The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sinus node dysfunction and bradycardia via a complete loss of HCN4 channel function, due to a dominant negative trafficking defect. Since other reported HCN4 channel mutations only have a minor defect in channel gating, mostly with an impaired cAMP modulation, it was our aim to characterize the disease causing mechanism of the D553N mutation in more detail. Utilizing fluorescence microscopy and FACS analysis, we surprisingly found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Consistent with the cell surface expression of D553N mutants, it was possible to functionally characterize the D553N mutants inXenopusoocytes and mammalian cells. We show that D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. Similar as previously described for other HCN4 channel mutations, we observed a defect in the cAMP regulation of the channel. Cell attached patch clamp recordings show that the single channel conductance of D553N mutants is not altered. However, even under physiological cAMP levels the open probability of D553N channels was strongly reduced. Our data suggest a C-linker mediated gating defect, including a reduction in open-probability of D553N channels and an impaired cAMP regulation. We hypothesize that the D553N mutation stabilizes a salt bridge important for the closed state of the channel. Our study supports the role of the C-linker in regulating HCN channel gating.