Behavioral motor dysfunction in Kv3-type potassium channel-deficient mice

The voltage-gated potassium channels Kv3.1 and Kv3.3 are expressed in several distinct neuronal subpopulations in brain areas known to be involved in motor control such as cortex, basal ganglia and cerebellum. Depending on the lack of Kv3.1 or Kv3.3 channel subunits, mutant mice show different Kv3-null allele-dependent behavioral alterations that include constitutive hyperactivity, sleep loss, impaired motor performance and, in the case of the Kv3.1/Kv3.3 double mutant, also severe ataxia, tremor and myoclonus (Espinosa et al. 2001, J Neurosci 21, 6657-6665, Genes, Brain Behav 3, 90-100). The lack of Kv3.1 channel subunits is mainly responsible for the constitutively increased locomotor activity and for sleep loss, whereas the absence of Kv3.3 subunits affects cerebellar function, in particular Purkinje cell discharges and olivocerebellar system properties (McMahon et al. 2004, Eur J Neurosci 19, 3317-3327). Here, we describe two sensitive and non-invasive tests to reliably quantify normal and abnormal motor functions, and we apply these tests to characterize motor dysfunction in Kv3-mutant mice. In contrast to wildtype and Kv3.1-single mutants, Kv3.3-single mutants and Kv3 mutants lacking three and four Kv3 alleles display Kv3-null allele-dependent gait alterations. Although the Kv3-null allele-dependent gait changes correlate with reduced motor performance, they appear to not affect the training-induced improvement of motor performance. These findings suggest that altered cerebellar physiology in the absence of Kv3.3 channels is responsible for impaired motor task execution but not motor task learning.     

Transmembrane biogenesis of Kv1.3

Using a combination of protease protection, glycosylation, and carbonate extraction assays, we have characterized the topogenic determinants encoded by Kv1.3 segments that mediate translocation events during endoplasmic reticulum (ER) biogenesis. Transmembrane segments S1, S2, S3, S5, and S6 initiate translocation, only S1 and S2 strongly (>60%) anchor themselves in the membrane, S5 exhibits signal anchor activity and contains a cryptic cleavage site, and S3 and S6 fail to integrate into the membrane. Elongation of each single-transmembrane construct to include multiple transmembrane segments alters integration and translocation efficiencies, indicating that multiple topogenic determinants cooperate during Kv1. 3 topogenesis and assembly. Several surprising findings emerged from these studies. First, in the presence of T1, the N-terminal recognition domain, S1 was unable to initiate either translocation or membrane integration. As a result, S2 likely functions as the initial signal sequence to establish Kv1.3 N-terminus topology. Second, S4 independently integrates into the membrane. Third, S6 plus the C-terminus of Kv1.3 is a secretory protein but can be converted to a membrane-integrated protein with a correctly oriented, cytosolic C-terminus by linking S6 to S5 and the pore loop. These results have implications for the role of the N-terminus in Kv biogenesis and on the mechanisms of dominant negative suppression of Kv1.3 by truncated Kv1.3 fragments [Tu et al. (1996) J. Biol. Chem. 271, 18904-18911].     

Kv1.5 association modifies Kv1.3 traffic and membrane localization

Kv1.3 activity is determined by raft association. In addition to Kv1.3, leukocytes also express Kv1.5, and both channels control physiological responses. Because the oligomeric composition may modify the channel targeting to the membrane, we investigated heterotetrameric Kv1.3/Kv1.5 channel traffic and targeting in HEK cells. Kv1.3 and Kv1.5 generate multiple heterotetramers with differential surface expression according to the subunit composition. FRET analysis and pharmacology confirm the presence of functional hybrid channels. Raft association was evaluated by cholesterol depletion, caveolae colocalization, and lateral diffusion at the cell surface. Immunoprecipitation showed that both Kv1.3 and heteromeric channels associate with caveolar raft domains. However, homomeric Kv1.3 channels showed higher association with caveolin traffic. Moreover, FRAP analysis revealed higher mobility for hybrid Kv1.3/Kv1.5 than Kv1.3 homotetramers, suggesting that heteromers target to distinct surface microdomains. Studies with lipopolysaccharide-activated macrophages further supported that different physiological mechanisms govern Kv1.3 and Kv1.5 targeting to rafts. Our results implicate the traffic and localization of Kv1.3/Kv1.5 heteromers in the complex regulation of immune system cells.     

