Essential for calcium homeostasis, TRPV5 and TRPV6 are calcium-selective channels belonging to the transient receptor potential (TRP) gene family. In this study, we investigated the evolutionary history of these channels to add an evolutionary context to the already available physiological information. Phylogenetic analyses revealed that paralogs found in mammals, sauropsids, amphibians, and chondrichthyes, are the product of independent duplication events in the ancestor of each group. Within amniotes, we identified a traceable signature of three amino acids located at the amino-terminal intracellular region. The signature correlates with both the duplication events and the phenotype of fast inactivation observed in mammalian TRPV6 channels. Electrophysiological recordings and mutagenesis revealed that the signature sequence modulates the phenotype of fast inactivation in all clades of vertebrates but reptiles. A transcriptome analysis showed a change in tissue expression from gills, in marine vertebrates, to kidneys in terrestrial vertebrates. Our results highlight a cytoplasmatic structural triad composed by the Helix-Loop-Helix domain, the S2-S3 linker, and the TRP domain helix that is important on modulating the activity of calcium-selective TRPV channels. In vertebrates, epithelial calcium absorption is a crucial physiological process needed to maintain Ca 2+ homeostasis. Epithelia consist of a continuous layer of individual cells where calcium absorption is maintained by two routes, the transcellular and paracellular pathways 1,2. Tight junctions regulate the passive passage of ions and molecules through the paracellular pathway 2. Calcium permeation through this pathway constitutes a major route for Ca 2+ absorption under normal physiological conditions 1. On the other hand, the transcellular pathway implicates controlled Ca 2+ movement through epithelial barriers and occurs against a concentration gradient in normal conditions. It is an active and saturable process, maintained by an array of transporters, pumps, and ion channels 1. TRPV5 and TRPV6 are calcium-selective ion channel members of the Transient Receptor Potential (TRP) gene family 3. These proteins, expressed at the apical membrane of Ca 2+ transporting epithelia, serve as entry channels in transepithelial Ca 2+ transport 3. In agreement with their physiological role, TRPV6-EphB6 double knockout mice have impaired Ca 2+ homeostasis, evidenced by poor weight gain, decreased bone mineral density, and reduced fertility 4. However, more recent research using TRPV6 null mice suggests that the channel becomes relevant only when dietary calcium supply is low 5. On the other hand, TRPV5 null mice present less severe physiological consequences, nonetheless, the absence of TRPV5 channels causes hypercalciuria, compensatory hyperabsorption of dietary calcium, and abnormal bone thickness 6. At negative membrane potentials, calcium-selective

Important for calcium homeostasis, TRPV5 and TRPV6 are calcium-selective channels belonging to the transient receptor potential (TRP) gene family. In this study, we investigated the evolutionary history of these channels to add an evolutionary context to the already available physiological information. Phylogenetic analyses revealed that paralogs found in mammals, sauropsids, amphibians, and chondrichthyans, are the product of independent duplication events in the ancestor of each group. Within amniotes, we identified a traceable signature of three amino acids located at the amino-terminal intracellular region (HLH domain). The signature correlates well with both the duplication events and the phenotype of fast inactivation observed in mammalian TRPV6 channels. Electrophysiological recordings and mutagenesis suggest that calcium-induced fast inactivation represents an evolutionary innovation that emerged independently after gene duplication.

TRPV5 and TRPV6 are calcium-selective ion channels expressed at the apical membrane of epithelial cells. Important for systemic calcium (Ca2+) homeostasis, these channels are considered gatekeepers of this cation transcellular transport. Intracellular Ca2+ exerts a negative control over the activity of these channels by promoting inactivation. TRPV5 and TRPV6 inactivation has been divided into fast and slow phases based on their kinetics. While slow inactivation is common to both channels, fast inactivation is characteristic of TRPV6. It has been proposed that the fast phase depends on Ca2+ binding and that the slow phase depends on the binding of the Ca2+/Calmodulin complex to the internal gate of the channels. Here, by means of structural analyses, site-directed mutagenesis, electrophysiology, and molecular dynamic simulations, we identified a specific set of amino acids and interactions that determine the inactivation kinetics of mammalian TRPV5 and TRPV6 channels. We propose that the association between the intracellular helix-loop-helix (HLH) domain and the TRP domain helix (TDh) favors the faster inactivation kinetics observed in mammalian TRPV6 channels.