Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex.

Kv4 pore-forming subunits co-assemble with β-subunits including KChIP2 and DPP6 and the resultant complexes conduct cardiac transient outward K current (I). Compound NS5806 has been shown to potentate I in canine cardiomyocytes; however, its effects on I in other species yet to be determined. We found that NS5806 inhibited native I 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 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 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. Since I is a potential therapeutic target for treatment of multiple cardiovascular diseases, our data will facilitate the development of new therapeutic I modulators.