The TRPs channels N-and C- tails contain a number of conserved epitopes that specifically bind the intracellular modulators.  It was identified new binding sites for the calmodulin (CaM) and S100A1, located in the very distal part of the TRPM4 N-terminus. We have used chemically synthesised peptides of the TRPM4, mimicking the binding epitopes, along with fluorescence methods to determine and specify CaM- and S100A1- binding sites. It was recognized 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.

It was also investigated in silico whether TRPM4_CT can bind either Calmodulin (CaM) or S100A1. In canonical Ca2+-CaM peptide complexes, the peptide is anchored through interactions of two large hydrophobic residues to one of each Ca2+-CaM lobe. The N-terminal anchor forms extensive hydrophobic contacts with the surrounding residues of F92, L105, M124 and M144, lining a deep hydrophrobic pocket in the Ca2+-CaM C-lobe. These four residues form the tetrad. Similarly, there is  tetrad in the Ca2+-CaM N-lobe formed by F19, L32, M51 and M71. The relative sequence position of the anchor residues defines the binding mode of Ca2+-CaM-peptide complexes. We searched the PDB and selected representative structures representing various canonical binding motifs (1-5-10, 1-8-14, 1-10, 1-14, etc.). The TRPM4_CT peptide contains several 1-10 motifs and thus it is possible to model its binding to Ca2+-CaM. However, in the context of the entire TRPM4, it has to be taken into account that the hydrophobic amino acids from the N-terminus of TRPM4_CT, are in close contacts with the sensor domain formed by helices S1-S4. Therefore, it can not be ruled out that the TRP re-entrant (TRPM4_CT) is bound to calmodulin by only one CaM lobe. It opens the possibility that CaM could stimulate the oligomerization of TRP ion channels. Alternatively, CaM (and S100A1) could stimulate dislocation of the TRP re-entrant segment from the S1-S4 sensor. Then, CaM and S100A1 comparing to PIP2 would appear to have exactly the opposite effects on gating of TRPM channels.