Margatoxin

AIM:

The aim of the study was to determine the potential for K(V)1 potassium channel blockers as inhibitors of human neoinitimal hyperplasia.

METHODS AND RESULTS:

Blood vessels were obtained from patients or mice and studied in culture. Reverse transcriptase-polymerase chain reaction and immunocytochemistry were used to detect gene expression. Whole-cell patch-clamp, intracellular calcium measurement, cell migration assays, and organ culture were used to assess channel function. K(V)1.3 was unique among the K(V)1 channels in showing preserved and up-regulated expression when the vascular smooth muscle cells switched to the proliferating phenotype. There was strong expression in neointimal formations. Voltage-dependent potassium current in proliferating cells was sensitive to three different blockers of K(V)1.3 channels. Calcium entry was also inhibited. All three blockers reduced vascular smooth muscle cell migration and the effects were non-additive. One of the blockers (margatoxin) was highly potent, suppressing cell migration with an IC(50) of 85 pM. Two of the blockers were tested in organ-cultured human vein samples and both inhibited neointimal hyperplasia.

CONCLUSION:

K(V)1.3 potassium channels are functional in proliferating mouse and human vascular smooth muscle cells and have positive effects on cell migration. Blockers of the channels may be useful as inhibitors of neointimal hyperplasia and other unwanted vascular remodelling events.

Cheong A., et al. (2011) Potent suppression of vascular smooth muscle cell migration and human neointimal hyperplasia by KV1.3 channel blockers. Cardiovasc Res. PMID 20884640

Kv1.3 channels are known to modulate many aspects of neuronal function. We tested the hypothesis that Kv1.3 modulates the function of postganglionic sympathetic neurons. RT-PCR, immunoblot, and immunohistochemical analyses indicated that Kv1.3 channels were expressed in these neurons. Immunohistochemical analyses indicated that Kv1.3 protein was localized to neuronal cell bodies, processes, and nerve fibers at sympathetic neurovascular junctions. Margatoxin (MgTX), a specific inhibitor of Kv1.3, was used to assess the function of the channel. Electrophysiological analyses indicated that MgTX significantly reduced outward currents [P < 0.05; n = 18 (control) and 15 (MgTX)], depolarized resting membrane potential, and decreased the latency to action potential firing [P < 0.05; n = 11 (control) and 13 (MgTX)]. The primary physiological input to postganglionic sympathetic neurons is ACh, which activates nicotinic and muscarinic ACh receptors. MgTX modulated nicotinic ACh receptor agonist-induced norepinephrine release (P < 0.05; n >or= 6), and MgTX-sensitive current was suppressed upon activation of muscarinic ACh receptors with bethanechol (P < 0.05; n = 12). These data indicate that Kv1.3 affects the function of postganglionic sympathetic neurons, which suggests that Kv1.3 influences sympathetic control of cardiovascular function. Our data also indicate that modulation of Kv1.3 is likely to affect sympathetic control of cardiovascular function.

Doczi MA. et al. (2008) Kv1.3 channels in postganglionic sympathetic neurons: expression, function, and modulation. Am J Physiol Regul Integr Comp Physiol. PMID 18614767

Diphenyl phosphine oxide-1 (DPO-1) is a potent Kv1.5 channel inhibitor that has therapeutic potential for the treatment of atrial fibrillation. Many other Kv1.5 channel blockers also potently inhibit the Kv1.3 channel, but whether DPO-1 blocks Kv1.3 channels has not been investigated. The Kv1.3 channel is highly expressed in activated T cells, which is considered a favorable target for immunomodulation. Accordingly, we hypothesized that DPO-1 may exert immunosuppressive and anti-inflammatory effects by inhibiting Kv1.3 channel activity. In this study, DPO-1 blocked Kv1.3 current in a voltage-dependent and concentration-dependent manner, with IC₅₀ values of 2.58 µM in Jurkat cells and 3.11 µM in human peripheral blood T cells. DPO-1 also accelerated the inactivation rate and negatively shifted steady-state inactivation. Moreover, DPO-1 at 3 µM had no apparent effect on the Ca²⁺ activated potassium channel (K(Ca)) current in both Jurkat cells and human peripheral blood T cells. In Jurkat cells, pre-treatment with DPO-1 for 24 h decreased Kv1.3 current density, and protein expression by 48±6% and 60±9%, at 3 and 10 µM, respectively (both p<0.05). In addition, Ca²⁺ influx to Ca²⁺-depleted cells was blunted and IL-2 production was also reduced in activated Jurkat cells. IL-2 secretion was also inhibited by the Kv1.3 inhibitors margatoxin and charybdotoxin. Our results demonstrate for the first time that that DPO-1, at clinically relevant concentrations, blocks Kv1.3 channels, decreases Kv1.3 channel expression and suppresses IL-2 secretion. Therefore, DPO-1 may be a useful treatment strategy for immunologic disorders.

