KCNE2 Modulates The Function Of Transient Outword Potassium Current And Electrophysiological Properties In Cadiomyocytes

MiRPl is encoded by KCNE2, which contains 123 amino acids, also called KCNE2 protein. They do not produce any current by themselves. Heterologously expressed KCNE2 can associate with different a-subunits of voltage-dependent potassium channel participated in cardiac action potential repolarization, such as HERG (fast component of delayed rectifier potassium current, a subunit of Ikr), Kv4.x (x=2/3) and Kv3.4 (transient outward potassium current,a subunit of Ito), KCNQ1 (slow delayed rectifier potassium current, a subunit of Iks) and hyperpolarization-activated cyclic nucleotide gated cation channel HCNx (x=1/2) (cardiac pacemaker channel current, funny current, a-subunit of If) and play a regulatory role.KCNE2 gene mutations can lead to acquired and congenital long QT syndrome (LQTS), but the specific mechanism is unclear now. With a detailed study, the function of ion channel in cardiac hypertrophy models is in a variety of concern. The downregulation of Ito could be found in almost all cardiac hypertrophy models and the downregulation of Iks could be found in part of cardiac hypertrophy models. It has been considered that reconstruction of potassium channels is the result of cardiac hypertrophy. But in recent years, studies have shown that the downregulation of potassium channels are participated in cardiac hypertrophy. Asβsubunits oflkr, Ito and Iks channel, KCNE2 has been found decreased in dog myocardial infarction model, while KCNE2 expression increase in the cardiomyocytes of old rats and spontaneously hypertensive rats (SHR). Whether the changes of KCNE2 expression will play a role in the process of heart disease through affecting cardiac electrophysiological or not, yet to be further studied.Part 1:Functional regulation of Kv4.3 by I57T and V65M, two kinds of LQT6 associated mutations in KCNE2In this study, we used whole-cell patch clamp recording to study the effect of the two LQT6 related mutants I57T and V65M on Kv4.3 channel function in COS-7, and investigate the electrophysiological mechanisms of arrhythmia induced by LQT6. The results showed:1.1 Effect of 57T and V65M on Kv4.3 channel current density and gating kineticsCo-expression of Kv4.3 and KCNE2 current density had prone to decrease than that of Kv4.3 alone. I57T and V65M also decreased Kv4.3 channel current density and there is no significant difference.KCNE2 significantly slowed the rate of Kv4.3 channel activation and inactivation. The phenotype of I57T and Kv4.3 coexpressed channel was similar to that of Kv4.3 expressed alone. But TTP and T0.5 were significantly faster than that of Kv4.3 and KCNE2 coexpression channel. In contrast, not only the TTP and T0.5 of V65M and Kv4.3 coexpressed channel were significantly higher than that of Kv4.3 alone (P=0.008 and P =0.021, respectively), but also higher than that of Kv4.3 and KCNE2 coexpression, although there is no significant difference.1.2 Effect of I57T and V65M on voltage dependence of Kv4.3 channel current activationThe expression of KCNE2 caused a positive shift of Kv4.3 current voltage-dependent inactivation, P=0.020.I57T caused a slight negative shift of Kv4.3 channel inactivation, closer to the Kv4.3 channel (P=0.740). But it was significantly greater than Kv4.3 and KCNE2 co-expression (P=0.008). V65M made a significantly positive shift of Kv4.3 channel inactivation (P=0.001), but there was lack of significant difference compared with Kv4.3 and KCNE2 co-expression (P=0.327). K-values among the four groups were not statistically significant, indicating no change of mutant KCNE2 on ion selectivity of Kv4.3.1.3 Effect of I57T and V65M on voltage-dependence of Kv4.3 channel current recovery from inactivationKCNE2 accelerated Kv4.3 channel recovery from inactivation. And KCNE2 didn't induce an overshoot of Kv4.3 current. I57T didn't significantly slow down Kv4.3 and Kv4.3+KCNE2 channel recovery from inactivation. But V65M could further accelerated Kv4.3 channel recovery from inactivation.Conclusion:Co-expression of KCNE2 with Kv4.3 slowed the rate of activation and inactivation, and caused a positive shift of voltage-dependence of inactivation of Kv4.3 channel. Moreover, KCNE2 accelerated recovery of Kv4.3 channel from inactivation. In comparison with the effect of wild type KCNE2,the phenotype of I57T and Kv4.3 coexpressed channel was similar to that of Kv4.3 expressed alone. In contrast, V65M enhanced all the effects of KCNE2 on Kv4.3. The results suggest that I57T causes "loss of function" while V65M results in "gain of function" of KCNE2.Part 2:Co-expression of KCNE2 and KChIP2c modulates the electrophysiolo-gical properties of Kv4.2 current in cos-7 cellsSeveralβ-subunits have been suggested to modulate the electrophysiological properties of Ito in cardiac myocytes, including the obligatoryβsubunit KChIP2 and KCNE2. The present study is to investigate how Ito current is modulated both KChIP2 and KCNE2 are co-expressed. 