Ca²⁺ binding proteins interact with the N-terminal region of TRPM1

Michaela Jirku ¹, Kristyna Bousova ¹, Ladislav Bumba ², Jan Teisinger ¹

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

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

TRPM1 or melastatin channel (MLSN) belongs to melastatin subfamily of transient receptor potential channels (TRPs). These non-selective ion channels are responsible for entry of mono- and divalent cationts into the cell. They participate on many processes e.g. sensitivity to high and low temperatures, taste, pressure, vision and pain. Intracellularly located N- and C-tails are responsible for regulation of TRP channels, which carry binding sites for signal molecules like calmodulin (CaM) or S100A1.

TRPM1 is present in human melanocytes and retina. It seems that loss of TRPM1 correlates with increased aggressiveness in melanoma. TRPM1 is localized in bipolar cells in retina and participates in processes connected to vision. Mutations of TRPM1 gene are associated with congenital stationary night blindness in humans. There is currently a scarcity of structural / functional data on TRPM1 channel.

We studied possible interactions between CaM and S100A1 and the N- and C-termini of rat TRPM1. Using bioinformatic approach we identified CaM and S100A1 binding site in region 242-344 within the rat TRPM1 N-terminus (NT). The domain L₂₄₂-E₃₄₄ on NT was cloned into the pET32b vector and verified by sequencing. This construct was expressed in bacteria E. coli Rosetta cells and purified in two-step purification protocol using affinity chromatography and HPLC gel chromatography. Amino acid sequence was checked by MS MALDI-TOF. Steady-state fluorescence anisotropy and surface plasmon resonance measurements were used to test the binding of CaM and S100A1 to L₂₄₂-E₃₄₄. We determined several positive and hydrophobic residues to be responsible for binding of TRPM1-NT L₂₄₂-E₃₄₄ to CaM and S100A1. The results of the experiments also suggest that CaM and S100A1 bind to the same or overlapping binding site.

This project was supported by Grants GACR 301/10/1159, GACR 207/11/0717 and GACR - Project of Excellence in the Field of Neuroscience P304/12/G069.