Zhao N., et al. (2013) Potent suppression of Kv1.3 potassium channel and IL-2 secretion by diphenyl phosphine oxide-1 in human T cells. PLoS One. PMID 23717641

OBJECTIVE:

The transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation.

DESIGN:

Case-control study.

SUBJECTS AND METHODS:

We took peripheral blood samples from 10 healthy individuals and 9 recently diagnosed rheumatoid arthritis (RA) patients receiving no anti-rheumatic treatment. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2 and CD8 cells applying a novel flow cytometry approach. We also assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels.

RESULTS:

The peak of calcium influx in lymphocytes isolated from RA patients is reached more rapidly, indicating that they respond more quickly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. No difference was detected between Th1 and Th2 or CD4 and CD8 cells in the sensitivity to IKCa1 channel inhibition among lymphocytes of RA patients. However, specific inhibition of the Kv1.3 channel acts differentially on calcium influx kinetics in RA lymphocyte subsets. Th2 and particularly CD8 cells are inhibited more dominantly than Th1 and CD4 cells.

CONCLUSION:

The inhibition of Kv1.3 channels does not seem to be specific enough in peripheral RA lymphocytes, since anti-inflammatory Th2 cells are also affected to a noteworthy extent.

Toldi G., et al. (2013) The effects of Kv1.3 and IKCa1 potassium channel inhibition on calcium influx of human peripheral T lymphocytes in rheumatoid arthritis. Immunobiology. PMID 22705192

Leukocytes, such as lymphocytes and macrophages, predominantly express delayed rectifier K(+) channels (Kv1.3), and the channels play crucial roles in the activation and proliferation of the cells. Since lymphocytes are activated in patients with end-stage renal disease (ESRD), the channels expressed in those cells would contribute to the progression of renal fibrosis in advanced-stage chronic renal failure (CRF). In the present study, using a rat model with advanced CRF that underwent 5/6 nephrectomy followed by a 14-week recovery period, we examined the histopathological features of the kidneys and the leukocyte expression of Kv1.3-channels and cell cycle markers. Age-matched sham-operated rats were used as controls. In the cortical interstitium of advanced CRF rat kidneys, leukocytes proliferated in situ and overexpressed Kv1.3 channel protein in their cytoplasm. Treatment with margatoxin, a selective Kv1.3-channel inhibitor, significantly suppressed the number of leukocytes and the progression of renal fibrosis with a significant decrease in the cortical cell cycle marker expression. This study demonstrated for the first time that the number of leukocytes was dramatically increased in rat kidneys with advanced CRF. The overexpression of Kv1.3 channels in the leukocytes was thought to contribute to the progression of renal fibrosis by stimulating cell cycling and promoting cellular proliferation.

Kazama I., et al. (2012) Overexpression of Delayed Rectifier K(+) Channels Promotes In situ Proliferation of Leukocytes in Rat Kidneys with Advanced Chronic Renal Failure. Int J Nephrol. PMID 22701172

BACKGROUND AND PURPOSE:

The roles played by endothelium-derived NO and prostacyclin and by endothelial cell hyperpolarization in ACh-induced relaxation have been well characterized in arteries. However, the mechanisms underlying ACh-induced relaxation in veins remain to be fully clarified.

EXPERIMENTAL APPROACH:

ACh-induced smooth muscle cell (SMC) hyperpolarization and relaxation were measured in endothelium-intact and -denuded preparations of rabbit jugular vein.

KEY RESULTS:

In endothelium-intact preparations, ACh (≤ 10⁻⁸ M) marginally increased the intracellular concentration of Ca²⁺ ([Ca²⁺](i)) in endothelial cells but did not alter the SMC membrane potential. However, ACh (10⁻¹⁰ -10⁻⁸ M) induced a concentration-dependent relaxation during the contraction induced by PGF(2α) and this relaxation was blocked by the NO synthase inhibitor N(ω) -nitro-l-arginine. ACh (10⁻⁸ -10⁻⁶ M) concentration-dependently increased endothelial [Ca²⁺](i) and induced SMC hyperpolarization and relaxation. These SMC responses were blocked in the combined presence of apamin [blocker of small-conductance Ca²⁺-activated K⁺ (SK(Ca) , K(Ca) 2.3) channel], TRAM 34 [blocker of intermediate-conductance Ca²⁺ -activated K⁺ (IK(Ca) , K(Ca) 3.1) channel] and margatoxin [blocker of subfamily of voltage-gated K⁺ (K(V) ) channel, K(V) 1].

