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1, 2 , 3 , 3 , 4 , 4, 5 , * 1, 5
1  Instituto de Fisiologia, Facultad de Medicina, Universidad Austral de Chile
2  Escuela de Graduados, Facultad de Ciencias, Universidad Austral de Chile
3  Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia.
4  Center for Bioinformatics and Molecular Simulations (CBSM) University of Talca, Talca
5  Millennium Nucleus of Ion Channels-associated Diseases (MiNICAD)


TRPV5 and TRPV6 are highly calcium-selective channels from the Transient Receptor Potential (TRP) family1. These channels are considered gatekeepers of epithelial calcium transport, essential for calcium homeostasis1. At negative potentials, the channels exhibit a two-phase calcium-dependent inactivation where the slow component is shared and determined by the binding of Ca2+-Calmodulin complex to the C-terminal region of the channel (2,3). In contrast, the rapid phase of inactivation depends on the binding of calcium ions and allows differentiating both channels from a functional point of view; while TRPV6 shows a very robust inactivation, at the same calcium concentrations, the inactivation of TRPV5 conductance is modest4. The intracellular loop S2-S35 and residues downstream the transmembrane segment S66 has been associated to the differences observed in the kinetics of the rapid phase of inactivation. However, the exact location of the putative calcium-binding site and the molecular mechanism governing this process are not known. A thorough phylogenetic reconstruction in vertebrates suggest that the genes encoding for these channels duplicates more than once during evolution, naturally introducing the same set of mutations within a HLH domain located at the N terminal region. Further sequence analysis unveiled that the HLH domain acts as a fingerprinting in both channels. Molecular dynamics simulations, allowed us to identify a putative calcium-binding site that put together three different portions of the folded channel. By means of site-directed mutagenesis and patch clamp electrophysiology we reversed the phenotype of inactivation in these channels, confirming that the HLH sequence serves as modulator for the calcium-induced inactivation process. We conclude that subtle evolutionary-related variations within the binding region affect the phenotype of the fast inactivation phase.

Keywords: TRPV5 ; TRPV6; gene family evolution ; fast inactivation