['The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sino-atrial dysfunction via a pronounced dominant-negative trafficking defect. Since HCN4 mutations usually have a minor defect in channel gating, it was our aim to further characterize the disease causing mechanism of D553N.', 'Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N.', 'Surprisingly, we found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Co-expression of D553N with HCN4 also revealed no dominant-negative effect on wild-type channels. Consistent with the normal cell surface expression of D553N, it was possible to extensively characterize D553N mutants in Xenopus oocytes and mammalian cells. D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. While the regulation of D553N by tyrosine kinases is normal, we observed a change in the cAMP regulation which however cannot account for the strong loss-of-function of the mutant.'] Does the HCN4 channel mutation D553N associated with bradycardia have a C-linker mediated gating defect?