PIP2 interact with cytosolic N-terminal region of the melastatin channel TRPM1

Michaela Jirku1,2, Kristyna Bousova1, Ladislav Bumba3, Jiri Vondrasek4, Lucie Bednarova4, Jan Teisinger1

1Institute of Physiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague, Czech Republic
2Faculty of Science, Charles University in Prague, Albertov 6, 12843 Prague, Czech Republic
3Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 14220 Prague, Czech Republic
4Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nam. 2, 16637 Prague, Czech Republic


Transient receptor potential melastatin 1 (TRPM1) channel belongs to superfamily of ion channels that are mostly permeable to mono- and divalent cations and respond to various physiological stimuli like chemosensation, termosensation and mechanosensation. TRP channels have six transmembrane domains with a pore region between the fifth and the sixth segments. Cytosolic N-/C-tails are responsible for regulation of TRPs, which carry binding sites for signal molecules. [1, 2] 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 and loss of TRPM1 correlates with increased aggressiveness in melanoma. [3, 4]

In this study using bioinformatic approach we identified phosphatidylinositol 4,5-bisphosphate (PIP2) binding site in region A451-N566 within the TRPM1 N-terminus (NT). Fusion protein of TRPM1-NT was expressed in bacteria E. coli Rosetta cells and purified in two-step purification protocol using affinity and HPLC gel chromatography. Alanine substitution mutagenesis screening revealed the crucial amino acids for these interactions. The equilibrium dissociation constants were estimated using surface plasmon resonance measurement. The CD spectra were used to characterize the interactions as well.

We identified the PIP2-binding site and found mutations that decreased the affinity of the TRPM1-NT/PIP2 interaction. Our results suggest that the sequence of TRPM1-NT contains several basic amino acids which interact with anionic phospholipids. Moreover we have provided the structural insight to the TRPM1-NT/PIP2 interaction using computer ligand docking.

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4. M. Nakamura, R. Sanuki, T. R. Yasuma, A. Onishi, K. M. Nishiguchi, C. Koike, M. Kadowaki, M. Kondo, Y. Miyake, T. Furukawa, Mol Vis, 16, (2010), 425-37.


This project was supported by Grants GAUK 238214 and GACR - Project of Excellence in the Field of Neuroscience P304/12/G069.