Conserved Gating Elements in TRPC4 and TRPC5 Channels

Abstract

Background: Gating mechanisms of TRPC channels are mostly unknown. Results: Replacing the highly conserved glycine residue within the linker between transmembrane domains 4 and 5 by serine renders TRPC4 and TRPC5 channels constitutively active. Conclusion: TRPC channel opening seems to require similar constraints than the voltage-gated potassium channels. Significance: Novel mechanistic insights into structural requirements of TRPC channel gating are provided.

Trans-activation Response (TAR) RNA-binding Protein 2 Is a Novel Modulator of Transient Receptor Potential Canonical 4 (TRPC4) Protein

“…Bound proteins were eluted by 2ϫ concentrated denaturing sample buffer (60 l), followed by incubation at 65°C for 20 min. SDS-PAGE and Western blot analysis were performed as described previously (25). Proteins were detected with horseradish peroxidase-coupled secondary antibodies and Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences).…”

“…During the measurement, cells were incubated in the presence of 1 M thapsigargin (no Ca 2ϩ in the bath solution), 2 mM CaCl 2 , and 100 M carbachol. For the tetracycline-induced Flp-In TM -293 cells expressing the TRPC4 G503S or TRPC5 G504S mutants, 2 mM CaCl 2 was added to the bath solution after reaching a stable F340/F380 base line, and changes of cytosolic Ca 2ϩ were monitored as described previously (25).…”

“…Antibodies, Coimmunoprecipitation, and Western Blot Analysis-In-house-generated, affinity-purified antibodies for TRPC4 and TRPC5 (25) were used, as well as antibodies against FLAG M2 (Sigma-Aldrich), GST (GE Healthcare Biosciences), calmodulin (Zymed Laboratories Inc.), Dicer, anti-Dicer-1133 (catalog no. D38E7, Cell Signaling Technology), and anti-Dicer-Ct (an epitope corresponding to amino acids 1701-1912, mapping at the C terminus of human Dicer according to the manufacturer; catalog no.…”

“…Therefore, we made use of the TRPC4 G503S mutant, which renders the channel constitutively active (25). The measurable phenotype, in this case ensemble conductance in the absence of any agonist, comprises the typical, doubly rectifying TRPC4 current (Fig.…”

“…These results show that Tarbp2 also binds to TRPC5. We transfected the Tarbp2 cDNA into a tetracyclineinducible HEK293 cell line expressing TRPC5 G504S (25) that, like TRPC4 G503S , forms a constitutively active channel. Upon inducing TRPC5 G504S expression and addition of extracellular Ca 2ϩ (Fig.…”