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Abstract: FR-OR065

G-protein Pathway Suppressor 2 Abolished the WNK4-Mediated Inhibition of the Large Conductance Ca2+ Activated Potassium (BK) Channels

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Wang, Yunman, Emory University, Atlanta, Georgia, United States
  • Li, Ruidian, Emory University, Atlanta, Georgia, United States
  • Chen, Shan, Emory University, Atlanta, Georgia, United States
  • Xiao, Jia, Emory University, Atlanta, Georgia, United States
  • Shen, Saier, Emory University, Atlanta, Georgia, United States
  • Feng, Xiuyan, LSU Shreveport , Shreveport, Louisiana, United States
  • Cai, Hui, Emory University, Atlanta, Georgia, United States
Background

G-protein pathway suppressor 2 (GPS2) is a multifunctional protein and transcriptional regulation factor. We have recently reported that GPS2 enhances BK channel activity and its protein expression by reducing ERK1/2 signaling-mediated degradation of the channel. Previous studies reported that WNK4 inhibits BK channel activity and protein expression through stimulating ERK 1/2 signaling pathway. Our yeast-two hybrid screening data showed that WNK4 interacts with GPS2. Thus, we hypothesized that GPS2 increases BK protein expression through interfering with the WNK4-mediated effect on BK.

Methods

Cell culture, transfection, western blot analysis, co-immunoprecipitation, and cell surface biotinylation were used in the experiments.

Results

To determine whether GPS2 modulates the WNK4-mediated inhibitory effect on BK, we first wanted to confirm whether GPS2 interacts with WNK4. In HEK293 cells cotransfected with HA-WNK4 and either myc-vector or myc-GPS2, co-immunoprecipitation (co-IP) experiments showed that myc-GPS2 co-immunoprecipitates WNK4, whereas myc-vector does not. To further confirm their interaction, we performed the reciprocal co-IP experiments in HEK293 cells cotransfected with HA-GPS2 and either myc-vector or myc-WNK4. We found that myc-WNK4 co-immunoprecipitates HA-GPS2 while myc-vector does not. To further assess the effects of GPS2 on WNK4-mediated inhibitory effects on BK, we performed the western blot analysis in HEK293 cells cotransfected with BK and WNK4 in the presence or absence of GPS2. We found that WNK4 significantly inhibits BK protein expression as expected. However, in the presence of GPS2, BK protein expression is restored to the control level. We further did cell surface biotinylation experiments in Cos-7 cells co-transfected with BK and WNK4 in combination with increasing doses of GPS2. We showed that WNK4 significantly inhibits total and surface BK protein expressions and GPS2 reversed the WNK4-mediated inhibition of BK in a dose-dependent manner. We further showed that GPS2 reversed the WNK4-mediated inhibition of BK protein expression by partially reducing the BK degradation through a lysosomal pathway.

Conclusion

These data suggested that GPS2 abolishes the WNK4-mediated inhibition of BK by partially reducing BK degradation through a lysosomal pathway.

Funding

  • Veterans Affairs Support