Compound NS5806 Modulates The Kv4 Channel And Its Molecular Mechanism

Kv4 channels are the primary subunits of the rapidly activating and inactivating K⁺channels,contributing to the transient K⁺currents including somato dendritic subthreshold A-type current(I_SA)and cardiac transient outward K⁺current(I_to).Due to its unique kinetic features,Kv4 channels play a central role in controlling neuronal excitation and shaping the cardiac action potentials(AP).The I_SAA has been identified in many peripheral and central neurons and shown to be key determinants in shaping action potential waveforms and repetitive firing properties,as well as in the regulation of synaptic transmission and plasticity.In the heart,I_too underlies phase 1repolarization of cardiac AP and sets the plateau potential of the AP in the hearts of larger mammals.The reduction of the I_too density,accompanying with action potential duration(APD)prolongation is a consistent finding in many physiopathological conditions such as cardiac hypertrophy and heart failure(HF)and thus contributes to the arrhythmias.Accumulating evidence suggests that I_too reduction in HF is not simply a secondary change but could be a causative factor or promoter for HF development.Thus,enhancement of the I_too density is a promising strategy for HF treatment.It is generally accepted that there are two I_too components with distinct recovery kinetics:the fast(I_to,f)and slow(I_to,s)components.The Kv4.2(KCND2)and Kv4.3(KCND3)encode I_to,f,while Kv1.4(KCNA4)encodes I_to,so,s channels.In human and canine ventricular myocytes,functional I_to,fo,f channels are likely to be Kv4.3 homotetramers,because Kv4.2 is not expressed.Kv4channel properties are known to be modulated by its auxiliary subunits,such as K⁺channel-interacting proteins(KChIPs)or dipeptidyl peptidase-like proteins(DPPs).Within these,KChIP2 and DPP6 have been proposed as most likely candidates that coassemble with Kv4 subunits in the human heart.KChIP2 is a cytosolic Ca²⁺-binding ancillary subunit that interacts with the amino terminus of the Kv4a-subunit;it facilitates channel trafficking,slows the Kv4 current inactivation kinetics,accelerates the recovery from inactivation and shifts the voltage dependence of steady-state inactivation to more positive potentials.DPP6 is a single membrane-spanning protein that can accelerate both current inactivation and recovery kinetics and shifts both activation and inactivation curves of Kv4 channels to more negative potentials.Co-expression of K_V4.3 with KCh IP2 and DPP6 in heterologous systems results in currents with kinetics similar to those of I_too in human ventricular myocytes.Thus,Kv4-KChIP-DPP complex could be a valuable target for therapeutic approaches to cardiac diseases.A small molecule sulfonylurea compound NS5806,has been identified to potentate I_too in canine cardiac myocytes by slowing the current inactivation kinetics.Further studies have shown that current potentiation by NS5806depends on the presence of KChIPs in recombinant Kv4 channels.NS5806reverses the HF-associated reduction of I_too density in canine ventricular cardiomyocytes and,thus,may be of significant pharmacological value.However,the potentiating action of NS5806 on native I_too has been shown to vary in the different tissue.Given it potential therapeutic value,a better understanding of the molecular mechanisms underlying the modulation of Kv4channels by NS5806 is urgently required.I_too is the major component of cardiac action potentials repolarization in rodents and the murine cardiovascular system has become an important model for molecular studies of I_to(which is principally assembled from the Kv4.2?-subunit in mice).In addition,the generation of human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)has become another important resource for modeling human cardiac disease and for drug screening.In this study,we examined the effect of NS5806 on I_too in native mouse ventricular cardiomyocytes and in the hiPSC-CMs.Unexpectedly,we discovered that instead of potentiation,NS5806 decreased the amplitude of native I_too in both cell types with a significant acceleration of the current inactivation.We then tested the effects of NS5806 on cloned Kv4.3/DPP6/KChIP2 channels in heterogeneous expression system and discovered that two auxiliary subunits confer opposing effects of NS5806 on the Kv4.3 gating.We also quantified DPP6 and KCh IP2 protein abundance in mouse,canine and human cardiomyocytes and discovered that different expression level of these auxiliary subunits underlies divergent effects of NS5806 in these species.The results described here will provide the new insight for the therapeutic targeting of Kv4.Part 1 NS5806 suppressed I_too in mouse cardiomyocytes and