| Objective:Transient outward potassium current(Ito)plays an important role in maintainning cell membrane potential and regulating cell excitability.Ito's abnormality is the electrophysio-logical basis of many myocardial excitability-related diseases.Kv4.2(Voltage-gated potassi-um channel 4.2)is the core alpha subunit of rodent myocardial Ito channels,and KChIP2(Kv channel-interacting protein 2)is a key regulatory subunit of Kv4.2.It is known that the KChIP2 and location of channels in lipid rafts and the phosphorylation of Kv4.2 caused by sympathetic excitation can affect the stability of Kv4.2 on the cell membrane.They are close-ly related,but the molecular mechanism of correlation is not clear,this project aims to study the molecular mechanisms involved,so as to further reveal the molecular mechanisms regu-lating Kv4.2 functional activity in the body,and provide new cognitive vision and treatment ideas for myocardial excitability-related diseases.Methods:By constructing fluorescently-labeled Kv4.2,KChIP2 and lipid raft marker-GPI(Glycosylphosphatidylinositol)plasmids,first the co-localization technique was used to ana-lyze the localization relationship between the Ito subunits and the lipid raft in a heterologous expression system;further through drugs stimulation and site-directed mutagenesis simulate different phosphorylation states of Kv4.2,to study the effect of Kv4.2-S552 site phosphoryla-tion on its localization and distribution on cell membranes;Co-immunoprecipitation and FRET(fluorescence resonance energy transfer)techniques were used to determine the effect of Kv4.2 phosphorylation on the interaction between Kv4.2 and KChIP2;The patch-clamp technique was used to compare the differences in electrophysiological characteristics between wild-type and phosphorylated mutants;Kv4.2 was labeled on the cell membrane by exogenous insertion of HA tags,and compared the stability of Kv4.2 on the cell membrane between wild-type and phosphorylated mutants;The effects of sympathetic stimulation on the localiza-tion of Kv4.2 in lipid rafts and the stability on membranes were studied in cardiomyocytes of neonatal rats by extracting cell membrane lipid raft components and separating membrane components.Results:(1)In heterologous expression systems,Kv4.2 and KChIP2 exhibit different lipid raft localization distributions,and KChIP2 can help Kv4.2 localize to lipid raft microdomains;(2)Kv4.2 after PKA agonist(Forskolin)treatment the degree of colocalization with lipid rafts is reduced.In accordance with this,the mutant Kv4.2-S552D,which simulates the phosphoryla-tion state,also has a lower degree of colocalization with lipid rafts;(3)After treatment with Forskolin,the interaction between Kv4.2 and KChIP2 was significantly weakened,and Kv4.2-S552D compared with wild-type and non-phosphate mutants,the interaction was also significantly weakened;(4)It was found that the Kv4.2-S552D mutant had a lower Ito channel current amplitude and shifted the activation curve to the right than the wild-type and non-phosphate mutants;(5)Comparing the stability of wild and mutant Kv4.2 on the cell membrane,it was found that the mutants with the lowest degree of co-location with the lipid raft,Kv4.2-S552D,had the fastest reduction in fluorescence intensity on the membrane,indi-cating that it had the worst stability of its presence on the cell membrane;(6)Sympathetic stimulation in primary neonatal rat myocardial cells can cause a reduction in the distribution ratio of Kv4.2 between lipid rafts and non-lipid rafts,which is accompanied by a reduction in the distribution ratio between cell membranes and cytoplasmConclusion:(1)Kv4.2 itself cannot localize to lipid rafts.Interaction with KChIP2 can help Kv4.2 localize to lipid rafts on cell membranes;(2)After the Kv4.2-S552 site is phosphory-lated,the interaction between Kv4.2 and KChIP2 is weakened,which causes Kv4.2 to migrate out of the lipid raft region;(3)Whether in heterologous expression systems or in primary car-diomyocytes,the reduced localization of Kv4.2 in lipid rafts is accompanied by a decrease in its stability on the cell membrane,which affects the function of Ito. |
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