The Research Of Modulation Of HCN Channel By Extracellular PH

Background: The cardiovascular disease has become a major threat to people’s health in our country. About 1.5 million patients are died of cardiovascular disease including coronary heart disease and stroke every year. On the pathogenesis of myocardial infarction patients, the severe acidosis due to ischemia hypoxia, which can lead to circulation disorder and decrease the excretion of metabolic product, mainly influence on local tissue, especially for coronary artery smooth muscle and cardiac muscle cells [1,2]. Therefore, correct acidosis timely can greatly reduce the myocardial infarction myocardial cell damage in clinic.Therefore, revealed the mechanism of acidosis of myocardial cell injury, will help to offer a theoretical basis for some heart disease prognosis. In some diseases, acidosis will change myocardial sinus heart rate and decrease the cardiac output. The sinoatrial node is physiology pacemaker and dominate the heart beat by automatically depolarization of sinoatrial node cells.The ion currents participated in the sinoatrial node automatic depolarization has the following kinds:hyperpolarization activated introverted cationic current If, delayed rectifier potassium current Ik, inward calcium current ICa-T and sodium calcium exchange current INa/Ca,continuous ion current introversion IST, background current(including sodium and potassium flows) and type D ICa-L etc. If hyperpolarization-activated cyclic nucleotide-gated channel(HCN), which is mainly by sodium and potassium ion flow on and recent studies have found that it also has calcium ion flow components.Hyperpolarization-activated cyclic nucleotide-gated channel(HCN channel)mediated pacemaker current cause diastolic depolarization of sinoatrial node cells, thus play an important physiological role in the regulation of cardiac autonomic beat and rhythm of the heart. HCN channel in the body is high expression in cells of cardiac pacing. With the experimental results show that when the heart acidosis, the heart rate is slow, but the mechanism of the change in function of the HCN channel is unclear.HCN channel includes four subunits, in the heart including three subunits(HCN1,HCN2,HCN4), which may be the same polymers or the different polymers in the heart, therefore, this experiment using clawed frog oocyte establish express HCN channel of polymers with different expression model, to observe the effect of channel current of the extracellular acid liquid on HCN channel subunits with same polymers or different polymers.Aim:To observe the effect of different p H value on hyperpolarization—activated cyclic nucleotide—gated cation channel(HCN channel), we build HCN channel the different subunit expression in Xenopus laevis oocytes.Method: The two-electrode voltage clamp recording technique was applied to observe voltage-gated HCN ion channel currents expressed in Xenopus laevis oocytes without follicles. c DNAs, including HCN1, HCN2 and HCN3 transcribed in vitro. And then different c RNA was microinjected into the oocyte cytoplasm to express HCN channel currents. After 2-3 days, currents were recorded by the two-electrode voltage clamp and the effects of p H on HCN channel currents were observed.Result: The currents of HCN homopolymer and heteropolymer expressed in Xenopus laevis oocytes were recorded, which was inhibited by use of Cs Cl, a non-selective blocker of HCN channel. In the level of-140 m V, the amplitude of HCN1 channel currents was inhibited compared with control(83.4 % ± 9.5 %, n = 5,P < 0.001);and the inhibition rate of HCN2 channel current amplitude reached 99.7%±0.6%(n=4,P < 0.001).TRIP8 b is a auxiliary subunit that regulate the current amplitude of HCN1 in xenopus oocytes.The current amplitude of HCN1+TRIP8b( 1.4ng+1.4ng) group issignificant increase compare with HCN1(5.5ng) group.With the p H value decreased, the amplitude of HCN channel currents was inhibited in the level of-140 m V from p H 6.5to p H 5.0. The amplitude of HCN1 channel currents was inhibited compared with control(24.6% ± 8.1%, n=6,P<0.001);the amplitude of HCN2 channel current was inhibited compared with control(25.6% ± 6.8%,n=4,P<0.01);the amplitude of HCN1 and HCN2 heteropolymer channel current was inhibited compared with control(21.3%±6.5%, n=4,P<0.001);In a different alkaline solution,there is not significant difference in amplitude of different HCN subunit channel current. In the level of-140 m V, from p H 7.5 to p H 8.0, the amplitude of HCN1 channel current wasn’t inhibited compared with control(1.9% ± 0.1%, n=3,P>0.05);the amplitude of HCN1 and HCN2 heteropolymer wasn’t inhibited compared with control(5.6%±2.0%,n=3,P>0.05).Conclusion: HCN channel currents were blocked by application of HCN channel non–selective blocker in oocytes and it was demonstrated that HCN channel expressed in oocytes successfully. With decrease of PH value, the amplitude of HCN channel currents was inhibited. The The amplitude of HCN channel currents was not altered by the application of alkalinity PH. HCN channel currents were inhibited by decreased p H value was likely to involved in the change of heart rate in acidosis.