Extended Data Fig. 1: Consensus mutagenesis, protein biochemistry, and cryo-EM analysis of OCT1_CS.

a, FSEC traces showing strong monodisperse peaks for OCT1_CS-GFP and OCT2_CS-GFP, while WT hOCT1-GFP and WT hOCT2-GFP transfected HEK293T cells did not yield any discernable peak corresponding to target protein. Asterisk indicates target protein peak in FSEC. b, Map of all residues in OCT1_CS and OCT2_CS that deviate from WT hOCT1. The residues are colored based on their conservation score from MAFFT alignment. Blue spheres indicate mildly changed, while red spheres indicate drastic changes. c, Time-dependent accumulation of 10 nM [³H]-MPP⁺ in WT hOCT1 and OCT1_CS expressing oocytes (n = 3 biologically independent replicates per timepoint). d, Raw uptake values for controls in the OCT1 [¹⁴C]-metformin uptake experiments, corresponding to Fig. 1e (n = 3–4 biologically independent replicates, shown with mean ± s.e.m.). e, Raw uptake values for controls in the OCT2 [³H]-MPP⁺ uptake experiments, corresponding to Fig. 1f (n = 3 biologically independent replicates, shown with mean ± s.e.m.). f, [³H]-MPP+ uptake in oocytes expressing Y36C, F446I, and Y36C/F446I in the OCT1_CS-GFP background (n = 3 biologically independent replicates, individual values and mean ± S.E.M; water injected controls used for background correction, OCT1_CS uptake signal used for normalization). g, FSEC traces showing expression of selected OCT1_CS mutants in HEK293T cells. Asterisk indicates target protein peak in FSEC. h, Representative size-exclusion chromatography trace (left) and SDS-PAGE (right) of purified OCT1_CS or OCT2_CS samples used for cryo-EM grid preparation. The experiments were repeated independently for >10 times with similar results. Asterisks indicate target protein peak (in SEC) and band (in SDS-PAGE). i, Representative micrograph of a OCT1_CS sample. OCT2 _CS behaves similarly on cryo-EM grids. j, Representative 2D classes from a OCT1_CS dataset. k, Secondary structure topology of OCT1 and OCT2.

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