The Molecular Basis Underlying Regulation On HERG Potassium Channel By Different G Protein-coupled Receptors Through Specific PKC Isozymes

Arrhythmias,especially severe ventricular arrhythmias are leading causes of deaths in many cardiovascular diseases. Therefore, cardiovascular research has been focused on revealing the molecular basis of arrhythmia. Abnormality of struscture and/or dysfunction of ion channels result from either congenital genetic deficiency or acquired factors. Both the sympathetic nerve and renin-angiotensin system play pivotal role in maintaining cardiovascular homeostasis, but over-activation of which may contribute to cardiac arrhythmias in various cardiovascular diseases.The rapid component of the delayed rectifier K+ current, IKr, is the major repolarizing outward currents of ventricular action potentials in mammalian species, including humans. Human ether-a-go-go-related gene(HERG or KCNH2) encodes the pore forming subunit of the channel underlying IKr. Our previous study has shown that angiotensin II(Ang II) produces an inhibitory effect on IKr /h ERG currents via AT1 receptor linked to protein kinase C(PKC) pathway in ventricular myocytes.Stimulation of a1A-receptors by phenylephrine caused HERG current reduction through PKC pathway.PKC plays an important role in the cellular functional regulation and involves in the occurrence and development of many diseases. At present, it becomes an important target for new drug research and development. PKC isozymes are classified based on their structure and activation requirements into three subgroups: conventional PKCs(α、βI、βII、γ);novel PKCs(δ、ε、π、θ);and atypical PKCs(ζ、i/λ). As intracellular second messengers, PKC isozymes provide a link between autonomic stimulation and hormone in cardiac repolarization through regulation on ion channels. However, which isoform of PKC mediates the regulations of IKr by the stimulation of AT1 or a1A-receptors in ventricular myocytes is not known. In addition, the precise mechanism by which PKC regulates IKr in native cardiomycytes, i.e. either through direct PKC-dependent phosphorylation of the channel or an indirect mechanism remains to be fully elucidated.Therefore, by using molecular biological technique, this study was designed to define the PKC isoform mediating the regulations of IKr by the stimulation of AT1 or a1A-receptors and the interaction between HERG channel protein and PKC isoform.Objective:To detect which PKC isoform activated by the stimulation of AT1 or a1A-receptors and the co-localization between HERG channel protein and PKC isoform by using specific antibodies to phosphorylated PKC isoforms.Methods:In HEK cells transient transfection a1A or AT1 receptors, use A61603 or angiotensin II 10 minutes, than extraction cell membrane protein. The proteins were extracted by SDS-PAGE, then transferred to PVDF membranes, and blocked with 5% BSA for 2-4h. The membranes were incubated with primary antibodies against p-PKCa,p-PKCε and then with fluorescently labeled secondary antibodies.The anti-GAPDH antibody was used as internal loading control. Quantification of the signals was performed by using Odyssey 9120 Infrared Imaging System, and analysis of the band density was done with the software. The band intensities were normalized by GAPDH and summary data for interest protein abundance were presented as a percentage of the control group. All of data are presented as means ± SEM. The data analysis was performed with Origin software(version Pro 7.5). The n represents the repetitive times. The statistical significance of the differences between groups was evaluated using Student’s unpaired T test and differences with P <0.05 were considered statistically significant.Remove the guinea pig’s heart quickly,put it into Langendorff perfusion apparatus. First with Tyrode’s solution be stable after the perfusion, then perfusion A61603 or angiotensin II in 10 minutes, extraction of left ventricular cell membrane protein. The proteins were analysised by Western Blot. The data processing method is the same.After administration, extraction of left ventricular cell membrane proteins to do Co IP. The membranes were incubated with antibodies against p-PKCa,p-PKCε and HERG. Using Odyssey 9120 Infrared Imaging System to determine the expression of protein detection.Results:1 a1A and AT1 receptor agonist can activate different PKC isozymes.HERG-HEK293, joined the A61603 and using cell membrane protein. Western blot results showed that, compared with control group, the expression of p-PKCa increased about 1.2(P <0.05, n=3). However, compared to p-PKCε and control group, there is no significant change.Adult male guinea pig hearts were perfused with A61603, use the left ventricular myocardial membrane protein to do Western blot.The analysis showed compared with control group, p-PKCa protein expression increased about 1.1(P<0.05, n=3); the expression quantity of p-PKCε has some changes, but no statistical significance. The results show that, a1A receptor agonists can be selectively excited PKCa, the effect close to PMA; and for PKCε, a1A receptor agonists had no obvious effect.HERG-HEK293 joined the Ang II and using cell membrane protein.Western blot results showed that, compared with control group, p-PKCa increased about 1.2(P <0.05, n=3). Compared with control group, p-PKCε expression increased about 1.2(P <0.05, n=3).Adult male guinea pig hearts were perfused with Ang II, use the left ventricular myocardial membrane protein to do Western blot. Compared with control group, p-PKCa protein expression increased about 1.2(P <0.05, n=3); p-PKCε protein expression was about 1.3(P <0.05, n=3). Ang II effect on p-PKCa, p-PKCε protein expression changes were similar to PMA. These results confirmed that AT1 receptor agonists can be selectively excited PKCa and PKCε, but there are certain differences in arousal level.2 The relationship between the activation of PKCa, PKCε and HERG potassium channel.Adult male guinea pig hearts were perfused with A61603 and Ang II, use the left ventricular myocardial membrane protein to do Co IP. The experimental results suggested that p-PKCa, p-PKCε and HERG were co-localize.Conclusion:1 a1A receptor agonists can selectively excited PKCa; while for PKCε, a1A receptor agonists had no obvious effect; AT1 receptor agonists can be selectively excited PKCa and PKCε, but there are certain differences effect between the excited degree.In native cell, the influence to PKCε is stronger.2 a1A receptor agonists and AT1 receptor agonists lead to p-PKCa, p-PKCε and HERG potassium channel protein co-localize.