PKCα Isoform Mediates The Inhibition On Rapid Component Of The Cardiac Delayed Rectifier K⁺ Current By α_1A-adrenoreceptor Activation And Underlying Mechanism

The delayed rectifier K+ currents(IK) in mammalian ventricular myocytes consist of rapidly and slowely activating components(IKr and IKs, respectively).The human ether-a-go-go-related gene(HERG) encodes the pore-forming subunit of the channel responsible for IKr which has an important influence on maintaining myocardial action potential plateau phase and phase 3repolarization. The h ERG gene mutation is the molecular and genetic basis of channelopathies. Cardiovascular diseases such as hypertension, myocardial ischemia are associated with down-regulation of IKr which leds to acquired LQT syndrome.More and more studies have proved that phosphorylation is an effective way to regulate the function of h ERG channels, and then affect the electrophysiological properties of heart. And among those, the regulation of protein kinase C has been a focus. Based on their structure and activation requirements, protein kinase C(PKC) is classified into three subgroups:conventional PKCs(α, βI, βII, γ) that require calcium, diacylglycerol(DAG),phosphatidylserine(PS) for activation. Novel PKCs(δ, ε, π, θ) that can be activated by DAG and PS, but be insensitive to calcium. Atypical PKCs(ζ,?/λ), which are unresponsive to calcium or DAG, but require PS for activation.It is known that some PKC isoforms transformed to cell membrane after activation in cardiomyocytes to regulate the phosphorylation of ion channel,and became an important mediator of humoral factors and neurotransmitter.Some studies had revealed that different pathological stimuli such as ischemia and hypoxia, angiotensin II, oxidative stress(H2O2) activate distinct PKC isoforms, which may play specific roles in the regulation of ion channels.Makary et al. has reported that differential protein kinase C isoforms haveopposite action on acetylcholine-activated potassium channels. It has been revealed that the α1AAR activation acutely depresses h ERG current. Similarly,our previous study has found that AT1 R activation by Ang II acutely down-regulates h ERG current. Both α1AAR and AT1 R were Gq-protein-coupled receptors(GPCR), and PKC is the main mediator for the regulation of h ERG currents by GPCR activation. However, the modulation of the two GPCR on the h ERG channel shows a different manner. The activation of α1AAR rightly shifts the voltage-dependent activation curve, while the AT1 R does not. Thus, we speculate that α1AAR and AT1 R activation may modulate the channel through different PKC isoforms.To address the above hypothesis, the present study was designed to investigate the effects of α1AAR activator(A61603) on IKr/h ERG currents in native cardiomyocytes and heterologous expression system, to identify the PKC isoforms being involved, and analyzes the molecular mechanisms underlying the regulation of PKC isoforms by using electrophysiological technique and western blot.Part 1 Acute down-regulation of IKr by ?1A-adrenoceptor activationthrough PKC ? in ventricular myocytesObjective: To investigate the effects of ?1AAR activator on rapid component of delayed rectifier potassium current in adult guinea pig cardiocytes, and analyze the PKC isoforms being involved.Methods: Whole-cell patch-clamp technique was used to record IKr before and after 10 min incubation of ?1AAR activator(A61603) in left ventricular myocytes which were rapidly excised and freshly isolated by enzymatic method from male adult guinea pigs.Results:(1) A61603 decreased the amplitude of IKr in a concentration-dependent manner with an IC50 of 300 n M. The tail current density of IKr was decreased from 0.93 ± 0.04 p A/p F to 0.62 ± 0.07 p A/p F(P <0.01) at a prepulse potential of +40 m V after 10 min application of 1 μM A61603.(2) The ?1AAR antagonist WB4101 and non-selective PKC inhibitorBis-1 but not the PKA inhibitor H-89 depressed the inhibitory effect of A61603 on the amplitude of the IKr tail current, showing the specificity of the?1AAR mediated effects of A61603 mainly via PKC not PKA signaling pathway.(3) G?6976(selectively inhibiting PKC?, ?, and ?), G?6983(selectively inhibiting PKC?, ?, ?, δ, and ζ), and PKC alpha inhibitory peptide(αC2-4)significantly reduced the inhibitory effect of IKr by A61603, but the PKC εinhibitory peptide(?V1-2)showed no effect. These results suggest PKC alpha mediated the inhibitory effects of IKr by ?1AAR activation.Conclusions: The ?1AAR activation by A61603 produces an inhibitory effect on IKr currents via PKC? in freshly isolated ventricular myocytes.Part 2 Distinct effects of PKC isoforms activatory peptides on h ERGcurrent in heterologous expression systemObjective: To investigate the distinct effects of PKC isoforms activatory peptides on expressed h ERG current, and further verify the inhibition effects on cloned h ERG channel by ?1AAR activation and its PKC ?-dependent signaling pathway.Methods: Whole-cell patch-clamp technique was used to record h ERG current in human embryonic kidney(HEK) 293 cells co-transfected with human ether-a-go-go related gene(h ERG) encoding α-subunit of IKr and human ?1A-adrenoceptor(AR) gene. The A61603 was perfused with extracellular solution, the c PKC and PKC? activatory peptides were dissoluted into pipette solution.Results:(1) Both c PKC activatory peptide(c PKC-AP) and PKC ?activatory peptide( ?-AP) acutely depressed h ERG tail current. The V1/2shifted from-18.6 m V(control) to-9.5 m V(P < 0.01) after application of c PKC-AP, but the ?