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.