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 (KCa) 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.
References
[1]
Du YM, Zhang XX, Tu DN, Zhao N, Liu YJ, et al. (2010) Molecular determinants of Kv1.5 channel block by diphenyl phosphine oxide-1. J Mol Cell Cardiol 48: 1111–1120.
[2]
Lagrutta A, Wang J, Fermini B, Salata JJ (2006) Novel, potent inhibitors of human Kv1.5 K+ channels and ultrarapidly activating delayed rectifier potassium current. J Pharmacol Exp Ther 317: 1054–1063.
[3]
Regan CP, Wallace AA, Cresswell HK, Atkins CL, Lynch JJ (2006) In vivo cardiac electrophysiologic effects of a novel diphenylphosphine oxide IKur blocker, (2-Isopropyl-5-methylcyclohexyl) diphenylphosphine oxide, in rat and nonhuman primate. J Pharmacol Exp Ther 316: 727–732.
[4]
Stump GL, Wallace AA, Regan CP, Lynch JJ (2005) In vivo antiarrhythmic and cardiac electrophysiologic effects of a novel diphenylphosphine oxide IKur blocker (2-isopropyl-5-methylcyclohexyl) diphenylphosphine oxide. J Pharmacol Exp Ther 315: 1362–1367.
[5]
Pandit SV, Zlochiver S, Filgueiras-Rama D, Mironov S, Yamazaki M, et al. (2011) Targeting atrioventricular differences in ion channel properties for terminating acute atrial fibrillation in pigs. Cardiovasc Res 89: 843–851.
[6]
Liu HL, Lin JC (2004) A set of homology models of pore loop domain of six eukaryotic voltage-gated potassium channels Kv1.1-Kv1.6. Proteins 55: 558–567.
[7]
Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309: 897–903.
[8]
Decher N, Kumar P, Gonzalez T, Pirard B, Sanguinetti MC (2006) Binding site of a novel Kv1.5 blocker: a “foot in the door” against atrial fibrillation. Mol Pharmacol 70: 1204–1211.
[9]
Decher N, Pirard B, Bundis F, Peukert S, Baringhaus KH, et al. (2004) Molecular basis for Kv1.5 channel block: conservation of drug binding sites among voltage-gated K+ channels. J Biol Chem 279: 394–400.
[10]
Lewis RS, Cahalan MD (1995) Potassium and calcium channels in lymphocytes. Annu Rev Immunol 13: 623–653.
[11]
Chandy KG, Wulff H, Beeton C, Pennington M, Gutman GA, et al. (2004) K+ channels as targets for specific immunomodulation. Trends Pharmacol Sci 25: 280–289.
[12]
Varga Z, Hajdu P, Panyi G (2010) Ion channels in T lymphocytes: an update on facts, mechanisms and therapeutic targeting in autoimmune diseases. Immunol Lett 130: 19–25.
[13]
Rangaraju S, Chi V, Pennington MW, Chandy KG (2009) Kv1.3 potassium channels as a therapeutic target in multiple sclerosis. Expert Opin Ther Targets 13: 909–924.
[14]
Fasth AE, Cao D, van Vollenhoven R, Trollmo C, Malmstrom V (2004) CD28nullCD4+ T cells–characterization of an effector memory T-cell population in patients with rheumatoid arthritis. Scand J Immunol 60: 199–208.
[15]
Viglietta V, Kent SC, Orban T, Hafler DA (2002) GAD65-reactive T cells are activated in patients with autoimmune type 1a diabetes. J Clin Invest 109: 895–903.
[16]
Azam P, Sankaranarayanan A, Homerick D, Griffey S, Wulff H (2007) Targeting effector memory T cells with the small molecule Kv1.3 blocker PAP-1 suppresses allergic contact dermatitis. J Invest Dermatol 127: 1419–1429.
[17]
Beeton C, Wulff H, Barbaria J, Clot-Faybesse O, Pennington M, et al. (2001) Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc Natl Acad Sci USA 98: 13942–13947.
[18]
Beeton C, Wulff H, Standifer NE, Azam P, Mullen KM, et al. (2006) Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases. Proc Natl Acad Sci USA 103: 17414–17419.
[19]
Wang X, Liao Y, Zou A, Li L, Tu D (2007) Blockade action of ketanserin and increasing effect of potassium ion on Kv1.3 channels expressed in Xenopus oocytes. Pharmacol Res 56: 148–154.
[20]
Ahn HS, Kim SE, Jang HJ, Kim MJ, Rhie DJ, et al. (2007) Open channel block of Kv1.3 by rosiglitazone and troglitazone: Kv1.3 as the pharmacological target for rosiglitazone. Naunyn Schmiedebergs Arch Pharmacol 374: 305–309.
