14-3-3γ, a novel regulator of the large-conductance Ca2+-activated K+ channel

14-3-3 gamma 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 14-3-3 gamma is widely expressed in the mouse kidney. Therefore, we hypothesized that 14-3-3 gamma 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 14-3-3 gamma on BK channels both in vitro and in vivo. We demonstrated the interaction of 14-3-3 gamma with BK alpha-subunits (BK alpha) by coinununoprecipitation. In human embryonic kidney-293 cells stably expressing BK alpha, overexpression of 14-3-3 gamma significantly decreased BK channel activity and channel open probability. 14-3-3 gamma inhibited both total and cell surface BK alpha protein expression while enhancing ERK1/2 phosphorylation in Cos-7 cells cotransfected with flag-14-3-3 gamma and myc-BK. Knockdown of 14-3-3 gamma by siRNA transfection markedly increased BK alpha expression. Blockade of the ERK1/2 pathway by incubation with the MEK-specific inhibitor U0126 partially abolished 14-3-3 gamma-mediated inhibition of BK protein expression. Similarly. pretreatment of the lysosomal inhibitor bafilomycin A1 reversed the inhibitory effects of 14-3-3 gamma on BK protein expression. Furthermore, overexpression of 14-3-3 gamma significantly increased BK protein ubiquitination in embryonic kidney-293 cells stably expressing BK alpha. Additionally, 3 days of dietary K+ challenge reduced 14-3-3 gamma expression and ERK1/2 phosphorylation while enhancing renal BK protein expression and K+ excretion. These data suggest that 14-3-3 gamma modulates BK channel activity and protein expression through an ERK1/2-mediated ubiquitin-lysosomal pathway.