Impaired interaction between the slide helix and the C-terminus of Kir2.1: a novel mechanism of Andersen syndrome.
OBJECTIVE
Andersen syndrome (AS) is a rare genetic disease caused by mutations of the potassium channel Kir2.1 (KCNJ2). We identified two unrelated patients with mutations in the slide helix of Kir2.1 leading to AS. The functional consequences of these two mutations, Y68D and D78Y, were studied and compared with previously reported slide helix mutations.


METHODS
Channel function and surface expression were studied by voltage clamp recordings and a chemiluminescence assay in Xenopus laevis oocytes and by patch clamp recordings and fluorescence microscopy in HEK293 cells. In addition, a phosphatidylinositol bisphosphate (PIP(2)) binding assay and a yeast-two-hybrid assay were used to characterize the molecular mechanisms by which slide helix mutations cause AS.


RESULTS
Neither mutant channel produced any current, but both had dominant negative effects on Kir2.2, Kir2.3, and Kir2.4 channels. We show that Y68D, D78Y, and previously reported AS mutations are clustered on the hydrophilic, cytosolic side of the slide helix and traffic normally to the plasma membrane. The in vitro lipid binding assay indicated that Y68D or D78Y N-terminal peptides bind PIP(2) similar to wild-type peptides. Yeast-two-hybrid assays showed that AS-associated mutations disturb the interaction between the slide helix and the C-terminal domain of the channel protein.


CONCLUSIONS
Our experiments indicate a new disease-causing mechanism independent of trafficking and PIP(2) binding defects. Our findings suggest that the hydrophilic side of the slide helix interacts with a specific domain of the C-terminus facing the membrane. This interaction, which may be required for normal gating both in homomeric and heteromeric Kir2 channels, is disturbed by several mutations causing AS.
Decher
Niels
N
Nora Eccles Harrison CVRTI and Department of Physiology, University of Utah, Salt Lake City, UT 84112, USA. decher@staff.uni-marburg.de
Renigunta
Vijay
V
Zuzarte
Marylou
M
Soom
Malle
M
Heinemann
Stefan H
SH
Timothy
Katherine W
KW
Keating
Mark T
MT
Daut
Jürgen
J
Sanguinetti
Michael C
MC
Splawski
Igor
I
D000328:Adult; D050030:Andersen Syndrome / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010641:Phenotype; D024661:Potassium Channels, Inwardly Rectifying / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D014981:Xenopus
C489461:Kir2.1 channel; D024661:Potassium Channels, Inwardly Rectifying