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 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

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 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 AP2B1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 CAND1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Clathrin-H1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Drebrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Dynamin-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 EF2 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ƢII-spectrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ƢIII-spectrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-10 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-5a Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Myosin-HIIA Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Neurabin-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Neurabin-2 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ3 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin Speculation Prediction 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-4 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ2-actin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 OGDH Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SEC31A Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SNAP91 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 SRA-1 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 TUBGCP2 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 TUBGCP3 Affinity purification-mass spectrometry Endogenous protein Rat Brain Cerebral cortex lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Clathrin-H1 Speculation Prediction 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Drebrin Speculation Prediction 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Dynamin-1 Speculation Prediction 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ1 Speculation Prediction 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin Speculation Prediction 16025302
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  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 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Clathrin-H1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Drebrin Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Dynamin-1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC5 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Clathrin-H1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Drebrin Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 Dynamin-1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ1 Co-immunofluorescence staining HEK293 Human Full-length Not used 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ1 Co-immunoprecipitation Rat kidney lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NKAơ1 Co-immunoprecipitation Rat brain lysates 16025302
TRPC6 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin Co-immunoprecipitation Rat brain lysates 16025302
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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