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Kidney Week

Abstract: SA-PO1059

A Novel Molecule, Gephyrin, Functionally Associates with ClC-5 Chloride Channel in Response to Metabolic Acidosis in the Mouse Kidney

Session Information

  • Na+, K+, Cl-
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 703 Na+, K+, Cl- Basic

Authors

  • Ogawa, Miyuki, Kitasato University, SAGAMIHARA, KANAGAWA, Japan
  • Sakamoto, Hisato, Graduate School of Medical Science, Kitasato University, SAKAMIHARA, KANAGAWA, Japan
  • Itakura, Makoto, Kitasato University, SAGAMIHARA, KANAGAWA, Japan
Background

ClC-5 channel is co-localized with the V-ATPase in subapical endosomes of renal proximal tubule (PT) and α-intercalated cells. ClC-5 may play a crucial role in regulating both endocytosis and sorting of the acid transporters. However the regulatory molecule associated with ClC-5 has not been elucidated. This study aimed to identify the associated molecule and to examine its physiological roles in the regulation of intracellular sorting of ClC-5 in response to metabolic acidosis.

Methods

Following to immunoisolation of ClC-5-bearing vesicles using the magnetic beads coated with originally generated specific antibody (Ab), we identified specific binding molecules using the analysis by LC-MS and immunoprecipitation assays. To examine the physiological role of the molecule, we prepared the fractions enriched for plasma membrane (P1) and endosomal membrane (P2) using differential centrifugation under conditions with or without NH4Cl-induced acidosis. The protein abundances of transporters and associated molecule were assessed by Western blot. The co-localization of CLC-5 and associated proteins were also imaged using confocal microscopy.

Results

We identified gephyrin as a specific associated molecule with ClC-5 by LC-MS. Immunohistochemistry showed the predominant expression of gephyrin and its co-localized with ClC-5 in the apical membrane of PT compared with that of distal tubule. In addition, gephyrin was co-immunoprecipitated with specific Ab against ClC-5 using crude homogenates of mouse kidney. Mice given NH4Cl in drinking water developed metabolic acidosis within 2 days of acid intake. ClC-5 protein abundance was relatively decreased in P1 and increased in P2 after 6 days of acid loading. In contrast the protein abundances of gephyrin were increased by 230% in P1 and 120% in P2 under the same condition of acid loading.

Conclusion

ClC-5 might be functionally anchored by gephyrin in the apical membrane of PT. Furthermore, gephyrin may implicate in the self-assemble into a scaffold to reconstruct and strengthen plasticity following to the sorting of ClC-5 from plasma membrane to intracellular vesicles after acid loading.

Funding

  • Commercial Support