Involvement of tyrosine kinase in regulation of ion channel functions and crosstalk between signal transduction systems

人心肌延迟整流钾通道有I_lt；Kr&gt；和I_lt；Ks&gt；两种成分。Ilt；Kr&gt；通道的孔道亚基由HERG编码，I&lt；Ks&gt；通道由KvLQT1和minK两个分子实体组成。ET-1刺激共表达的人内皮素受体(HETR)对HERG电流无明显影响。但刺激共表达的hETR可明显抑制KvLQT1-mink电流。为探讨酪氨酸激酶是否参与hETR刺激对电流的抑制作用，用酪氨酸激酶抑制剂对表达KvLQT1-mink和hETR的卵母细胞进行预处理。用酪氨酸激酶抑制剂治疗并没有减弱hETR刺激的抑制作用，反而增强了它。HETR的刺激可以激活非洲爪哇卵母细胞中存在的Ca~(2+)和Gt~(2+)激活的Cl~-通道。刺激HETR后5min，氯离子电流的激活是短暂的，但明显。酪氨酸激酶抑制剂可明显抑制氯电流的增强，提示刺激hETR可引起酪氨酸激酶的激活。目前还没有确定hETR刺激后酪氨酸激酶的激活途径是什么。KvLQT1-mink、hETR和β_1肾上腺素能受体共表达，并观察受体刺激对KvLQT1-mink电流的影响。尽管已有报道使用化学试剂对蛋白激酶A(PKA)和蛋白激酶C(PKC)进行独占调制，但我们尚未观察到这种独占调制。细胞外酸中毒，已知发生在病理生理条件下，如缺血，对HERG电流的影响也被研究。酸中毒最显著的影响是加速了失活动力学。

Human cardiac delayed rectifier potassium channel has 2 components, I_<Kr> and I_<KS>. The poreforming subunit of I_<Kr> channel is encoded by HERG and I_<Ks> channel is composed of 2 molecular entities, KvLQT1 and minK.We have investigated on modulation of HERG and KvLQT1-minK channels using a Xenopus oocyte expression system. Stimulation of coexpressed human endothelin receptor (hETR) by ET-1 did not have obvious influence on HERG currents. However, stimulation of coexpressed hETR markedly inhibited KvLQT1-minK currents. To investigate whether tyrosine kinases are involved in the inhibition of the currents by hETR stimulation, oocytes expressing KvLQT1-minK and hETR were pretreated with tyrosine kinase inhibitors. Treatment with tyrosine kinase inhibitors did not attenuate the inhibitory effect of hETR stimulation, but rather enhanced it. Stimulation of hETR is known to activate Ca^<2+> activated Cl^- channel which is present in Xenopus oocytes. Activation of the Cl^- current was transient but obvious at 5 min after hETR stimulation. Tyrosine kinase inhibitors markedly inhibited the enhancement of the Cl^- currents, which suggested that stimulation of hETR caused activation of tyrosine kinases. It has not been determined yet what pathway is involved in activation of tyrosine kinases following hETR stimulation. KvLQT1-minK, hETR and β_1 adrenergic receptor were coexpressed and the effects of receptor stimulation on KvLQTl-minK currents were investigated. Although exclusive modulation of I_<Ks> current by protein kinase A (PKA) and protein kinase C (PKC) has been reported using chemical reagents, we have not observed the exclusive modulation. Effects of extracellular acidosis, which is known to occur in pathophysiological conditions such as ischemia, on HERG currents were also investigated. Most prominent effect of acidosis was acceleration of deactivation kinetics.