Abstract
Purpose: :
In retinal ON-bipolar cells, post-synaptic signal transduction is mediated by a G protein-coupled receptor cascade, which in turn controls the open state of a cation-selective channel. The transient receptor potential channel TRPM1 has recently been implicated in this pathway. TRPM1 is a 180 kDa protein which likely forms a multimeric channel complex. However, virtually nothing is known about the structure of the TRPM1 channel or its close relatives. To enable biochemical and structural studies, baculovirus-expressed TRPM1 was purified from insect cells.
Methods: :
TRPM1 with a C-terminal 1D4 epitope tag was affinity purified from Sf9 cells. Cell membranes were detergent solubilized and incubated with sepharose conjugated to the monoclonal antibody 1D4. After washing, TRPM1 was eluted from the resin with the nonapeptide corresponding to the 1D4 epitope. The purified protein was reconstituted into proteoliposomes by mixing the protein with solubilized lipids, then removing the detergent with Bio-Beads polystyrene adsorbent. For structural studies, the purified protein was mixed with amphipol, followed by detergent removal with Bio-Beads. Protein-amphipol complexes were examined by electron microscopy and single particle reconstruction.
Results: :
Both blue native PAGE and size exclusion chromatography indicate that the purified protein is a homogeneous oligomeric complex of approximately 500-600 kDa. Successful proteoliposome formation was confirmed by flotation of the protein in a sucrose gradient, and pull-down experiments with 1D4 antibody indicated symmetric orientation of the protein in the liposome membranes. Preliminary electron microscopy suggests that TRPM1 forms a complex with a small transmembrane domain and a larger basket-like cytoplasmic domain.
Conclusions: :
A single-step affinity purification scheme has been developed for TRPM1, with a yield of ~1-2 mg/L spinner culture. Future studies will focus on biochemical characterization of the purified protein and demonstration of channel function in proteoliposomes, as well as structure determination by electron microscopy.
Keywords: ion channels • bipolar cells • synapse