CONCLUSIONS AND IMPLICATIONS:

In rabbit jugular vein, NO plays a primary role in endothelium-dependent relaxation at very low concentrations of ACh (10⁻¹⁰ -10⁻⁸ M). At higher concentrations, ACh (10⁻⁸ -3 × 10⁻⁶ M) induces SMC hyperpolarization through activation of endothelial IK(Ca) , K(V) 1 and (possibly) SK(Ca) channels and produces relaxation. These results imply that ACh regulates rabbit jugular vein tonus through activation of two endothelium-dependent regulatory mechanisms.

Itoh T. et al. (2012) Characteristics of ACh-induced hyperpolarization and relaxation in rabbit jugular vein. Br J Pharmacol. PMID 22595036

The effect of the pore-blocking peptides charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK(v)1.3 wild-type and hK(v)1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation.

Nikouee A. et al. (2012) Charybdotoxin and margatoxin acting on the human voltage-gated potassium channel hKv1.3 and its H399N mutant: an experimental and computational comparison. J Phys Chem B. PMID 22490327

Lymphocytes are of rich in delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and the channels play crucial roles in the lymphocyte activation and proliferation. Since chloroquine, a widely used anti-malarial drug, exerts immunosuppressive effects, it will affect the channel currents in lymphocytes. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of chloroquine on the channels expressed in murine thymocytes. Published papers report that chloroquine will inhibit voltage-dependent K(+)-channel currents by plugging into the open-pore. We observed, indeed, that chloroquine suppressed the pulse-end currents of Kv1.3-channels at higher voltage steps. Surprisingly, however, we found that the drug enhanced the peak currents at both higher and lower voltage steps. Since chloroquine showed such biphasic effects on the thymocyte K(+)-channels, and since those effects were voltage dependent, we examined the effects of chloroquine on the activation and the inactivation of the channel currents. We noted that chloroquine shifted both the activation and the inactivation curves toward the hyperpolarizing potential, and that those shifts were more emphasized at lower voltage steps. We conclude that chloroquine facilitates both the activation and the inactivation of Kv1.3-channel currents in thymocytes, and that those effects are voltage dependent.

Kazama I. et al. (2012) Voltage-dependent biphasic effects of chloroquine on delayed rectifier K(+)-channel currents in murine thymocytes. J Physiol Sci. PMID 22328488

The ATP-sensitive K(+) channels opener (K(ATP)CO), P1075 [N-cyano-N’-(1,1-dimethylpropyl)-N″-3-pyridylguanidine], has been shown to cause relaxation of various isolated animal and human blood vessels by opening of vascular smooth muscle ATP-sensitive K(+) (K(ATP)) channels. In addition to the well-known effect on the opening of K(ATP) channels, it has been reported that vasorelaxation induced by some of the K(ATP)COs includes some other K(+) channel subtypes. Given that there is still no information on other types of K(+) channels possibly involved in the mechanism of relaxation induced by P1075, this study was designed to examine the effects of P1075 on the rat renal artery with endothelium and with denuded endothelium and to define the contribution of different K(+) channel subtypes in the P1075 action on this blood vessel. Our results show that P1075 induced a concentration-dependent relaxation of rat renal artery rings pre-contracted by phenylephrine. Glibenclamide, a selective K(ATP) channels inhibitor, partly antagonized the relaxation of rat renal artery induced by P1075. Tetraethylammonium (TEA), a non-selective inhibitor of Ca(2+)-activated K(+) channels, as well as iberiotoxin, a most selective blocker of large-conductance Ca(2+) -activated K(+) (BK(Ca)) channels, did not abolish the effect of P1075 on rat renal artery. In contrast, a non-selective blocker of voltage-gated K(+) (K(V)) channels, 4-aminopyridine (4-AP), as well as margatoxin, a potent inhibitor of K(V)1.3 channels, caused partial inhibition of the P1075-induced relaxation of rat renal artery. In addition, in this study, P1075 relaxed contractions induced by 20 mM K(+) , but had no effect on contractions induced by 80 mM K(+). Our results showed that P1075 induced strong endothelium-independent relaxation of rat renal artery. It seems that K(ATP), 4-AP- and margatoxin-sensitive K(+) channels located in vascular smooth muscle mediated the relaxation of rat renal artery induced by P1075.

