Abstract
Transient receptor potential (TRP) channels are a group of essential cation channels involved in many important sensory signal transduction processes, such as light, temperature, tastes and pressure sensing. Drosophila TRP channel is the first discovered family member and plays important roles in photo-transduction in Drosophila. Calmodulin (CaM), an important downstream effector of Ca2+ signal, was considered as a vital regulator of TRP activities. In this study, we discovered a novel Ca2+ dependent CaM binding site (TRP 783-862) in between the previously reported two calmodulin binding sites (CBSs). The isothermal titration calorimetry (ITC) and the size exclusion chromatography coupled with multi-angle static light scattering (SEC-MALS) results showed that the dissociation constant (Kd) between TRP 783-862 and Ca2+-CaM is 0.10 ± 0.04 μM and their binding stoichiometry is 1:1. In addition, the shortest Ca2+-CaM interaction region and core CaM binding sequences in TRP 783-862 were dissected by the boundary mapping and mutagenesis experiments. More interestingly, by comparing the circular dichroism (CD) spectra before and after Ca2+-CaM binding, the TRP 783-862 fragment showed Ca2+-CaM binding dependent secondary structure changes, indicating that the interaction between CaM and Drosophila TRP channel may have a conformational impact on TRP structure. In summary, by identifying and characterizing a novel CaM binding site in TRP C-terminus, our findings provided a biochemical and structural basis for further in vivo functional studies of Ca2+-mediated TRP channel regulation through CaM/TRP interaction.
