Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines.
The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.
Hirota
Yutaka
Y
Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science, Nishimachi 86, Yonago 683, Japan.
Kurata
Yasutaka
Y
Kato
Masaru
M
Notsu
Tomomi
T
Koshida
Shunya
S
Inoue
Toshiaki
T
Kawata
Yasushi
Y
Miake
Junichiro
J
Bahrudin
Udin
U
Li
Peili
P
Hoshikawa
Yoshiko
Y
Yamamoto
Yasutaka
Y
Igawa
Osamu
O
Shirayoshi
Yasuaki
Y
Nakai
Akira
A
Ninomiya
Haruaki
H
Higaki
Katsumi
K
Hiraoka
Masayasu
M
Hisatome
Ichiro
I
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D051666:Kv1.5 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D032342:Muscle Cells / Q000378:metabolism; D051381:Rats
D018840:HSP70 Heat-Shock Proteins; D051666:Kv1.5 Potassium Channel