Kv4 pore‐forming subunits co‐assemble with β‐subunits including KChIP2 and DPP6 and the resultant complexes conduct cardiac transient outward K ~(+) current ( I _(to)). Compound NS5806 has been shown to potentate I _(to) in canine cardiomyocytes; however, its effects on I _(to) in other species yet to be determined. We found that NS5806 inhibited native I _(to) in a concentration‐dependent manner (0.1~30?μM) in both mouse ventricular cardiomyocytes and human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs), but potentiated I _(to) in the canine cardiomyocytes. In HEK293 cells co‐transfected with cloned Kv4.3 (or Kv4.2) and β‐subunit KChIP2, NS5806 significantly increased the peak current amplitude and slowed the inactivation. In contrast, NS5806 suppressed the current and accelerated inactivation of the channels when cells were co‐transfected with Kv4.3 (or?Kv4.2), KChIP2 and another β‐subunit, DPP6‐L (long isoform). Western blot analysis showed that DPP6‐L was dominantly expressed in both mouse ventricular myocardium and hiPSC‐CMs, while it was almost undetectable in canine ventricular myocardium. In addition, low level of DPP6‐S expression was found in canine heart, whereas levels of KChIP2 expression were comparable among all three species. siRNA knockdown of DPP6 antagonized the I _(to) inhibition by NS5806 in hiPSC‐CMs. Molecular docking simulation suggested that DPP6‐L may associate with KChIP2 subunits. Mutations of putative KChIP2‐interacting residues of DPP6‐L reversed the inhibitory effect of NS5806 into potentiation of the current. We conclude that a pharmacological modulator can elicit opposite regulatory effects on Kv4 channel complex among different species, depending on the presence of distinct β‐subunits. These findings provide novel insight into the molecular design and regulation of cardiac I _(to). Since I _(to) is a potential therapeutic target for treatment of multiple cardiovascular diseases, our data will facilitate the development of new therapeutic I _(to) modulators.
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