SPR STUDIES ON THE INTERACTIONS OF TRP CHANNELS AND THEIR ACTIVITY MODULATORS

B. Holakovska, L. Grycova, J. Teisinger

Department of Protein Structures, Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, Praha 4, 142 20

Transient receptor potential (TRP) channels consist of a diverse group of cation channels that contains more than 30 members. The channels participate in many sensory and physiological processes. The vanilloid receptor (TRPV1) is one of the best characterized members of the TRPV subfamily. This nonselective cation channel serves as a polymodal receptor for various potentially harmful signals. Activation is caused by diverse stimuli, such as noxious heat (>43 ºC), low pH (<5.4) and chemicals such as capsaicin. TRPM3 is one of the least investigated proteins of the TRP family of ion channels. It was described to be involved in Ca2+ homeostasis in kidney cells and to be activated by noxious heat. Intracellular termini of TRP channels are involved in regulation of their activity via binding of intracellular ligands as CaM or phosphatidylinositol – 4,5 - bisphosphate (PIP2). In order to characterize complex forming we used surface plasmon resonance (SPR) as a suitable tool. Employing two different types of SPR protocols, we have explored interactions of C-terminal regions of TRPV1 with phospholipids namely L777-S820 and two novel binding sites of TRPM3 for CaM namely A35-K124 and H291-G382. All SPR measurements assessing the PIP2 and protein equilibrium dissociation constant were performed at 25 °C using a liposome-coated NLC chip in ProteOn XPR36 Protein Interaction Array System (Bio-rad, Hercules, CA, USA. All SPR measurements characterizing TRPM3 / CaM interactions were perfomed on CaM coated GLC chip on ProteOn XPR36 (Bio-Rad, Hercules, CA, USA). TRPV1 showed relatively high affinity and selectivity toward PIP2, KD 1.88 +/- 0.46 µM. TRPM3 constructs A35-K124 and H291-G382 bound CaM with high affinity, KD 0.198 +/- 0.018 and 0.481 +/- 0.074 µM respectively. Site directed mutagenesis experiments further revealed that basic residues within these binding sites may play a crucial role in TRPV1 and TRPM3 channels binding to PIP2 and to CaM respectively.

This project was supported by grants GACR 301/10/1159, GACR P205/10/P308 and Research project No. AV0Z50110509.