N dodecyl β d maltoside

Reagents were obtained from the following sources: Ni Sepharose 6 Fast Flow (Cytiva, 17531802), Glutathione Sepharose 4 Fast Flow (Cytiva, 17513201), tris(2-carboxyethyl)phosphine (Thermo, 75259), EDTA-free protease inhibitor cocktail (Roche, 11873580001), PreScission Protease (Absin, abs01243), Anapoe-X-100 (Anatrace, 9002-93-1), ATP (Roche, 11140965001), MgOAc (Sigma-Aldrich, M0631), L-glutamic acid potassium (Sigma-Aldrich, G1501), biotinylated anti-His antibody (Bioss Antibodies, bs-0287R-bio), streptavidin (Sangon Biotech, A610492), LD555-MAL (Lumidyne, 04), LD655-MAL (Lumidyne, 10), GDP (Sigma-Aldrich, G7127), GTPγS (Roche, 10220647001), GMppNHp (Sigma-Aldrich, G0635), mPEG-SVA-5000 (Laysan Bio, 170-106), Biotin-PEG-SVA-5000 (Laysan Bio, 170-124), benzoic acid (Sigma-Aldrich, 242381), protocatechuate 3,4-dioxygenase (Sigma-Aldrich, P8279), and n-dodecyl-β-D-maltoside (Avanti Polar Lipids, 850520). All lipids were obtained from Avanti Polar Lipids: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, 850457); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, 840034); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, 810144); and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-PE, 810150).

Purification of neuronal IP₃R1 reconstituted was performed as previously described^{20 (link),67 (link)}. Briefly, rat cerebellar membranes were solubilized in 2 mM Lauryl Maltose Neopentyl Glycol (LMNG, Anatrace) and 0.1% (w/v) l-α-phosphatidylcholine (PC, Sigma) for 2 h at 4 °C. Non-solubilized material were cleared by ultracentrifugation (100,000 x g) and the supernatant was applied to an immunoaffinity matrix pre-coupled with monoclonal antibodies (10 mg; T433 epitope of IP₃R1) in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.02 mM LMNG. Reconstitution of IP₃Rs into lipid-nanodiscs occurred “on-column” as we described in ref. 20 (link): 1.2 mM POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, Avanti Polar Lipids) prepared in 3% DDM (n-dodecyl-β-d-maltoside, Avanti Polar Lipids, Inc), 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM DTT, 1 mM EDTA was added to the IP₃R1-bound immunoaffinity column matrix followed by the addition of 0.6 mg/ml MSP1E3D1 (Cube Biotech). Detergent removal was achieved by the addition of 0.25 mg/ml activated Bio-Beads-SM2 (BioRad) for 16 h at 4 °C. IP₃R1 was eluted with 500 μM of T433 peptide and concentrated using a 100 kDa Amicon centricon (Millipore).