[21]
Pang B, Zheng H, Shin DH, Jung KC, Ko JH, et al. (2010) TNF-alpha inhibits the CD3-mediated upregulation of voltage-gated K+ channel (Kv1.3) in human T cells. Biochem Biophys Res Commun 391: 909–914.
[22]
DeCoursey TE, Chandy KG, Gupta S, Cahalan MD (1984) Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis? Nature 307: 465–468.
[23]
Matteson DR, Deutsch C (1984) K channels in T lymphocytes: a patch clamp study using monoclonal antibody adhesion. Nature 307: 468–471.
[24]
Garcia-Calvo M, Leonard RJ, Novick J, Stevens SP, Schmalhofer W, 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 268: 18866–18874.
[25]
Cahalan MD, Chandy KG (2009) The functional network of ion channels in T lymphocytes. Immunol Rev 231: 59–87.
[26]
Grissmer S, Lewis RS, Cahalan MD (1992) Ca2+-activated K+ channels in human leukemic T cells. J Gen Physiol 99: 63–84.
[27]
Ghanshani S, Wulff H, Miller MJ, Rohm H, Neben A, et al. (2000) Up-regulation of the IKCa1 potassium channel during T-cell activation. Molecular mechanism and functional consequences. J Biol Chem 275: 37137–37149.
[28]
Fanger CM, Rauer H, Neben AL, Miller MJ, Rauer H, et al. (2001) Calcium-activated potassium channels sustain calcium signaling in T lymphocytes. Selective blockers and manipulated channel expression levels. J Biol Chem 276: 12249–12256.
[29]
Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, et al. (2000) Design of a potent and selective inhibitor of the intermediate-conductance Ca2+-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci USA 97: 8151–8156.
[30]
Robbins JR, Lee SM, Filipovich AH, Szigligeti P, Neumeier L, et al. (2005) Hypoxia modulates early events in T cell receptor-mediated activation in human T lymphocytes via Kv1.3 channels. J Physiol 564: 131–143.
[31]
Villalonga N, David M, Bielanska J, Gonzalez T, Parra D, et al. (2010) Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels. Biochem Pharmacol 80: 858–866.
[32]
Choi JS, Hahn SJ, Rhie DJ, Yoon SH, Jo YH, et al. (1999) Mechanism of fluoxetine block of cloned voltage-activated potassium channel Kv1.3. J Pharmacol Exp Ther 291: 1–6.
[33]
Ahn HS, Kim SE, Choi BH, Choi JS, Kim MJ, et al. (2007) Calcineurin-independent inhibition of KV1.3 by FK-506 (tacrolimus): a novel pharmacological property. Am J Physiol Cell Physiol 292: C1714–C1722.
[34]
Robe RJ, Grissmer S (2000) Block of the lymphocyte K+ channel mKv1.3 by the phenylalkylamine verapamil: kinetic aspects of block and disruption of accumulation of block by a single point mutation. Br J Pharmacol 131: 1275–1284.
[35]
Choi JS, Hahn SJ, Rhie DJ, Jo YH, Kim MS (1999) Staurosporine directly blocks Kv1.3 channels expressed in Chinese hamster ovary cells. Naunyn Schmiedebergs Arch Pharmacol 359: 256–261.
[36]
Verheugen JA, Vijverberg HP, Oortgiesen M, Cahalan MD (1995) Voltage-gated and Ca2+-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium. J Gen Physiol 105: 765–794.
[37]
Rus H, Pardo CA, Hu L, Darrah E, Cudrici C, et al. (2005) The voltage-gated potassium channel Kv1.3 is highly expressed on inflammatory infiltrates in multiple sclerosis brain. Proc Natl Acad Sci USA 102: 11094–11099.
[38]
Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, et al. (2003) The voltage-gated Kv1.3 K+ channel in effector memory T cells as new target for MS. J Clin Invest 111: 1703–1713.
[39]
Regan CP, Stump GL, Wallace AA, Anderson KD, McIntyre CJ, et al. (2007) In vivo cardiac electrophysiologic and antiarrhythmic effects of an isoquinoline IKur blocker, ISQ-1, in rat, dog, and nonhuman primate. J Cardiovasc Pharmacol 49: 236–245.
[40]
Panyi G, Possani LD, Rodriguez DLVR, Gaspar R, Varga Z (2006) K+ channel blockers: novel tools to inhibit T cell activation leading to specific immunosuppression. Curr Pharm Des 12: 2199–2220.
