Pflügers Archiv

, Volume 451, Issue 1, pp 87–98 | Cite as

Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na+/K+-ATPase

  • Monu Goel
  • William Sinkins
  • Andrew Keightley
  • Michael Kinter
  • William P. SchillingEmail author
Ion Channels, Transporters


Mammalian transient receptor potential canonical (TRPC) genes encode a family of nonselective cation channels that are activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C. In Drosophila photoreceptor cells, TRP channels are found in large, multimolecular signaling complexes in association with the PDZ-containing scaffolding protein, INAD. A similar mammalian TRPC “signalplex” has been proposed, but has yet to be defined. In the present study, affinity-purified polyclonal antibodies against TRPC5 and TRPC6 were used to immunoprecipitate signalplex components from rat brain lysates. Immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and sequenced by mass spectrometry. Proteins identified in the immunoprecipitates included cytoskeletal proteins spectrin, myosin, actin, drebrin, tubulin, and neurabin; endocytic vesicle-associated proteins clathrin, dynamin and AP-2; and the plasmalemmal Na+/K+-ATPase (NKA) pump. Several of these interactions were confirmed by reciprocal immunoprecipitation followed by Western blot analysis. In lysates from rat kidney, TRPC6, but not TRPC3, was found to coimmunoprecipitate with the NKA pump. Likewise, TRPC6, stably expressed in human embryonic kidney (HEK) cells, coimmunoprecipitated with endogenous NKA and colocalized with the pump to the plasmalemma when examined by immunofluorescence microscopy. Cell surface biotinylation experiments in intact HEK cells, confirmed that both the Na+ pump and TRPC6 were present in the surface membrane and appeared to interact. Lastly, TRPC6 coimmunoprecipitated with the NKA pump when the proteins were coexpressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. These observations suggest that TRPC6 and the Na+ pump are part of a functional complex that may be involved in ion transport and homeostasis in both the brain and kidney.


Dendritic Spine Human Embryonic Kidney Cell Transient Receptor Potential Canonical Transient Receptor Potential Canonical Channel Mammalian Transient Receptor Potential 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported by NIH grant GM52019.


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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Monu Goel
    • 3
  • William Sinkins
    • 3
  • Andrew Keightley
    • 2
  • Michael Kinter
    • 2
  • William P. Schilling
    • 1
    • 3
    Email author
  1. 1.Rammelkamp Center for Education and Research Room R-322MetroHealth Medical CenterClevelandUSA
  2. 2.Department of Cell Biology, Lerner Research InstituteCleveland Clinic FoundationClevelandUSA
  3. 3.Department of Physiology and BiophysicsCase Western Reserve University School of MedicineClevelandUSA

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