Kv1.3/Kv1.5 heteromeric channels compromise pharmacological responses in macrophages

Voltage-dependent K(+) (Kv) channels are involved in the immune response. Kv1.3 is highly expressed in activated macrophages and T-effector memory cells of autoimmune disease patients. Macrophages are actively involved in T-cell activation by cytokine production and antigen presentation. However, unlike T-cells, macrophages express Kv1.5, which is resistant to Kv1.3-drugs. We demonstrate that mononuclear phagocytes express different Kv1.3/Kv1.5 ratios, leading to biophysically and pharmacologically distinct channels. Therefore, Kv1.3-based treatments to alter physiological responses, such as proliferation and activation, are impaired by Kv1.5 expression. The presence of Kv1.5 in the macrophagic lineage should be taken into account when designing Kv1.3-based therapies.     

Immunosuppressive effects of a Kv1.3 inhibitor

The voltage gated potassium channel (Kv1.3) has been shown to play a role in immune responsiveness. Blockade of the channel led to diminution of T cell activation and delayed type hypersensitivity. Previous in vitro studies of the blockade were focused on T cell activation and proliferation. In this study we examined other T and monocytic cell mediated events to glean the extent of the immunosuppressive effects of a Kv1.3 specific inhibitor, Margatoxin (MgTX). We found that MgTX inhibited the intracellular production of Th-1 as well as Th-2 cytokines. MgTX can also inhibit IL-2 production and proliferation of T cells upon stimulation with anti-CD3 and VCAM-1. Furthermore, a redirected cytolytic activity was also inhibited by MgTX. However, MgTX did not inhibit generation of CTL to EBV transformed lymphoma cells or antibody-dependent cellular cytolysis mediated by monocytes. It appears that a Kv1.3 blockade does not affect all immune responses, particularly those of innate immunity.     

Multiple binding sites for melatonin on Kv1.3

Melatonin is a small amino acid derivative hormone of the pineal gland. Melatonin quickly and reversibly blocked Kv1.3 channels, the predominant voltage-gated potassium channel in human T-lymphocytes, acting from the extracellular side. The block did not show state or voltage dependence and was associated with an increased inactivation rate of the current. A half-blocking concentration of 1.5 mM was obtained from the reduction of the peak current. We explored several models to describe the stoichiometry of melatonin-Kv1.3 interaction considering one or four independent binding sites per channel. The model in which the occupancy of one of four binding sites by melatonin is sufficient to block the channels gives the best fit to the dose-response relationship, although all four binding sites can be occupied by the drug. The dissociation constant for the individual binding sites is 8.11 mM. Parallel application of charybdotoxin and melatonin showed that both compounds can simultaneously bind to the channels, thereby localizing the melatonin binding site out of the pore region. However, binding of tetraethylammonium to its receptor decreases the melatonin affinity, and vice versa. Thus, the occupancy of the two separate receptor sites allosterically modulates each other.     

Reduced Kv1.3 Potassium Channel Expression in Human Prostate Cancer

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = −0.25, P = 0.003) as well as tumor stage (r = −0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.     

Reduced Kv1.3 potassium channel expression in human prostate cancer

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = -0.25, P = 0.003) as well as tumor stage (r = -0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.     

KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels

Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 potassium channels. The inhibition is found on channels expressed heterologously in both Xenopus oocytes and mammalian HEK293 cells. mKCNE4 does not inhibit Kv1.2, Kv1.4, Kv1.5, or Kv4.3 homomeric complexes, but it does significantly reduce current through Kv1.1/Kv1.2 and Kv1.2/Kv1.3 heteromeric complexes. Confocal microscopy and Western blotting reveal that Kv1.1 is present at the cell surface together with KCNE4. Real-time RT-PCR shows a relatively high presence of mKCNE4 mRNA in several organs, including uterus, kidney, lung, intestine, and in embryo, whereas a much lower mRNA level is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials.     

Benzamide derivatives as blockers of Kv1.3 ion channel

The voltage-gated potassium channel, Kv1.3, is present in human T-lymphocytes. Blockade of Kv1.3 results in T-cell depolarization, inhibition of T-cell activation, and attenuation of immune responses in vivo. A class of benzamide Kv1.3 channel inhibitors has been identified. The structure-activity relationship within this class of compounds in two functional assays, Rb_Kv and T-cell proliferation, is presented. In in vitro assays, trans isomers display moderate selectivity for binding to Kv1.3 over other Kv1.x channels present in human brain.     

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.     

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.     

