Characterization of the S100A1 Protein Binding Site on TRPC6 C-Terminus

Abstract

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the “canonical TRP” family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

Shared CaM‐ and S100A1‐binding epitopes in the distal TRPM4 N terminus

“…The dissociation constants of TRPM4np WT with CaM and S100A1 were in a comparable micromolar range for both complexes, which corresponds to the data of previous binding affinities measured for other TRP/CaM and TRP/S100A1 complexes [33,34,37]. Using steady-state fluorescence anisotropy measurements, we have determined the dissociation constants of the TRPM4np/CaM and TRPM4np/S100A1 complexes with clusters of hydrophobic L134, L138, V143 and basic R139, R140, R144 residues involved in the interactions.…”

“…Using steady-state fluorescence anisotropy measurements, we have determined the dissociation constants of the TRPM4np/CaM and TRPM4np/S100A1 complexes with clusters of hydrophobic L134, L138, V143 and basic R139, R140, R144 residues involved in the interactions. The Ca 2+ dependence of TRP/CaM or TRP/S100A1 interactions was validated in our previous experiments [33][34][35]37], which made it possible to use buffers with 2 mM of CaCl 2 in our binding assays. Using steadystate fluorescence anisotropy measurements, we determined the dissociation constants of the TRPM4np/ CaM and TRPM4np/S100A1 complexes.…”

“…It is plausible to expect that CaM exerts multiple regulatory roles in TRPMs and uses different mechanisms for the activation/inhibition channel function. The shared CBP-binding epitopes have been reported for the TRPM3, TRPC6 and TRPV1 [33,36,37,47,48]. There is clear evidence that the Fig.…”

“…CaM becomes more extended with each pair of EF-hands upon Ca 2+ binding, exposing a hydrophobic patch via CaM a-helix flexible linker that is available to interact with a target receptor domain [28]. This first step reveals the positive amino-acid residues eligible to form a specific interaction with the ligand [33][34][35][36][37][38][39][40]. Both monomer subunits are composed of four helices containing two EF-hand calcium-binding loops [29].…”

S100A1 and S100B: Calcium Sensors at the Cross‐Roads of Multiple Chondrogenic Pathways

“…Calmodulin regulates the function of numerous TRP channels via binding to multiple binding sites on the N-and C-intracellular tails of TRPC, TRPV, and TRPM (Holakovska et al, 2012). S100 proteins bind to similar structural motifs as calmodulin, and indeed overlapping S100A1 and calmodulin binding sites were found on the N-tail of the TRPM subfamily (Holakovska et al, 2012), and on the C-tail of TRPC6 (Bily et al, 2013) and TRPV1 (Grycova et al, 2015). A binding site for S100A1 in TRPV4 has not been described so far.…”