Localization of calmodulin and PIP2 binding sites on the C-termini of the transient receptor potential cation channel TRPM5

J. Bily, H. Janouskova, J. Teisinger

Institute of Physiology, Academy of Science of the Czech Republic

Transient receptor potential (TRP) channels are involved in the perception of a wide range of physical and chemical stimuli, including temperature, pain, taste, light, osmolarity changes and pheromones. Recent studies have indicated that members of the TRP family of ion channels can function as calcium influx channels both in excitable and non-excitable tissues. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. On the basis of structural information the TRP family is subdivided in three main subfamilies: the TRPC (canonical) group, the TRPV (vanilloid) group and the TRPM (melastatin) group.

TRPM5 channel is member of the TRPM ion channel family. Members of TRPM family are divided into 4 groups - TRPM1+3, TRPM2+8, TRPM4+5, TRPM6+7. TRPM4+5 are closely related cation channels that are ubiquitously expressed. TRPM5 is found mainly in the intestine, taste buds, pancreas, stomach, lung, testis and brain. Channels TRPM4+5 are impermeable to Ca2+, but they however play roles in Ca2⁺ modulation. TRPM4 channel is activated by Ca2⁺ is often via complex signaling cascades including Ca2⁺-Calmodulin binding. Characterization of binding site has not been determined in case of TRPM5. The series of different length of highly purified fusion proteins of C-terminus of TRPM5 was performed to get sufficient amount of soluble proteins. To study the location of Calmodulin binding site on the C-terminal segments of TRPM5 was used the fluorescence spectroscopy measurements. Similarly was identified PIP₂ binding site in C-termini of TRPM5 using side directed mutagenesis. It could be deduced that both sites (CAM and PIP₂) were overlapped in C-terminus of TRPM5. This project can help us understand the regulation of TRPM5 ion channel.

Supported by GA CR 301/10/1159 and GAAV IAA600110701 and Centre of Neurosciences No. LC554.