-AP had no effects on V1/2(P > 0.05). c PKC-AP and ?-AP depressed h ERG channel maximal conductance(Gmax) to 56.8% and 80.6%of control respectively.(2) The ?1A-AR activator A61603 acutely depressed h ERG tail current in HEK293 cells. The V1/2 shifted from-16.9 m V(before) to-11.8 m V(after)(P< 0.01), and the Gmax decreased to 70.8% of control. The effects of A61603 on h ERG tail current were similar to that of c PKC-AP.(3) The activation time constant were significantly slowed at different voltages in presence of c PKC-AP or ?-AP. Both the fast and slow components of the deactivation were significantly inhibited by c PKC-AP, only the slow components of the deactivation were inhibited by ?-AP. The time constants of recovery from inactivation and the steady-state inactivation curve were not affected neither by c PKC-AP nor ?-AP(P > 0.05).Conclusions: Both c PKC and PKC ? activatory peptides acutely depressed h ERG tail current. c PKC, not PKC ? activatory peptide rightly shifted the voltage-dependent activation curve. Similarly, A61603 shifted V1/2towards positive potentials. The results suggested the c PKC isoforms were involved in the regulation of ?1AAR on IKr/h ERG currents.Part 3 Mechanism underlying the downregulation of Ikr/h ERG current byprotein kinase C?Objective: To investigate the selective activation of PKC isoforms after application of ?1AAR activator in adult guinea pig cardiocytes, and the regulation mechanism of PKC isoforms on h ERG channel in HEK293 cells.Methods: The freshly isolated adult guinea pig ventricular cardiocytes were incubated with ?1AAR activator A61603 for 10 minutes, the total proteins were extracted by SDS-PAGE, then transferred to NC membranes,and blocked with 5% fat-free milk for 2-4h. The membranes were incubated with primary antibody against PKC ?(Phospho-Tyr657) or PKC ?(Phospho-Ser729) and then with fluorescently labeled secondary antibodies.Whole-cell patch-clamp technique was used to record currents in human embryonic kidney(HEK) 293 cells co-transfected with h ERG- ? PKC(an h ERG mutant in which 17 of the 18 predicted PKC acceptor serines/threonines were changed to alanine except T74) and human α1A-adrenoceptor gene, to investigate the effects of A61603 and PKC isoforms activatory peptides on h ERG-?PKC currents and analyzes the molecular mechanism. The A61603 was perfused with extracellular solution, the PKC ? and PKC ? activatorypeptides were dissoluted into pipette solution.Results:(1) Western blot analysis showed that incubation of A61603(1μM) for 10 minutes increased p-PKC? protein expression to 2.72 ± 0.25(P <0.01), the p-PKC ? had no change(P > 0.05). The results illuminated that activation of ?1AAR associated with PKC? phosphorylation.(2) c PKC activatory peptide(c PKC-AP) depressed h ERG-?PKC currents acutely in HEK293 cells, while PKC ? activatory peptide( ?-AP) had no influence on the tail currents. The two PKC isoform activatory peptides did not affect the V1/2 of h ERG-?PKC channel.(3) c PKC activatory peptide(c PKC-AP) acutely depressed h ERG-WT and h ERG-?PKC tail currents. The decrease percents were 39.3% and 37.4%respectively at a prepulse potential of 0 m V in HEK293 cells, and the Gmax changes were not different either, which suggested that c PKC-AP inhibited h ERG current by T74 sit phosphorylation directively or other indirect phosphorylation mechanism. The decrease percents of PKC ? activatory peptide( ?-AP) on h ERG-WT was 29.3%, and it had no effects on h ERG-?PKC tail currents(P > 0.05). ?-AP depressed h ERG-WT Gmax to80.6% of control, but had no influence on the Gmax of h ERG-?PKC channel(P > 0.05). The results suggested that ?-AP depressed the h ERG tail currents via direct phosphorylation of the channel.(4) The ?1AAR activator A61603 acutely depressed h ERG- ? PKC tail currents in HEK293 cells, and the decrease percent of tail current density were31.8% at a prepulse potential of 0 m V, it had no difference with the decrease percent(28.4%) on h ERG-WT tail currents. Obviously, the depression effects of A61603 on h ERG currents still reserved after the 17 phosphorylation sites mutation, which were similar to the effects of c PKC activatory peptide.Although the c PKC include PKCα, βI, βII, and γ, the c PKC activatory peptide mainly activated PKC ? isoform since it showed similar effects on h ERG currents with A61603 which has been proved to mediate h ERG currents via PKC?.Conclusions: ?1AAR selectively activated PKC? isoform. The regulationmechanisms of h ERG currents by PKCα and PKCε isoforms were different: it was possible that PKC ? depressed the h ERG current via direct phosphorylation of T74 sit of h ERG channel or other indirect phosphorylation mechanism, while PKC ? depressed h ERG tail currents via direct phosphorylation of h ERG channel.Summary: 1 The ?1A-adrenoceptor activator A61603 decreased the amplitude of IKr/h ERG current in a concentration-dependent manner in freshly isolated ventricular myocytes and heterogeneous expression system, and the effects were mediated by PKC?.2 The ?1A-adrenoceptor activation selectively induced the activation of PKCα isoform.3 The regulatory mechanisms of h ERG currents by PKCα and PKCεisoforms were different: it was possible that PKC ? depressed the h ERG current via direct phosphorylation of T74 sit of h ERG channel or other indirect phosphorylation mechanism, while PKC ? depressed h ERG tail currents via direct phosphorylation of h ERG channel.