The Interaction And Its Mechanism Of I_Ca,L And I_Kr

Background and purpose:Heart’s most basic electrical activity is action potential(AP),which is formed by a variety of ion current.AP includes in depolarization and repolarization.The process of repolarization is more complicated and more important to action potential duration(APD).In recent years, many studies have shown that the interaction of cardiac ion current.The cardiac L-type calcium channel is voltage-dependent channel and its current is part of action potential 2 phase(plateau).The rapidly activated delayed rectifier potassium channel(HERG) also is the cardiac voltage-dependent channel and its current is part of action potential 3 phase(Rapid repolarization phase).We studied the electrophysiological characteristics of Ica-l and IKr of ventricular myocyte of newborn rat and Ica-l and IKr expressed by Cav1.2 and HERG to determine the existence of interaction of the two current.The First Part The electrophysiological property of the ICa,L and IKr of newborn rat’s ventricular myocyteMethods:The ventricular myocyte of newborn wistar rats were enzymatically disassociated by the method of enzyme digestion with differential attachment.Whole cell-patch clamp recording technique was used to record Ica-l and IKr.Under the effect of D o f e t i l i d e(Potassium channel blockers),observing the change of I C a, L. Un d e r t h e e ff e c t o f Ba y K 8 6 4 4( C a l c i u m c h a n n e l a g o n i s t s)、C d C l 2( C a l c i u m c h a n n e l blockers)、W7( C a l mo d u l i n i n h i b i t o r s) 、 K N 9 3( C a l mo d u l i n k i n a s e 2 i n h i b i t o r s), observing the change of IKr.Results:1.After the addition of D o f e t i l i d e, The peak current of I C a, L in normal ventricular myocyte group was(-9.76±0.37)p A/p F compared to(-11.62 ±0.41)p A/p F with significant difference(P<0.05). The Steady-state inactivation curve V1/2-21.21±0.97 m V,k 9.85±0.90 m V,compared to V1/2-25.85±0.91 m V, k 6.50±0.84 m V with significant difference(P<0.05). 2. After the addition of Ba y K 8 6 4 4, The peak current of I K r in normal ventricular myocyte group was(18.79±0.32)p A/p F compared to(14.09 ±0.52)p A/p F with significant difference(P<0.05). 3. After the addition of C d C l 2 、 W7 、 K N 9 3, The peak current of I K r in normal ventricular myocyte group was(18.79 ±1.32)p A/p F compared to(26.13±2.35)p A/p F、(26.43±1.72)p A/p F、(25.93±1.88)p A/p F with significant difference(P<0.05).Conclusions:1. After D o f e t i l i d e reduce the current density of I C a, L and doesn’t influence the steady-state activation curve.,but move the steady-state inactivation curve to the right.That is to say IK r can change the peak of I C a, L and change the process of the inactivation,but doesn’t influence the process of the activation.2. Increase I C a, L can decrease the current density of I K r,but doesn’t change the steady-state activation curve. That is to say the I C a, L can change the peak of I K r,but doesn’t influence the process of the activation3. After the addition of C d Cl 2 、W7、KN93,all increase the current density of I K r,but doesn’t change the steady-state activation curve. That is to say the I C a, L maybe influence I K r through C a2 +/ Ca M/Ca MKII.The Second Part The electrophysiological property of the Cav1.2 and HERG after the transfectionMethods:The Cav1.2 and HERG channel expression system was made using Immunomagnetic Beads positive mark method. Through the determination of the I-V curve, the steady-state activation curve and the steady-state inactivation curve, comparing the difference of the WT-Type and Co-transfection.Results:1.After co-transfecting with Cav1.2,the peak current of H E R G in WT group was(27.79±1.87)p A/p F、(42.6±1.52)p A/p F compared to(20.48±1.57)p A/p F、(34.72±1.62) p A/p F with significant difference(P<0.05). 2. The Steady-state activation curve is not significant difference. 3. After co-transfecting with HERG,the peak current of C a v 1. 2 in WT group was(-11.31±0.51)p A/p F compared to(-8.31 ±0.52)p A/p F with significant difference(P<0.05). 4. The Steady-state inactivation curve moved the V1/2 to the left 4.8m V,K decrease 3.1m V with significant difference(P<0.05).Conclusions:1. the current density of co-transfected HERG channel current is smaller than that of the wild-type, but doesn’t change the characteristics of the inward rectifier and the steady-state activation curve is nearly the same. That is to say synergistic effect of C a v 1. 2 channel protein can significantly change the peak of the HERG channel current,but doesn’t influence the process of the activation2. the current density of co-transfected Cav1.2 channel current is smaller than that of the wild-type and the steady-state activation curve is nearly the same,but the steady-state inactivation curve move to the left. That is to say synergistic effect of HE R G channel protein can significantly change the peak of the Cav1.2 channel current and doesn’t influence the process of the activation,but change the process of the inactivation3. The dynamics change of co-transfected HERG and Cav1.2 channel indicates direct interaction and influence.