Novakovic A., et al. (2012) Different potassium channels are involved in relaxation of rat renal artery induced by P1075. Basic Clin Pharmacol Toxicol. PMID 22225832

The 39 amino acid peptide, margatoxin (MgTX), a potent inhibitor of the voltage-activated potassium channel (Kv 1.3) in human T lymphocytes, was synthesized by a solid phase technique. Formation of the disulfide bridges was rapid at pH 8.2. The final product was purified to homogeneity and was physically and biologically indistinguishable from the toxin prepared biosynthetically. The disulfide bridge pairing was similar to that found previously for the related toxin-charybdotoxin (3): from Cys7 to Cys29, from tested for inhibition of 125I margatoxin binding to voltage-activated potassium channels. The results indicate that the three C-terminal residues of MgTX are important for the efficient toxin binding to Kv1.3.

Bednarek, M. A., et al. (1994) Chemical synthesis and structure-function studies of margatoxin, a potent inhibitor of voltage-dependent potassium channel in human T lymphocytes, Biochem Biophys Res Commun.PMID: 8297371

The solution structure of the 39-residue peptide margatoxin, a scorpion toxin that selectively blocks the voltage-gated potassium-channel Kv1.3, has been determined by NMR spectroscopy. The toxin was isotopically labeled with 13C and 15N and studied using two-dimensional homonuclear and three- and four-dimensional heteronuclear NMR spectroscopy. The final structure was determined using 501 constraints, comprising 422 NOE constraints, 60 dihedral angle constraints, 9 disulfide constraints, and 10 hydrogen bond constraints. Structures were initially determined with the program PEGASUS and subsequently refined with X-PLOR. The average rms deviation from a calculated average structure for the backbone atoms of residues 3-38 is 0.40 A. A helix is present from residues 11 to 20 and includes two proline residues at positions 15 and 16. A loop at residues 21-24 leads into a two-strand antiparallel sheet from residues 25 to 38 with a turn at residues 30-33. Residues 3-6 run adjacent to the 33-38 strand but do not form a canonical beta-strand. The two additional residues of margatoxin, relative to the related toxins charybdotoxin and iberiotoxin, insert in a manner that extends the beta-sheet by one residue. Otherwise, the global structure is very similar to that of these two other toxins. The longer sheet may have implications for channel selectivity.

Johnson, B. A., et al. (1994) Determination of the three-dimensional structure of margatoxin by 1H, 13C, 15N triple-resonance nuclear magnetic resonance spectroscopy, Biochemistry.PMID: 7999764

A novel peptidyl inhibitor of K+ channels has been purified to homogeneity from venom of the new world scorpion Centruroides margaritatus. The primary structure of this 39-amino-acid peptide, which we term margatoxin (MgTX), was determined by amino acid compositional analysis and peptide sequencing. Margatoxin potently inhibits binding of radiolabeled charybdotoxin (ChTX) to voltage-activated channels in brain synaptic plasma membranes. Like ChTX, MgTX blocks the n-type current of human T-lymphocytes (Kv1.3 channel), but compared to ChTX, is 20-fold more potent (half-block at approximately 50 pM), has a slower dissociation rate, and has no effect on calcium-activated channels. To demonstrate that these characteristics are due solely to the purified toxin, recombinant MgTX was expressed in Escherichia coli as part of a fusion protein. After cleavage and folding, purified recombinant MgTX displayed the same properties as native peptide. Replacement of the COOH-terminal histidine residue of MgTX with asparagine resulted in a peptide with a 10-fold reduction in potency. This was due to a faster apparent dissociation rate, suggesting that the COOH-terminal amino acid may play an important role in the binding of MgTX to the Kv1.3 channel. MgTX displays significant sequence homology with previously identified K+ channel inhibitors (e.g. ChTX, iberiotoxin, noxiustoxin, and kaliotoxin). However, given its potency and unique selectivity, MgTX represents an especially useful tool with which to study the physiologic role of Kv1.3 channels.

Garcia-Calvo, et al. (1993) Purification, characterization, and biosynthesis of margatoxin, a component of Centruroides margaritatus venom that selectively inhibits voltage-dependent potassium channels, J Biol Chem. PMID: 8360176