In the mammalian brain TRPC channels, a family of Ca2+-permeable cation channels, are involved in a variety of processes from neuronal growth and synapse formation to transmitter release, synaptic transmission and plasticity. The molecular appearance and operation of native TRPC channels, however, remained poorly understood. Here we used high-resolution proteomics to show that TRPC channels in the rodent brain are macro-molecular complexes of more than 1 MDa in size that results from co-assembly of the tetrameric channel core with an ensemble of interacting proteins (interactome). The core(s) of TRPC1, 4, 5-containing channels are mostly heteromers with defined stoichiometries for each subtype, while TRPC 3, 6, 7 preferentially form homomers. In addition, TRPC1, 4, 5-channels may co-assemble with the metabotropic glutamate receptor mGluR1 thus guaranteeing both specificity and reliability of channel activation via the phospholipase-Ca2+ pathway. Our results unveil the subunit composition of native TRPC channels and resolve molecular details underlying their activation.