Author(s): Shang-Zhong Xu [1, 4, 5]; Piruthivi Sukumar [1, 4]; Fanning Zeng ; Jing Li ; Amit Jairaman ; Anne English ; Jacqueline Naylor ; Coziana Ciurtin ; Yasser Majeed ; Carol J. Milligan ; Yahya M. Bahnasi ; Eman Al-Shawaf ; Karen E. Porter ; Lin-Hua Jiang ; Paul Emery ; Asipu Sivaprasadarao ; David J. Beech (corresponding author) 
Mammalian homologues of Drosophila melanogaster transient receptor potential (TRP) are a large family of multimeric cation channels that act, or putatively act, as sensors of one or more chemical factor [1, 2]. Major research objectives are the identification of endogenous activators and the determination of cellular and tissue functions of these channels. Here we show the activation of TRPC5 (canonical TRP 5) homomultimeric and TRPC5-TRPC1 heteromultimeric channels [3, 4, 5] by extracellular reduced thioredoxin, which acts by breaking a disulphide bridge in the predicted extracellular loop adjacent to the ion-selectivity filter of TRPC5. Thioredoxin is an endogenous redox protein with established intracellular functions, but it is also secreted and its extracellular targets are largely unknown [6, 7, 8, 9]. Particularly high extracellular concentrations of thioredoxin are apparent in rheumatoid arthritis [8, 10, 11, 12], an inflammatory joint disease that disables millions of people worldwide . We show that TRPC5 and TRPC1 are expressed in secretory fibroblast-like synoviocytes from patients with rheumatoid arthritis, that endogenous TRPC5-TRPC1 channels of the cells are activated by reduced thioredoxin, and that blockade of the channels enhances secretory activity and prevents the suppression of secretion by thioredoxin. The data indicate the presence of a previously unrecognized ion-channel activation mechanism that couples extracellular thioredoxin to cell function.
TRPC5 is markedly activated by extracellular lanthanide ions [4, 14, 15]. The effects of these ions depend on a glutamic acid residue at position 543 (ref. 14) in the predicted extracellular loop adjacent to the ion pore (Supplementary Figs 1 and 2). This structural feature may therefore have functional importance in enabling extracellular factors to activate the channels. Because lanthanides are unlikely to be physiological activators, we were interested in alternatives and developed a hypothesis based on amino acid sequence alignment, which showed two cysteine residues near glutamic acid 543 that are conserved in TRPC5, TRPC4 and TRPC1 (Supplementary Fig. 2), a subset of the seven TRPC channels [1, 2, 3, 4, 5]. TRPC5 and TRPC4 have similar functional properties  and both form heteromultimers with TRPC1 (refs 3-5), a subunit that has weak targeting to the plasma membrane when expressed in isolation [3, 16].
Pairs of cysteine residues may be covalently linked by a disulphide bridge that can be cleaved by reduction. We therefore applied the chemical reducing agent dithiothreitol (DTT) to HEK-293 cells expressing TRPC5 (refs 15, 16). There was channel activation with the characteristic current-voltage ( I -V ) relationship of TRPC5 and blocking by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of TRPC5 (ref. 5) (Fig. 1a, b, d). Current recovered on wash-out of DTT (data not shown). Similarly, the membrane-impermeable disulphide reducing agent Tris (2-carboxyethyl) phosphine...