Calmodulin and S100A1 protein interact with the intracellular termini of the TRPM4 channel

Kristyna Bousova ¹, Michaela Jirku ¹, Ladislav Bumba ², Jiri Vondrasek ³, Jan Teisinger ¹

¹Institute of Physiology, Academy of Sciences of the Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic

³Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech republic

TRPM4 belongs to the large family TRP channels, a group of non-selective cation permeable channels. This channel participates in processes ongoing in neurons, cardiomyocytes, T-cells, etc.. It has been proven link between defects of TRPM4 receptor and progressive familial heart block type 1B. TRP channels consist of six transmembrane helices and a pore-forming loop between S5 and S6, with different lengths of intracellular amino and carboxy termini. [1, 2] It has been shown that TRPM4 activity could be modulated by intracellular calcium binding proteins calmodulin (CaM) and S100A1. [3]

In this study, one CaM/S100A1 binding site was localized in TRPM4 N-terminal region (NT) S₅₈₃-A₆₆₈. Another CaM/S100A1 binding site was determined in C–terminus domain (CT) V₁₀₅₀-S₁₁₁₄. The results from the previous experiments suggest that CaM and S100A1 could bind to the same or overlapping binding site. [3] Fusion proteins of the corresponding lengths were expressed in E.coli and purified by affinity and gel permeation chromatography. Sequences of the proteins were verified by Mass Spectrometry. To characterize CaM/S100A1 binding site on intracellular termini regions of the TRPM4 surface plasmon resonance measurements were used. Potential changes in secondary structure TRPM4/CaM complex were checked by the circular dichroism. De Novo molecular models of TRPM4 termini combined with ligand docking by CaM provides a structural insight into the TRPM4/CaM binding. Several positively charged residues were identified to be responsible for binding of CaM and S100A1 within the mentioned domains. The binding of both domains to CaM and S100A1 respectively is Ca²⁺ dependent.

We thank Dr. Lucie Bednarova from the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic for assistance with CD spectra measurements.

This project was supported by Grants GACR 301/10/1159, GACR 207/11/0717 and GAUK 842313.

1. Krause M., et al., J. Clin. Invest., 119 (2009), 2737-2744.

2. Nilius B., et al., J. Biol. Chem., 278 (2003), 30813-20. 

3. Holakovska B., et al., J. Biol. Chem., 287 (2012), 16645-55.