A Potent and Selective Peptide Blocker of the Kv1.3 Channel: Prediction from Free-Energy Simulations and Experimental Confirmation

The voltage-gated potassium channel Kv1.3 is a well-established target for treatment of autoimmune diseases. ShK peptide from a sea anemone is one of the most potent blockers of Kv1.3 but its application as a therapeutic agent for autoimmune diseases is limited by its lack of selectivity against other Kv channels, in particular Kv1.1. Accurate models of Kv1.x-ShK complexes suggest that specific charge mutations on ShK could considerably enhance its specificity for Kv1.3. Here we evaluate the K18A mutation on ShK, and calculate the change in binding free energy associated with this mutation using the path-independent free energy perturbation and thermodynamic integration methods, with a novel implementation that avoids convergence problems. To check the accuracy of the results, the binding free energy differences were also determined from path-dependent potential of mean force calculations. The two methods yield consistent results for the K18A mutation in ShK and predict a 2 kcal/mol gain in Kv1.3/Kv1.1 selectivity free energy relative to wild-type peptide. Functional assays confirm the predicted selectivity gain for ShK[K18A] and suggest that it will be a valuable lead in the development of therapeutics for autoimmune diseases.     

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     

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].     

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.     

Potent selective inhibitors of protein kinase C

A series of potent, selective inhibitors of protein kinase C has been derived from the structural lead provided by the microbial broth products, staurosporine and K252a. Our inhibitors block PCK in intact cells (platelets and T cells), and prevent the proliferation of mononuclear cells in response to interleukin 2 (IL2).     

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.     

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.     

Identification of a new class of inhibitors of the voltage-gated potassium channel,Kv1.3, with immunosuppressant properties

The voltage-gated potassium channel, K(v)1.3, is a novel target for development of immunosuppressants. Using a functional (86)Rb(+) efflux assay, a new class of high-affinity K(v)1.3 inhibitors has been identified. The initial active in this series, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone (PAC), which is representative of a disubstituted cyclohexyl (DSC) template, displays a K(i) of ca. 300 nM and a Hill coefficient near 2 in the flux assay and in voltage clamp recordings of K(v)1.3 channels in human T-lymphocytes. PAC displays excellent specificity as it only blocks members of the K(v)1 family of potassium channels but does not affect many other types of ion channels, receptors, or enzyme systems. Block of K(v)1.3 by DSC analogues occurs with a well-defined structure-activity relationship. Substitution at the C-1 ketone of PAC generates trans (down) and cis (up) isomer pairs. Whereas many DSC derivatives do not display selectivity in their interaction with different K(v)1.x channels, trans DSC derivatives distinguish between K(v)1.x channels based on their rates of C-type inactivation. DSC analogues reversibly inhibit the Ca(2+)-dependent pathway of T cell activation in in vitro assays. Together, these data suggest that DSC derivatives represent a new class of immunosuppressant agents and that specific interactions of trans DSC analogues with channel conformations related to C-type inactivation may permit development of selective K(v)1.3 channel inhibitors useful for the safe treatment of autoimmune diseases.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

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.     

N-glycosylation promotes the cell surface expression of Kv1.3 potassium channels

The voltage-gated potassium channel Kv1.3 plays an essential role in modulating membrane excitability in many cell types. Kv1.3 is a heavily glycosylated membrane protein. Two successive N-glycosylation consensus sites, N228NS and N229ST, are present on the S1-S2 linker of rat Kv1.3. Our data suggest that Kv1.3 contains only one N-glycan and it is predominantly attached to N229 in the S1-S2 extracellular linker. Preventing N-glycosylation of Kv1.3 significantly decreased its surface protein level and surface conductance density level, which were ～?49% and ～?46% respectively of the level of wild type. Supplementation of N-acetylglucosamine (GlcNAc), l-fucose or N-acetylneuraminic acid to the culture medium promoted Kv1.3 surface protein expression, whereas supplementation of d-glucose, d-mannose or d-galactose did not. Among the three effective monosaccharides/derivatives, adding GlcNAc appeared to reduce sialic acid content and increase the degree of branching in the N-glycan of Kv1.3, suggesting that the N-glycan structure and composition had changed. Furthermore, the cell surface half-life of the Kv1.3 surface protein was increased upon GlcNAc supplementation, indicating that it had decreased internalization. The GlcNAc effect appears to apply mainly to membrane proteins containing complex type N-glycans. Thus, N-glycosylation promotes Kv1.3 cell surface expression; supplementation of GlcNAc increased Kv1.3 surface protein level and decreased its internalization, presumably by a combined effect of decreased branch size and increased branching of the N-glycan.     

