Instability of KCNE1-D85N that causes long QT syndrome: stabilization by verapamil.
BACKGROUND
A KCNE1 polymorphism, D85N, causes long QT syndrome (LQTS) with a decrease in the slowly activating delayed-rectifier K(+) channel current (IKs ). We examined impacts of D85N polymorphism on KCNE1 protein stability and functions, and tested the ability of various drugs to modify them.


METHODS
KCNE1-D85N or the wild-type protein was coexpressed in COS7 cells with KCNQ1 to form K(+) channels. Expression, degradation, and intracellular localization of KCNE1 proteins, as well as the currents conferred by KCNQ1/KCNE1 complexes, were determined using immunoblots, immunofluorescence, and patch-clamp techniques.


RESULTS
The protein level of KCNE1-D85N was lower than that of the wild-type, in spite of the comparable levels of their mRNA. KCNE1-D85N was highly ubiquitinated and rapidly degraded as compared to the wild-type; a proteasome inhibitor, MG132, inhibited its degradation and increased its steady-state level. Both KCNE1-D85N and the wild-type proteins were co-immunoprecipitated with KCNQ1. Immunofluorescent signals of KCNE1-D85N accumulated in the endoplasmic reticulum and Golgi apparatus, with reduced levels on the cell membrane. Patch-clamp experiments demonstrated that the membrane current corresponding to IKs was much smaller in cells expressing KCNE1-D85N than in those expressing the wild-type. Verapamil (0.5-10 μM) increased the protein level of KCNE1-D85N, decreased its ubiquitination, slowed its degradation, and enhanced KCNQ1/KCNE1-D85N channel currents. Pretreatment with amiodarone abolished these effects of verapamil.


CONCLUSIONS
KCNE1-D85N is less stable than the wild-type protein, and is rapidly degraded through the ubiquitin-proteasome system. Verapamil may be of a therapeutic value in LQTS patients via preventing degradation of KCNE1-D85N.
Department of Pediatrics, Faculty of Medicine, Tottori University, Yonago, Japan.
Shinji
S
Sakata
Yasutaka
Y
Kurata
Peili
P
Li
Tomomi
T
Notsu
Kumi
K
Morikawa
Junichiro
J
Miake
Katsumi
K
Higaki
Yasutaka
Y
Yamamoto
Akio
A
Yoshida
Yasuaki
Y
Shirayoshi
Kazuhiro
K
Yamamoto
Minoru
M
Horie
Haruaki
H
Ninomiya
Susumu
S
Kanzaki
Ichiro
I
Hisatome
D002121:Calcium Channel Blockers / Q000494:pharmacology* / Q000627:therapeutic use*; D002478:Cells, Cultured; D006801:Humans; D008133:Long QT Syndrome / Q000188:drug therapy* / Q000235:genetics*; D011110:Polymorphism, Genetic; D024642:Potassium Channels, Voltage-Gated / Q000187:drug effects* / Q000235:genetics*; D014700:Verapamil / Q000494:pharmacology* / Q000627:therapeutic use*
D002121:Calcium Channel Blockers; C489622:KCNE1 protein, human; D024642:Potassium Channels, Voltage-Gated; D014700:Verapamil
D85N polymorphism; KCNE1; amiodarone; delayed-rectifier K+ channel current; long QT syndrome; verapamil