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

Abstract

The transient receptor potential channel of melastatin 4 (TRPM4) belongs to a group of large ion receptors that are involved in countless cell signalling cascades. This unique member is ubiquitously expressed in many human tissues, especially in cardiomyocytes, where it plays an important role in cardiovascular processes. Transient receptor potential channels (TRPs) are usually constituted by intracellular N- and C- termini, which serve as mediators affecting allosteric modulation of channels, resulting in the regulation of the channel function. The TRPs tails contain a number of conserved epitopes that specifically bind the intracellular modulators. Here, we identify new binding sites for the calmodulin (CaM) and S100 calcium-binding protein A1 (S100A1), located in the very distal part of the TRPM4 N terminus. We have used chemically synthesized peptides of the TRPM4, mimicking the binding epitopes, along with fluorescence methods to determine and specify CaM- and S100A1-binding sites. We have found that the ligands binding epitopes at the TRPM4 N terminus overlap, but the interacting mechanism of both complexes is probably different. The molecular models supported by data from the fluorescence method confirmed that the complexes formations are mediated by the positively charged (R139, R140, R144) and hydrophobic (L134, L138, V143) residues present at the TRPM4 N terminus-binding epitopes. The data suggest that the molecular complexes of TRPM4/CaM and TRPM4/S100A1 would lead to the modulation of the channel functions.

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

“…They are generally considered as relatively low specificity proteins, with dozens of interaction partners, among them they are unable to maintain a high selectivity [15]. In this study we determined the interaction profile of the full human S100 family (termed here as the S100ome) against a set of diverse known S100 partners (and some of their paralogs) systematically, including kinases such as RSK1 [16] and its paralogs MK2 and MNK1; cytoskeletal elements such as CapZ [17] (commonly known as TRTK12), NMIIA [18], ezrin [19], FOR20 and its paralog FOP [20]; membrane proteins such as NCX1 [15] and TRPM4 [21]; and other signaling proteins such as the tumor suppressor p53 [22][23][24], SIP [15] and MDM4 [23].…”

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4

“…The FA increased due to a slower rotational diffusion of the formed complexes, and the fraction bound (F b ) of M4nt_WT and M4ct_WT could be calculated ( Figure 2 A, Figure 3 A, Figure 4 A, Figure 5 A). Since the specific interactions of M4nt_WT and M4ct_WT with CaM/S100A1 were calcium-dependent [ 36 , 37 , 38 , 39 , 40 ], all samples were measured in the presence of 200-μM CaCl 2 . The fluorescence lifetimes τ i of all peptides and their complexes (and all the following characterised peptides/complexes) were constant during all the experiments, with the values close to 3 ns.…”

“…The most reasonable complex identified by the ClusPro protein–protein docking process revealed that the M4nt_WT peptide was bound to the main binding site of S100A1 [ 40 , 43 ]. During subsequent MD simulations, M4nt_WT retained the canonical alpha-helical conformation, and numerous stabilising salt bridges were established ( Figure 3 C–F).…”

“…Two potential binding epitopes for the endogenous modulators of TRPM4 have been identified in the N- and C-termini of TRPM4-representing peptides. The binding affinities of the M4nt_WT and M4ct_WT peptides to endogenous modulators were examined in vitro, which revealed a typical micromolar range of K D values [ 36 , 40 , 46 , 47 , 48 ]. The N-terminal binding site (M4nt_WT) is apparently able to bind CaM and S100A1; moreover, the C-terminal binding site (M4ct_WT) binds PIP 2 as well.…”

Role of the TRPM4 Channel in Cardiovascular Physiology and Pathophysiology

“…Mutations in the C-terminal binding site of CaM can significantly reduce TRPM4 current amplitude and promote faster current decay. This suggests that the C-terminal binding site of CaM is vital for the Ca 2+ sensitivity of TRPM4 [ 8 , 75 ].…”