2.1 Effects of coexpression of KCNE2 and KChIP2c on Kv4.2 current density and gating kineticsKChIP2c significantly increased Kv4.2 current density by 2.3 fold. In comparison with the effect of KChIP2c alone, coexpression of KCNE2 did not change the current density. The activation and inactivation rates of Kv4.2 were slowed down by the expression of KChIP2c alone. When coexpressed with KChIP2c, KCNE2 did not further slowed Kv4.2 activation rate, but slowed Kv4.2 inactivation kinetics.2.2 Effects of coexpression of KCNE2 and KChIP2c on voltage-dependence of Kv4.2 current activationExpression of KChIP2c caused 8.0 mV positive shift of Kv4.2 current activation. When KCNE2 was coexpressed, the effect of KChIP2c on voltage-dependence of Kv4.2 current activation was weakened, only 2.7 mV positive shift versus Kv4.2 group.2.3 Effects of coexpression of KCNE2 and KChIP2c on voltage-dependence of Kv4.2 current inactivationIn comparison with the effect of KChIP2c, coexpression of KCNE2 caused no further alteration in voltage-dependence of inactivation.2.4 Effects of coexpression of KCNE2 and KChIP2c on voltage-dependence of Kv4.2 current recovery from inactivationKChIP2c accelerated Kv4.2 recovery from inactivation. When KCNE2 was coexpressed, the recovery was further accelerated. It is noteworthy that coexpression of KCNE2 induced an overshoot of Kv4.2 current during recovery from inactivation. Conclusion:KChIP2c and KCNE2 are simultaneously involved in the recapitulation of the electrophysiological properties of native Ito in cardiac myocytes.Coexpression of KCNE2 failed to change some of the current properties in comparison with the current modulated by KChIP2c alone which suggests that coexpression of KChIP2c and KCNE2 might change the pattern of KCNE2 and/or KChIP2c modulation of Kv4.2 currents.Part 3:Effects of changes in the expression of KCNE2 on the function of ion channel and electrophysiological properties in cardiomyocytesHere, we changed the expression of KCNE2 using adenovirus vector and siRNA interference technology in cultured cardiac myocytes to investigate the role of KCNE2 protein in cardiac electrophysiology. The results showed that:3.1 Effects of changes in the expression of KCNE2 on Ito current3.1.1 Effects of changes in the expression of KCNE2 on Ito current density and gating kineticsAdKCNE2 group and siKCNE2 group had no significant difference on Ito current density compared with their corresponding control group.KCNE2 significantly slowed down Ito channel activation and inactivation kinetics. Meanwhile, TTP and T0.5 in shRNA group were significantly slower than that in siKCNE2 group.3.1.2 Effects of changes in the expression of KCNE2 on voltage-dependence of Ito current inactivationcaused a positive shift of Ito channel inactivation. In contrast, siKCNE2 caused a negative shift in the voltage-dependence of inactivation.3.1.3 Effects of changes in the expression of KCNE2 on expression of Ito a subunitsAlthough changes in KCNE2 expression slightly affected the expression of Kv4.3 and Kv4.2 protein in membrane compared with their corresponding control group, but there was no significant difference (P> 0.05). 3.2 Effects of changes in the expression of KCNE2 on ICa-L3.2.1 Effects of changes in the expression of KCNE2 on ICa-L currentOverexpression of KCNE2 significantly increased L-type calcium current. Calcium current density at+10 mV in AdKCNE2 group increased by about 37.5% compared with that in AdGFP group (P=0.024). And current density in siKCNE2 group decreased by about 28.9% of that in shRNA group, P=0.000.3.2.2 Effects of changes in the expression of KCNE2 on Cav1.2α1c expression, a subunits of ICa-LChanges in KCNE2 expression significantly affected Cav1.2α1c expression compared with their corresponding control group. There was about 28% increase in AdKCNE2group compared with AdGFP group. In contrast, siKCNE2 decreased about by 34%.3.3 Effects of changes in the expression of KCNE2 on IK1The change does not affect the reversal potential and rectification properties of IK1.IK1 current density at the voltage range of-120 mV to-90 mV in AdKCNE2 group increased slightly compared with AdGFP group, but there was no statistically significance.3.4 Effects of changes in the expression of KCNE2 on action potential duration(APD)APD in AdKCNE2 group was significantly longer than the AdGFP group. RMP does not change between the two groups. APD in siKCNE2 group was significantly shorter than that in shRNA group, while the RMP has not changed.3.5 Effects of changes in the expression of KCNE2 on total protein content and ANP mRNA expressionThe results showed that overexpression and inhibition of KCNE2 caused a marked increase in the total protein content in cardiommyocytes. Compared with their corresponding control group there was increase by 45.5% and 29.7%, respectively. Realtime-PCR results showed that compared with the respective control group, overexpression and inhibition of KCNE2 significantly increased transcription of ANP mRNA levels.3.6 The level of KCNE2 protein of membrane from ventricular myocardium of SD rat of 3 age groupsWe used western blot to detect expression of KCNE2 protein on ventricular cardiomypcytes membrane.20-24 months old SD rats were used as the elderly group, 3-4 months old SD rats as adult group, and SD neonatal rats born in 1-3days as young group. The result showed that, there was significant increase in the expression of KCNE2 protein in ventricular membrane with age increasing, which was the same as the result reported in literature.Conclusion:Changes of KCNE2 expression affected transient outward potassium current kinetics gating and voltage-dependence, thus making the corresponding action potential duration changed. But changes of KCNE2 expression had no effect on Itoαsubunit expression in the membrane, which indicated that as theβregulatory subunit, the regulation role of KCNE2 on Ito channel occurred after assembling with Kv4 proteins into the complex in the cell membrane, not affecting the transport process of the Kv4 proteins to the membrane. Surprisingly, changes of KCNE2 expression affected the ICa-L current and the expression of a subunit protein Cavl.2α1c. The mechanism should be studied in detail. The effect of changes in KCNE2 protein on cardiac hypertrophy and the significant increase in the expression of KCNE2 in the aged heart further show that KCNE2 plays an important role in cardiac electrophysiology stability of normal and disease heart.