The Effects Of PTEN On H₂O₂-Induced BK_Ca Channel Activation

Large conductance calcium and voltage-dependent potassium(BK_Ca) channels are ubiquitously distributed and play a pivotal role in physiological and pathological condition, such as vascular tone,neuronal secretion.In general,the BK_Ca channel function is regulated by oxidation,phosphorylation,intracellular calcium concentration and others. H₂O₂ is one of members of Reactive Oxygen Species(ROS) and also plays an important role in physiological and pathological processes.It not only causes oxidative damages to cellular function,but also acts as a second messenger.PTEN negatively controls the activity of PI3K to regulate cellular function,such as cell size,cell growth,apoptosis and migration.In this study,the relationship between H₂O₂,BK_Ca channels and PTEN was elucidated by studying the effect of H₂O₂ on BKca channel activity,PTEN and its lipid phosphatase activity on H₂O₂- induced BK_Ca channel activation.This study is to prove the molecular mechanism of H₂O₂-induced BK_Ca channel activation or vasodilation and provide evidences for discovering the mechanism of oxidation induced vascular diseases.In this study,BK_Ca channel encoded by mouse Slo was heterologously expressed in HEK 293 cells and the typical BKc,channel currents were recorded to study the effects of H₂O₂ on BK_Ca channel in inside-out and cell-attached configurations.In inside-out configurations,our results showed that BK_Ca channel current was inhibited after exposure to H₂O₂ at 0μmol Ca²⁺ bath solutions,and the inhibition could be reversed partially by DTT.However,the effects of H₂O₂ and DTT on BK_Ca channel were abolished at 10μmol Ca²⁺ bath solutions.These data suggested that the effect of H₂O₂ on BK_Ca channel is in a voltage and Ca²⁺-dependent manner and H₂O₂ could decrease the Ca²⁺ sensitivity of channels by thiol oxidation.In cell-attached configuration,we found that NP_o of single BK_Ca channels were significantly increased after application of H₂O₂.However,the specific sulfhydryl oxidant DTNB inhibited BK_Ca channel NP_o in two different configurations,suggesting that the regulation of BK_Ca channels by DTNB and H₂O₂ is different.It also elucidate that the effects of H₂O₂ on BK_Ca channels depend on different action modes and H₂O₂ concentrations.To explore the effect of PTEN on H₂O₂-induced BK_Ca channel activation,the PTEN-Tdimer2 plasmids were constructed in our studies.The PTEN-Tdimer2 plasmids and mSlo in the presence or absence of hβ1 were coexpressed in HEK 293 cells and the typical BK_Ca channel currents were recorded in cell-attached configuration.Contrary to H₂O₂-induced BK_Ca activation,the BK_Ca channel currents and conductance in the absence or presence of hβ1 were not changed in PTEN overexpressing cells during the initial 10 min treatment with H₂O₂.It not only suggested that PTEN inhibited H₂O₂-induced BK_Ca channel activation,but also verified that the inhibition of PTEN was not affected byβ1. Similarly,the typical BK_Ca channel currents were recorded in cell-attached configuration when HEK293 cells expressing mSlo channels were pre-incubated with the PI3K inhibitor LY294002 or Wortmannin for 1 hour.The results showed that the effect of LY294002 or Wortmannin on H₂O₂-induced BK_Ca channel activation was same as PTEN,suggesting that PI3K activity is involved in H₂O₂-induced BK_Ca channel activation.In order to elucidate the effects of PI3K/PTEN pathway on H₂O₂- induced BK_Ca channel activation in physiological and pathological processes,H₂O₂-induced relaxation of isolated rat thoracic aortas was investigated.The results showed that H₂O₂-induced relaxation of aortal rings was decreased by PI3K inhibitor LY294002.However,the effect of LY294002 was abolished in the presence of BK_Ca channel inhibitor Iberiotoxin.It suggested that PI3K activity is involved in H₂O₂-induced relaxation of aorta tings via BK_Ca channel activation.To further elucidate the mechanism of PTEN on the inhibiton of H₂O₂-induced BK_Ca channel activation,mSlo was coexpressed with catalytically inactive PTEN_C124S/ PTENG_G129E mutants that lack lipid phosphatase activity.We found that the mutants produced no regulation on the H₂O₂-induced BK_Ca channel activation,suggesting that PTEN regulates H₂O₂-induced BK_Ca channels activation by acting as a phosphatidylinositol 3-phosphatase.Meanwhile,the p-AKT expression in PTEN_wt, PTEN_C124S and PTEN_G129E overexpressing cells after H₂O₂ application was investigated by Western Blot analysis.We found that the p-AKT expression inhibited by PTEN was not changed after H₂O₂ addition for 10 min.However,the inhibition was changed after H₂O₂ addition for 30 min.The results suggested that the change of p-AKT expression may be through oxidation of PTEN by H₂O₂.The cytoplasmic free calcium concentrations ([Ca²⁺]_i) in PTEN_wt,PTEN_C124S and PTEN_G129E overexpressing cells afeer H₂O₂ application were also detected by laser scanning confocal microscopy.We found that the increase of[Ca²⁺]_i induced by H₂O₂ was also inhibited in PTEN_wt overexpressing cells. However,the results were not detected in PTEN_C124S and PTEN_G129E overexpressing cells. These data suggested that the lipid phosphatase activity of PTEN is involved in H₂O₂-induced BK_Ca channel activation,p-AKT and[Ca²⁺]_i may be important downstream signal molecules for H₂O₂-induced BK_Ca channel activation.