Canonical Transient receptor potential channel TRPC6 and S100A1 protein as binding partners

Bily J. ¹, Janouskova H. ¹, Bumba L. ² and Teisinger J. ¹

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

²Institute of Microbiology Academy of Science of the Czech Republic

S100A1 is a calcium-binding protein which undergoes large conformational change upon binding calcium interacts with numerous target proteins involved in signaling pathways e.g. calcium signaling, neurotransmitter release or cooperation with cytoskeletal and filament associated proteins. Superfamily of TRP channels can function as calcium influx channels in eukaryotic cells, sensory receptor cells and play role in response to external signal. TRPC6 is Ca²⁺ permeable non-selective cation channel and its activity is modulated by multiple factors (calmodulin, PIP₂ etc).

We indentified the binding site for S100A1 protein on C-terminus of the transient receptor potential from canonical subfamily TRPC6 by using methods of fluorescence anisotropy and surface plasmon resonance. Part of C – terminus of TRPC6 (amino acids 801-878) was subcloned in PET42b expression vector with histidine tag. Point mutations of several amino acids residues for alanine were performed. Recombinant protein TRPC6 and all mutants were purified using chelating Sepharose fast flow and by HPLC chromatography (Superdex 75). The cluster of basic charged amino residues R852A 859A R860A R864A was involved in the binding of S100A1 protein on basis the fluorescence anisotropy measurements. It can be concluded that putative bunding site on TRPC6 C- tail binds S100A1 in a similar way like another calcium binding protein calmodulin.

Supported by GACR 301/10/1159