hiPSC-CMsObjective:To investigate the inhibitory effect of NS5806 on I_too in mouse cardiomyocytes and hiPSC-CMs.Methods:Electrophysiological patch-clamp was used to record the outward potassium current in mouse,canine left ventricular myocytes and hiPSC-CMs,and distinguish the inhibitory current component.Results:NS5806(10-30?M)significantly inhibited the outward potassiumcurrentinmouseleftventricularmyocytesina concentration-dependent manner.The outward potassium current density was significant decrease from 73.0±2.3 pA/pF to 38.1±1.4 pA/pF at+60mV and accelerated the current decay(n=25,P<0.01).The inhibit effect of NS5806 on outward potassium current lead to the extension of QT interval(QTc)in isolatedperfusedmousehearts,andtheprolongedQTcina concentration-dependent manner.NS5806 decreased the current amplitude both I_to,fo,f and I_k,slow,slow in a concentration-dependent manner;the IC₅₀0 values for the inhibition of I_to,fo,f and I_k,slow,slow were 12.5±0.2?M and 2.7±0.7?M respectively.To identify whether the inhibitory effect of NS5806 on Kv4 family channel just limited to rodents,we investigated the effect of NS5806 on I_too in hiPSC-CMs.NS5806 induced prominent inhibitory effect in hiPSC-CMs and with an IC₅₀of 8.3±1.9?M.Given the striking difference between the effects of NS5806 in canine cardiomyocytes and murine and human cardiomyocytes,we tested the effect of NS5806 on the I_too in canine cardiomyocytes to confirm its potentiating effect under our experimental conditions.Indeed,the I_too amplitude was enhanced and the current decay was delayed in the presence of 10?M NS5806,which concurs with the previous reports.Conclusion:The effects of NS5806 on I_too in different species are different.NS5806 inhibit the I_too in mouse left ventricular myocytes and hiPSC-CMs and accelerate the current inactivation.But,NS5806 increases the I_too in canine ventricular cardiomyocytes and slows current decay.Part 2 The role of DPP6 in the inhibitory action of NS5806Objective:To explore the molecular correlative(s)of opposite effects of NS5806 on the cardiac I_too in different species.Methods:Cloned Kv4 channels co-express with different stoichiometry auxiliary subunits KChIP2 and DPP6 in HEK293 cells.Western-blot detected the protein abundance of DPP6 in ventricular myocardium of mouse,canine and hi PSC-CMs and knocked-down DPP6 expression in hiPSC-CMs using a specific RNA interference approach.Results:In heterogenous expression system,NS5806 significant increase the Kv4/KChIP2 channels current and slow the current activation.These finding were quite consistent with the previous reports.However,NS5806decreased the Kv4/KChIP2/DPP6 channels current and accelerate the current activation.Kv4.3/DPP6 channels were unaffected by NS5806,suggesting that an interplay between KChIP2 and DPP6 is necessary to confer the inhibition.NS5806 significantly decreased the peak current of I_{Kv4.3/KChIP2/DPP6},and the effect was more prominent when the expression of DPP6 was increased by changing Kv4.3/KChIP2/DPP6 transfection plasmid ratio from 1:1:1 to1:1:3.The steady-state inactivation was left shifted with a change of V_1/2/2 from-28.8±0.4 mV to-42.5±0.2 mV,and the recovery from inactivation was accelerated from 30.9±5.1 ms to 14.4±2.7 ms in the presence of NS5806.The DPP6 protein was much more abundantly expressed in mouse ventricular tissue and hiPSC-CMs compared to canine heart whereas the expression level of KChIP2 protein was comparable between these species.The knock down of DPP6 in hiPSC-CMs the I_too displayed significantly slower decay kinetics.The knockdown of DPP6 significantly alleviated the response to NS5806,albeit the compound still produced a modest suppression of the current in hiPSC-CMs.Conclusion:DPP6 subunit played a key role in the inhibitory action of NS5806.Part 3 Possible molecular mechanism underlying the inhibitory response to NS5806Objective:To elucidate the molecular mechanism underlying different pharmacological respond of native I_too on NS5806 in different species.Methods:The interaction between DPP6 and KChIP2 was analyzed by CO-IP and computer simulation.Results:The co-immunoprecipitation assay was verified the physical interaction between DPP6 and KChIP2.Moreover,the computer simulation of the molecular docking between DPP6 and KCh IP2 also indicates the possibility of combining.Mutation the N-terminal of DPP6 disrupted the interactionbetweenDPP6andKChIP2,successfullychangedthe pharmacological respond of the Kv4 channels to NS5806 in heterogenous expression system.Conclusion:Our data provided the evidence that DPP6 subunits play a key role in the inhibitory action of NS5806 on native Ito and the association between DPP6 and KChIP2 conferred the inhibitory effect of NS5806 on Kv4 channel.