Modulatory Effect Of Kv1.3Potassium Channel Blocking Drugs On Human T Cell Activation

Part ⅠPotent suppression of Kv1.3potassium channel and IL-2secretion by diphenyl phosphine oxide-1in human T cellsPurpose:Diphenyl phosphine oxide-1(DPO-1) is a novel and potent blocker of Kv1.5channel promising to be used in the therapy of atrial fibrillation (AF), which can selectively prolong the atrial effective refractory period. Kv1.3channel predominantly expresses in T cells and take part in the activation, migration, proliferation and cytokine secretion of T cells. Selective blockade of Kv1.3channel serves as a good target for immunomodulatory therapies. The present study was carried out to investigate the effect of DPO-1on Kv1.3channel and IL-2secretion in human T cells.Methods:Whole-cell patch-clamp was applied to record Kv1.3and Ca2+-activated potassium (Kca) channel currents in human peripheral blood T cells and Jurkat cells, real-time PCR and western blotting to detect Kv1.3channel expression level, fluo-3to measure the intracellular Ca2+and ELISA to quantity IL-2production in Jurkat cells.Results:DPO-1inhibited the Kv1.3channel current in a voltage-dependent and concentration-dependent manner, with IC50values of2.58μmol/L in Jurkat cells and 3.11μmol/Lin human peripheral blood T cells. DPO-1also accelerated the inactivation rate and negatively shifed the steady-state inactivation curves. Morever, DPO-1at3μmol/L had no apparent effect on Kca channel current in both Jurkat cells and human peripheral blood T cells. In Jurkat cells, pretreatment with DPO-1for24h decreased Kvl.3channel current density, and protein expression by48±6%,60±9%, respectively. In addition, Ca2+influx into Ca2+-depleted cells was blunted and IL-2production was also reduced in active Jurkat cells.Conclusions:Kv1.5channel blocker DPO-1can also blocks Kv1.3channel, reduces Kv1.3channel expression andinhibits Ca2+influx, IL-2secretion in Jurkat cells. The immunomodulatory function of DPO-1may additionally benefit its application in the treatment of AF. Part Ⅱ Acacetin blocks Kv1.3channels and inhibits human T cell activationBackground:Acacetin, a natural flavonoid compound extracted from thetraditional Chinese medicine Xuelianhua, was prospective for AF antiarrhythmia therapy. Furthermore, it has been proved to exert anti-inflammatory andimmunomodulatory effect, but the mechanism requires further investigation.The present study was designed tocharacterize the inhibition of Kv1.3channels by Acacetin in human T cells and examine its role in T cell activation.Methods:Whole-cell patch-clamp was applied to record Kvl.3and Ca2+activated-potassium (KCa) currents in human peripheral blood T cells or Jurkat cells. Real-time PCR and western blot were carried out to detect Kv1.3expression as well as NFAT1, NF-κB activity. Fluo-4, CCK-8kit and ELISA kit were used to measure Ca2+influx, proliferation, and IL-2secretion, respectively.Results:Acacetin decreased Kv1.3currents, accelerated the decay rate of current inactivation and negatively shifted the steady-state inactivation-curves in a concentration-dependent manner. The block ofKvl.3by Acacetin was time-and voltage-dependent.IC50for peak and steady state currents was21.09±2.75μmol/Land3.63±0.25μmol/L, respectively.However,30μmol/L Acacetin had no apparent effect on KCa current.Furthermore, Acacetin significantly inhibited the protein expression level of Kv1.3channels. In addition, paralleling Kvl.3inhibition, Acacetin also inhibited Ca2+influx, Ca2+-activated transcription factors NFAT1, NF-κB p65/p50, proliferation as well as IL-2production in human T cells.siRNA against Kv1.3reduced the inhibitory effect of Acacetin on IL-2secretion.Conclusions:Acacetin blocks Kvl.3channel and exerts the immunomodulatory and anti-inflammatory actionsin human T cells, which may benefit for its application in atrial fibrillation and other T cell-mediated inflammatory disorders. PART Ⅲ Immunomodulatory effect of Lovastatin in human T cells through blocking Kv1.3channelsBackground:Lovastatin is a member of statins, inhibitors of3-hydroxy-3-methyl-glutarylCoA (HMG-CoA), can reduce the formation of intermediaries in the mevalonate pathway, then exert immunomodulatory effect. Recently, statins have been the "upstream therapy" for AF, and also extenuatethe T cells-mediated autoimmune diseases. But the immunomodulatory mechanisms require further investigation. Kv1.3potassium channels play important roles in the activation, migration, proliferation and cytokine secretion of T cells. Selective blockade of Kv1.3channels has been an attractive target in the immunomodulatory therapy. The present study was designed to examine the block effect of Lovastatin on Kv1.3channels in human T cells, to clarify its new immunomodulatory mechanism in T cells.Methods:Whole-cell patch-clamp was applied to record Kv1.3and KCacurrents in Jurkat cells. Real-time PCR and western blot were used to detect Kv1.3expression. Fluo-4, CCK-8kit and ELISA kit were used to measure Ca2+influx, T cells proliferation, and IL-2 secretion, respectively.Results:Lovastatin inhibited Kv1.3currents in a concentration-and voltage-dependent manner, and the ICso for peak and steady-state currents was39.81±5.11and6.92±0.95μmol/L, respectively.Lovastatin also accelerated the decay rate of current inactivation and negatively shifted the steady-state inactivationcurves in a concentration-dependent manner, withouteffect on the activation curves.However,30μmol/LLovastatinhad no apparent effect on KCa current.24h incubation with Lovastatin had no effect onthe mRNA and protein expression level of Kvl.3channels. In addition, paralleling Kv1.3inhibition, Acacetin also inhibited Ca2+influx, T cell proliferation as well as IL-2production.Mevalonate application partially reversed the inhibition of Lovastatin on IL-2secretion. At last, siRNA against Kvl.3reduced the inhibitory effect of Lovastatin on IL-2secretion.Conclusions:Lovastatin probably exerts immunodulatory effect through Kvl.3channels in human T cells, which may be a new mechanism for its therapy effect in atrial fibrillation and other T cell-mediated autoimmune diseases.