Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity

Abstract

Crucial cysteine residues can be involved in the modulation of protein activity via the modification of thiol (-SH) groups. Among these reactions, disulfide bonds (S-S) play a key role in the folding, stability, and activity of membrane proteins. However, the regulation of extracellular cysteines in classical transient receptor potential (TRPC) channels remains controversial. Here, we examine the functional importance of the extracellular disulfide bond in TRPC5 in modulating channel gating and trafficking. Specifically, we investigated TRPC5 activity in transiently transfected HEK293 cells with wild-type (WT) or cysteine (C553 and C558) mutants in the pore loop. Using reducing agents, we determined that a disulfide linkage mediates the tetrameric formation of the TRPC5 channel. By measuring the TRPC5 current, we observed that C553S or C558S mutants completely lose channel activity induced by lanthanides or receptor stimulation. Co-expression of TRPC5 (WT) with mutants demonstrated a dominant-negative function in mutants, which inhibited the activity of TRPC5 (WT). We generated TRPC5-TRPC5 dimers and observed reduced activity of WT-mutant (C553S or C558S) dimers compared to WT-WT dimers. When pretreated with reducing agents for 12 h, the TRPC5 current decreased due to a reduction in membrane TRPC5 distribution. In addition, we identified a reduced expression of C553S mutant in plasma membrane. We analyzed a dimeric interaction of wild-type and mutant TRPC5 using co-immunoprecipitation and FRET method, indicating a weak interaction between dimeric partners. These results indicated that the disulfide bond between conserved extracellular cysteines, especially C553, is essential for functional TRPC5 activity by channel multimerization and trafficking.

Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s

“…The I-V curve showed a typical doubly rectifying shape, consistent with previous reports (Fig. 3B) [2324252627]. The current at -60 mV increased from -10.…”

Functional and Structural Divergence in Human TRPV1 Channel Subunits by Oxidative Cysteine Modification

“…Ion channels are known to possess disulfide bonds to provide a structural scaffold or redox sensitivity. These include ion channels such as NMDA receptor, CLIC1, Kir2.2, cardiac ryanodine receptor, TWIK-1 channel, and TRPC5 (47)(48)(49)(50)(51)(52)(53). However, to our knowledge this is the first observation in which an ion channel uses the redox heterogeneity of a single specific Cys residue (Cys-258) to provide both oxidation sensitivity and protein stability.…”

Identification of a Membrane-targeting Domain of the Transient Receptor Potential Canonical (TRPC)4 Channel Unrelated to Its Formation of a Tetrameric Structure

“…2C and 4B). In general, making a dimer or tetramer construct takes a long time, and dimer or tetramer constructs usually do not work well due to the linker length or the flexibility of the linker (22). To combine the imaging methods and the FRET methods was useful for finding a membranetargeting domain or tetrameric assembly domain.…”

“…Collected proteins were then analyzed by Western blot. The experiment was performed as previously described in detail (22).…”