Pflugers Arch. 2005 Oct;451(1):87-98. Epub 2005 Jul 16.

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

Goel, M., Sinkins, W., Keightley, A., Kinter, M., Schilling, W. P.,
["Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA."]
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.
PMID: 16025302External
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Screening Toggle
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPC5 Link AP2B1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link CAND1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Clathrin-H1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Drebrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Dynamin-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link EF2 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Myosin-10 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Myosin-5a Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Myosin-HIIA Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Neurabin-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link Neurabin-2 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link NKAα1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link NKAα3 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link OGDH Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link SEC31A Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link SNAP91 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link SRA-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link TUBGCP2 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link TUBGCP3 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link α-actinin Speculation Prediction 16025302
TRPC5 Link α-actinin-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link α-actinin-4 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link α2-actin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link αII-spectrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link βII-spectrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link βIII-spectrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC6 Link Clathrin-H1 Speculation Prediction 16025302
TRPC6 Link Drebrin Speculation Prediction 16025302
TRPC6 Link Dynamin-1 Speculation Prediction 16025302
TRPC6 Link NKAα1 Speculation Prediction 16025302
TRPC6 Link α-actinin Speculation Prediction 16025302
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Arrow2 Screening Arrow2 Validation: In vitro validation Arrow2 Validation: In vivo validation Arrow2 Characterization Arrow2 Functional consequence Arrow2 top
Validation: In vivo validation Toggle
  Assay with endogenous proteins Assay with overexpressed proteins Reference
Cell or tissue Cell or tissue TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Species Region
TRPC5 Link Clathrin-H1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link Drebrin Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link Dynamin-1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link NKAα1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link α-actinin Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link Clathrin-H1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link Drebrin Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link Dynamin-1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link NKAα1 Co-immunofluorescence staining HEK293 Human Full-length Not used 16025302
TRPC6 Link NKAα1 Co-immunoprecipitation Rat kidney lysates 16025302
TRPC6 Link NKAα1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link α-actinin Co-immunoprecipitation Rat brain lysates 16025302
(Link: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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