Characterization of the calmodulin and S100A1 binding domains on the N-terminus of TRPM1 receptor

Michaela Jirku¹, Kristyna Bousova¹, Ladislav Bumba², Jiri Vondrasek³, Jan Teisinger¹

¹Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic

³Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nam. 2, 166 37 Prague, Czech Republic

Transient receptor potential melastatin 1 (TRPM1) belongs to the TRP family of non-selective cation permeable channels. TRPM1 is expressed in human melanocytes and bipolar cells in retina and participates in processes connected to vision. Mutations of TRPM1 gene are associated with congenital stationary night blindness in humans. [1, 2]

It is assumed that TRPM1 channel has six transmembrane domains with a pore domain between the fifth and the sixth segments. 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. [3]

In this study two independent CaM /S100A1 binding sites on the intracellular N-terminus of rat TRPM1 were characterized. Using bioinformatic approach we identified Ca²⁺-dependent CaM /S100A1 binding sites in regions L242-E344 and A451-N566. Several basic and hydrophobic amino acid residues responsible for binding in these regions of TRPM1 to CaM and S100A1 were determined.

Fusion proteins were expressed in E.coli and purified according to the two-step purification protocol. Amino acid sequence was checked by mass spectroscopy. Fluorescence anisotropy, surface plasmon resonance and measurement of CD spectra were used to characterize the interactions between the binding sites of TRPM1 N-terminus and CaM /S100A1. Interactions were modeled using molecular molecular modeling. Experimental data also indicate that CaM and S100A1 bind to the same or overlapping binding sites.

[1] Zhu M. X., Pflugers Arch 451: 105-15, 2005

[2] Nakamura M. et al., Mol Vis 16: 425-437, 2010

[3] Holakovska B. et al., J Biol Chem 287: 16645-16655, 2012

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.

The authors thank Dr. L. Bednarova from the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic for her assistance with CD spectra measurements.