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Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation. [ABSTRACT FROM AUTHOR]