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14-3-3γ is a small protein regulating its target proteins through binding to phosphorylated serine/threonine residues. Sequence analysis of large-conductance Ca2+-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 mycBK. 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. [ABSTRACT FROM AUTHOR]

G protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor that is involved in the G protein MAPK signaling pathway. It has been shown that the MAPK signaling pathway plays an important role in the regulation of renal large-conductance Ca2+- activated potassium (BK) channels. In this study, we investigated the effects of GPS2 on BK channel activity and protein expression. In human embryonic kidney (HEK) BK stably expressing cells transfected with either GPS2 or its vector control, a single-cell recording showed that GPS2 significantly increased BK channel activity (NPo), increasing BK open probability (Po), and channel number (N) compared with the control. In Cos-7 cells and HEK 293 T cells, GPS2 overexpression significantly enhanced the total protein expression of BK in a dose-dependent manner. Knockdown of GPS2 expression significantly decreased BK protein expression, while increasing ERK1/2 phosphorylation. Knockdown of ERK1/2 expression reversed the GPS2 siRNA-mediated inhibition of BK protein expression in Cos-7 cells. Pretreatments of Cos-7 cells with either the lysosomal inhibitor bafilomycin A1 or the proteasomal inhibitor MG132 partially reversed the inhibitory effects of GPS2 siRNA on BK protein expression. In addition, feeding a high-potassium diet significantly increased both GPS2 and BK protein abundance in mice. These data suggest that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel. [ABSTRACT FROM AUTHOR]

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20- related proline alanine-rich kinase) or WNK4-extracellular signalregulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone- mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2- specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)- 2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs. [ABSTRACT FROM AUTHOR]