Native Kv1.3 Channels are Upregulated by Protein Kinase C

The voltage-gated potassium channel, Kv1.3, which is highly expressed in a number of immune cells, contains concensus sites for phosphorylation by protein kinase C (PKC). In lymphocytes, this channel is involved in proliferation—through effects on membrane potential, Ca²⁺ signalling, and interleukin-2 secretion—and in cytotoxic killing and volume regulation. Because PKC activation (as well as increased intracellular Ca²⁺) is required for T-cell proliferation, we have studied the regulation of Kv1.3 current by PKC in normal (nontransformed) human T lymphocytes. Adding intracellular ATP to support phosphorylation, shifted the voltage dependence of activation by +8 mV and inactivation by +17 mV, resulting in a 230% increase in the window current. Inhibiting ATP production and action with ``death brew' (2-deoxyglucose, adenylylimidodiphosphate, carbonyl cyanide-m-chlorophenyl hydrazone) reduced the K⁺ conductance (G _K ) by 41 ± 2%. PKC activation by 4β-phorbol 12,13-dibutyrate, increased G _K by 69 ± 6%, and caused a positive shift in activation (+9 mV) and inactivation (+9 mV), which resulted in a 270% increase in window current. Conversely, several PKC inhibitors reduced the current. Diffusion into the cell of inhibitory pseudosubstrate or substrate peptides reduced G _K by 43 ± 5% and 38 ± 8%, respectively. The specific PKC inhibitor, calphostin C, potently inhibited Kv1.3 current in a dose- and light-dependent manner (IC₅₀∼ 250 nm). We conclude that phosphorylation by PKC upregulates Kv1.3 channel activity in human lymphocytes and, as a result of shifts in voltage dependence, this enhancement is especially prevalent at physiologically relevant membrane potentials. This increased Kv1.3 current may help maintain a negative membrane potential and a high driving force for Ca²⁺ entry in the presence of activating stimuli. Received: 12 July 1996/Revised: 21 October 1996     

Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes

Mitochondria have been shown to play a pivotal role in apoptotic signalling in various cell types. We have recently reported that in lymphocytes the voltage-gated potassium channel Kv1.3, known to reside in the plasma membrane, is active also in the inner mitochondrial membrane. Upon induction of apoptosis, outer-membrane inserted Bax binds to and inhibits Kv1.3 resulting in hyperpolarization, an increase in reactive oxygen species production and cytochrome c release. In cells lacking Kv1.3 these events do not take place. Here, we present new data which further corroborates an important role of this channel in the sequence of events leading to Bax-induced cytochrome c release. Recombinant Kv1.3, when pre-incubated with Bax, prevents the actions of Bax at the level of mitochondria. Furthermore, we report the presence of Kv1.3 protein in mitochondria from PC3 and MCF-7 cancer cells, suggesting that this channel might play a role in the apoptotic signalling not only in lymphocytes but also in other cells.     

Pegylation: a method for assessing topological accessibilities in Kv1.3.

Each subunit of a voltage-gated potassium channel (Kv) contains six putative transmembrane segments, S1-S6, and a cytosolic N-terminal recognition domain, T1. Although it is well-established that Kv channels are tetrameric structures, the protein-protein, protein-lipid, and protein-aqueous interfaces are not precisely mapped. The topological accessibility of specific amino acids may help to identify these border residues. Toward this end, a variant of the substituted-cysteine-accessibility method that relies on mass-labeling of accessible SH groups with a large SH reagent, methoxy-polyethylene glycol maleimide, and gel shift assay has been used. Pegylation of full-length Kv1.3, as well as Kv1.3 fragments, integrated into microsomal membranes, allows topological characterization of the 12 native cysteines (C1-C12), as well as cysteines engineered into a T1-T1 interface. Cysteines engineered into the T1-T1 interface had lower rates of pegylation than cytosolic-facing cysteines, namely, C5 in the T1 domain and C10-C12 in the C terminus.     

Assembly and suppression of endogenous Kv1.3 channels in human T cells

The predominant K+ channel in human T lymphocytes is Kv1.3, which inactivates by a C-type mechanism. To study assembly of these tetrameric channels in Jurkat, a human T-lymphocyte cell line, we have characterized the formation of heterotetrameric channels between endogenous wild-type (WT) Kv1.3 subunits and heterologously expressed mutant (A413V) Kv1.3 subunits. We use a kinetic analysis of C-type inactivation of currents produced by homotetrameric channels and heterotetrameric channels to determine the distribution of channels with different subunit stoichiometries. The distributions are well- described by either a binomial distribution or a binomial distribution plus a fraction of WT homotetramers, indicating that subunit assembly is a random process and that tetramers expressed in the plasma membrane do not dissociate and reassemble. Additionally, endogenous Kv1.3 current is suppressed by a heterologously expressed truncated Kv1.3 that contains the amino terminus and the first two transmembrane segments. The time course for suppression, which is maximal at 48 h after transfection, overlaps with the time interval for heterotetramer formation between heterologously expressed A413V and endogenous WT channels. Our findings suggest that diversity of K+ channel subtypes in a cell is regulated not by spatial segregation of monomeric pools, but rather by the degree of temporal overlap and the kinetics of subunit expression.