The HCN4 channel mutation D553N associated with bradycardia has a C-linker mediated gating defect.
OBJECTIVE
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.


METHODS
Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N.


RESULTS
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.


CONCLUSIONS
The pronounced current reduction and the regular surface expression indicate a major gating defect of the C-linker gate. We hypothesize that the D553N mutation stabilizes a previously reported salt bridge important for the gating of the channel.
Institute of Physiology and Pathophysiology, Vegetative Physiology Group, Philipps University of Marburg, Germany.
Michael F
MF
Netter
Marylou
M
Zuzarte
Günter
G
Schlichthörl
Nikolaj
N
Klöcker
Niels
N
Decher
D000818:Animals; D001919:Bradycardia / Q000235:genetics*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015640:Ion Channel Gating; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014981:Xenopus
C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009124:Muscle Proteins; D015221:Potassium Channels