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

人心脏延迟整流钾通道有2个组分，I_2<Kr>和<KS>I_3。I_<Kr>channel由HERG编码，<Ks>由KvLQT 1和minK两个分子实体组成，我们利用爪蟾卵母细胞表达系统研究了HERG和KvLQT 1-minK通道的调控。ET-1刺激共表达的人内皮素受体（hETR）对HERG电流无明显影响。然而，共表达的hETR的刺激显着抑制KvLQT 1-minK电流。为了研究酪氨酸激酶是否参与hETR刺激对电流的抑制，用酪氨酸激酶抑制剂预处理表达KvLQT 1-minK和hETR的卵母细胞。用酪氨酸激酶抑制剂处理并没有减弱hETR刺激的抑制作用，反而增强了它。已知hETR的刺激可以激活存在于爪蟾卵母细胞中的Ca^2+激活的Cl^-通道。Cl^-电流的激活是短暂的，但在hETR刺激后5分钟明显。酪氨酸激酶抑制剂显著抑制Cl^-电流的增强，这表明hETR的刺激引起酪氨酸激酶的激活。尚未确定hETR刺激后酪氨酸激酶的激活涉及何种途径。共表达KvLQT 1-minK、hETR和β_1肾上腺素能受体，观察受体刺激对KvLQT 1-minK电流的影响。虽然<Ks>蛋白激酶A（PKA）和蛋白激酶C（PKC）对电流的专一性调节已有报道，但我们还没有观察到这种专一性调节。细胞外酸中毒，这是已知发生在病理生理条件下，如缺血，对HERG电流的影响也进行了研究。酸中毒最显著的影响是加速失活动力学。

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.