The transient receptor potential (TRP) superfamily is a large class of ion channels that are widely expressed and involved in a myriad of biological processes. This project focuses on the TRP vanilloid 5 and 6 (TRPV5 & TRPV6) channels, which are responsible for calcium (Ca2+) transport in epithelial cells of the kidney and intestine. They form a distinctive category within the TRP family based on their high selectivity for Ca2+ ions together with a Ca2+-dependent inactivation mechanism that is regulated by calmodulin (CaM). Despite the long-established role of CaM with its two Ca2+-binding lobes independently influencing voltage-gated ion channels (known as ‘calmodulation’), there is no consensus on such TRP channel regulation. Functional consequences of bilobal CaM binding are not understood. My group elucidated the 3D structure of TRPV5 in complex with CaM (PNAS 2019), offering intriguing and unique insights to unravel CaM regulation at single molecule level. Aim and Approach: This project aims to integrate the novel structural data with functional analyses to provide in-depth insights into the intermolecular control of TRPV5/TRPV6 channels, by addressing the following objectives: 1) Ca2+-dependence of CaM binding to TRPV5/6 – Investigate bilobal CaM regulation of channel function by fluorescence-lifetime imaging (FLIM)-based FRET (fluorescence resonance energy transfer) measurements, Fura-2 Ca2+ imaging, and electrophysiology. 2) Stoichiometry of the channel-CaM interaction – Study CaM binding composition through single molecule photobleaching by total internal reflection fluorescence microscopy (TIRF) and bio-layer interferometry (BLI). 3) Auxiliary role of CaM in channel trafficking – Delineate consequences of TRPV5/6-CaM binding for cellular trafficking by using fluorescent timer proteins, as well as functional and biochemical assays. Impact: This project will yield fundamental breakthroughs in the structure-function relationship of the Ca2+-selective TRPV5/6 channels, and challenge current definitions of CaM regulation. We aim to establish a calmodulation model for TRPV5/6 channels that answers yet unresolved questions on Ca2+-dependent regulation and binding kinetics. These insights together with the established techniques can ultimately be extrapolated to the complete TRP channel field to advance studies on channel diversity and their (patho)physiology in humans.