Modulation of the Kv1.3 Potassium Channel by Receptor Tyrosine Kinases

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (α-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the α-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium 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.     

Potent ACE inhibitors from 5-hydroxy indanone derivatives

A novel triazole derivatives(±)-2-(hydroxymethyl)-7,8-dihydro-1H-indeno[5,4-b]furan-6(2H)-one (12a–j) were designed and synthesized by the reaction between racemic azide and terminal acetylenes under click chemistry reaction conditions followed by biological evaluation as angiotensin converting enzyme (ACE) inhibitors. β-Amino alcohol derivatives of 1-indanone (15a–l) were synthesized from 5-hydroxy indanone, it was reacted with epichlorohydrin and followed by oxirane ring opening with various piperazine derivatives. Among the newly synthesized compounds 12b (IC₅₀: 1.388024 µM), 12g (IC₅₀: 1.220696 µM), 12j (IC₅₀: 1.312428 µM) and 15k (IC₅₀: 1.349671 µM) and 15l (IC₅₀: 1.330764 µM) emerged as most active non-carboxylic acid ACE inhibitors with minimal toxicity comparable to clinical drug Lisinopril.     

Potent alpha-glucosidase inhibitors purified from the red alga Grateloupia elliptica

Diabetes mellitus is a most serious and chronic disease whose incidence rates are increasing with incidences of obesity and aging of the general population over the world. One therapeutic approach for decreasing postprandial hyperglycemia is to retard absorption of glucose by inhibition of α-glucosidase. Two bromophenols, 2,4,6-tribromophenol and 2,4-dibromophenol, were purified from the red alga Grateloupia elliptica. IC₅₀ values of 2,4,6-tribromophenol and 2,4-dibromophenol were 60.3 and 110.4 μM against Saccharomyces cerevisiae α-glucosidase, and 130.3 and 230.3 μM against Bacillus stearothermophilus α-glucosidase, respectively. In addition, both mildly inhibited rat-intestinal sucrase (IC₅₀ of 4.2 and 3.6 mM) and rat-intestinal maltase (IC₅₀ of 5.0 and 4.8 mM). Therefore, bromophenols of G. elliptica have potential as natural nutraceuticals to prevent diabetes mellitus because of their high α-glucosidase inhibitory activity.     

Potent inhibitors of hepatitis C core dimerization as new leads for anti-hepatitis C agents

New indoline alkaloid-type compounds which inhibit HCV production by infected hepatoma cells have been identified. These compounds, dimeric-type compounds of previously known inhibitors, display double digit nanomolar IC₅₀ and EC₅₀ values, with cytotoxicity CC₅₀ indexes higher than 36 μM, thus providing ample therapeutic windows for further development of HCV drugs.     

Structure acquisition of the T1 domain of Kv1.3 during biogenesis

The T1 recognition domains of voltage-gated K(+) (Kv) channel subunits form tetramers and acquire tertiary structure while still attached to their individual ribosomes. Here we ask when and in which compartment secondary and tertiary structures are acquired. We answer this question using biogenic intermediates and recently developed folding and accessibility assays to evaluate the status of the nascent Kv peptide both inside and outside of the ribosome. A compact structure (likely helical) that corresponds to a region of helicity in the mature structure is already manifest in the nascent protein within the ribosomal tunnel. The T1 domain acquires tertiary structure only after emerging from the ribosomal exit tunnel and complete synthesis of the T1-S1 linker. These measurements of ion channel folding within the ribosomal tunnel and its exit port bear on basic principles of protein folding and pave the way for understanding the molecular basis of protein misfolding, a fundamental cause of channelopathies.