Open Access14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel
Author(s) -
Shan Chen,
Feng Xu,
Xinxin Chen,
Zhizhi Zhuang,
Jiumei Xiao,
Haian Fu,
Janet D. Klein,
Xiaonan H. Wang,
Robert S. Hoover,
Douglas C. Eaton,
Hui Cai
Publication year - 2020
Publication title -
american journal of physiology. renal physiology./american journal of physiology. renal physiology
Language(s) - English
Resource type - Journals
eISSN - 1931-857X
pISSN - 1522-1466
DOI - 10.1152/ajprenal.00584.2019
Subject(s) - bk channel , immunoprecipitation , gene knockdown , microbiology and biotechnology , western blot , hek 293 cells , transfection , chemistry , phosphorylation , biology , biochemistry , membrane potential , gene
14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca 2+ -activated K + (BK) channels revealed a putative 14-3-3 binding site in the COOH-terminal region. Our previous data showed that 14-3-3γ is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3γ has a novel role in the regulation of BK channel activity and protein expression. We used electrophysiology, Western blot analysis, and coimmunoprecipitation to examine the effects of 14-3-3γ on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3γ with BK α-subunits (BKα) by coimmunoprecipitation. In human embryonic kidney-293 cells stably expressing BKα, overexpression of 14-3-3γ significantly decreased BK channel activity and channel open probability. 14-3-3γ inhibited both total and cell surface BKα protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3γ and myc-BK. Knockdown of 14-3-3γ by siRNA transfection markedly increased BKα expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3γ-mediated inhibition of BK protein expression. Similarly, pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3γ on BK protein expression. Furthermore, overexpression of 14-3-3γ significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BKα. Additionally, 3 days of dietary K + challenge reduced 14-3-3γ expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K + excretion. These data suggest that 14-3-3γ modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.