1.3. Because Kv1.3-specific pharmacological blockade selectively inhibited anti-CD3-mediated proliferation, whereas naive T cells and T(CM) cells escaped inhibition due to up-regulation of KCa3.1, this difference indicated a potential for selective targeting of the T(EM) population. We examined the effects of pharmacological Kv1.3 blockers and a dominant-negative Kv1.x construct on T cell subsets to assess the specific effects of Kv1.3 blockade. Our studies indicated both T(CM) and T(EM) CD4+ T cells stimulated with anti-CD3 were inhibited by charybdotoxin, which can block both KCa3.1 and Kv1.3, whereas margatoxin and Stichodactyla helianthus toxin, which are more selective Kv1.3 inhibitors, inhibited proliferation and IFN-gamma production only in the T(EM) subset. The addition of anti-CD28 enhanced proliferation of freshly isolated cells and rendered them refractory to S. helianthus, whereas chronically activated T(EM) cell lines appeared to be costimulation independent because Kv1.3 blockers effectively inhibited proliferation and IFN-gamma regardless of second signal. Transduction of CD4+ T cells with dominant-negative Kv1.x led to a higher expression of CCR7+ T(CM) phenotype and a corresponding depletion of T(EM). These data provide further support for Kv1.3 as a selective target of chronically activated T(EM) without compromising naive or T(CM) immune functions. Specific Kv1.3 blockers may be beneficial in autoimmune diseases such as multiple sclerosis in which T(EM) are found in the target organ.