D553N is a missense mutation in the C-linker of HCN4 channel protein. It was found in a patient with severe cardiac arrhythmias including long QT syndrome, ventricular tachycardia polymorphisms, and bradycardia induced by sinus node dysfunction. Although it has been implicated that the cardiac arrhythmia caused by this mutant was due to its trafficking defect, no analysis reveals the underlying mechanism in the subcellular level. Here, we demonstrated that the trafficking defective mutant HCN4 D553N is restricted in the endoplasmic reticulum (ER) and perinuclear region. Two days after transfection D553N with the ER maker KKXX in the HEK293 cells, colocalization of these two proteins could be observed. Further, we employed the membrane surface dye FM1-34 to show that D553N cannot colocalize with the membrane dye FM1-34. On the contrary, wild type HCN4 channels colocalized with the dye FM1-34 on the cell membrane which serves as the control for the comparison. In conclusion, our data further demonstrate the subcellular location that the D553N mutant is retained in the ER, resulting in loss-of-function of this mutant at the cell membrane.
