Rationale:The hyperpolarization-activated current I h that is generated by hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) plays a key role in the control of pacemaker activity in sinoatrial node cells of the heart. By contrast, it is unclear whether I h is also relevant for normal function of cardiac ventricles.Objective: To study the role of the HCN3-mediated component of ventricular I h in normal ventricular function. Methods and Results:To test the hypothesis that HCN3 regulates the ventricular action potential waveform, we have generated and analyzed a HCN3-deficient mouse line. At basal heart rate, mice deficient for HCN3 displayed a profound increase in the T-wave amplitude in telemetric electrocardiographic measurements. Action potential recordings on isolated ventricular myocytes indicate that this effect was caused by an acceleration of the late repolarization phase in epicardial myocytes. Furthermore, the resting membrane potential was shifted to more hyperpolarized potentials in HCN3-deficient mice. Cardiomyocytes of HCN3-deficient mice displayed approximately 30% reduction of total I h . At physiological ionic conditions, the HCN3-mediated current had a reversal potential of approximately ؊35 mV and displayed ultraslow deactivation kinetics. Conclusions:

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The L-type calcium channel (LTCC) has a variety of physiological roles that are critical for the proper function of many cell types and organs. Recently, a member of the zinc-regulating family of proteins, ZnT-1, was recognized as an endogenous inhibitor of the LTCC, but its mechanism of action has not been elucidated. In the present study, using two-electrode voltage clamp recordings in Xenopus oocytes, we demonstrate that ZnT-1-mediated inhibition of the LTCC critically depends on the presence of the LTCC regulatory ␤-subunit. Moreover, the ZnT-1-induced inhibition of the LTCC current is also abolished by excess levels of the ␤-subunit. An interaction between ZnT-1 and the ␤-subunit, as demonstrated by co-immunoprecipitation and by fluorescence resonance energy transfer, is consistent with this result. Using surface biotinylation and total internal reflection fluorescence microscopy in HEK293 cells, we show a ZnT-1-dependent decrease in the surface expression of the pore-forming ␣ 1 -subunit of the LTCC. Similarly, a decrease in the surface expression of the ␣ 1 -subunit is observed following up-regulation of the expression of endogenous ZnT-1 in rapidly paced cultured cardiomyocytes. We conclude that ZnT-1-mediated inhibition of the LTCC is mediated through a functional interaction of ZnT-1 with the LTCC ␤-subunit and that it involves a decrease in the trafficking of the LTCC ␣ 1 -subunit to the surface membrane.