Human disease-causing mutations disrupt an N-C-terminal interaction and channel function of bestrophin 1.
Mutations in the human bestrophin 1 (hBest1) chloride channel cause Best vitelliform macular dystrophy. Although mutations in its transmembrane domains were found to alter biophysical properties of the channel, the mechanism for disease-causing mutations in its N and C termini remains elusive. We hypothesized that these mutations lead to channel dysfunction through disruption of an N-C-terminal interaction. Here, we present data demonstrating that hBest1 N and C termini indeed interact both in vivo and in vitro. In addition, using a spectrum-based fluorescence resonance energy transfer method, we showed that functional hBest1 channels in the plasma membrane were multimers. Disease-causing mutations in the N terminus (R19C, R25C, and K30C) and the C terminus (G299E, D301N, and D312N) caused channel dysfunction and disruption of the N-C interaction. Consistent with the functional and biochemical results, mutants D301N and D312N clearly reduced fluorescence resonance energy transfer signal, indicating that the N-C interaction was indeed perturbed. These results suggest that hBest1 functions as a multimer in the plasma membrane, and disruption of the N-C interaction by mutations leads to hBest1 channel dysfunction.
Qu
Zhiqiang
Z
From the Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322; Department of Physiology, Qingdao University School of Medicine, Qingdao, Shandong 266071, China. Electronic address: zqu@emory.edu.
Cheng
Wei
W
Department of Physiology and Membrane Biology, University of California, School of Medicine, Davis, California 95616.
Cui
Yuanyuan
Y
From the Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322.
Cui
Yuanyuan
Y
Department of Physiology and Membrane Biology, University of California, School of Medicine, Davis, California 95616.
Zheng
Jie
J
Department of Physiology and Membrane Biology, University of California, School of Medicine, Davis, California 95616.
D000075002:Bestrophins; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007668:Kidney / Q000166:cytology; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism* / Q000503:physiopathology; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
C113133:BEST1 protein, human; D000075002:Bestrophins; D018118:Chloride Channels; D005136:Eye Proteins; D021122:Protein Subunits