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.

Exercise is a great medicine to improve health and reduce the risk for cardiovascular diseases. However, high-volume high-intensity exercise training can acutely damage cardiac tissue, whereas prolonged exercise training may induce scar tissue. It is unknown how exercise may induced these deleterious effects, and these processes cannot be assessed in human and animal studies. Therefore, we aim to develop a unique exercise-on-a-chip model by exposing cultivated heart cells to simulated exercise conditions. This novel model would be a big step towards unravelling how extreme exercise may hurt the heart, and what can be done to avoid such side effects.

Kidney stones affect around 1 in 10 people worldwide. It can be treated, but recurrence rates are high (>30%) resulting in increased risk of chronic kidney disease. An improved, possibly preventive treatment is needed. Our research aims to develop a novel drug to prevent kidney stone formation, based on our body’s calcium regulation. Calcium, obtained from our diet and essential for i.a. bone, can precipitate in our kidneys. This may lead to crystal formation, and ultimately kidney stone development. We propose to stop this process and prevent stone formation by local calcium buffering in the kidney via an original peptide-drug.

Energy for Life: Coupling Magnesium and ATP regulation Cells require energy for growth and normal functioning. In the cell, energy is stored in the form of ATP molecules, which our bound to magnesium ions. When ATP production increases, the cell requires more magnesium. Recent results from my research team demonstrate that magnesium uptake in cells is facilitated by CNNM-TRPM7 magnesium channels. In the project, I will investigate how the activity of the channel is regulated by the energy needs of the cell. The importance of magnesium for the production and storage of energy will be elucidated.

The present project investigates the unique channel-kinase TRPM6 (Transient Receptor Potential Melastatin 6) that belongs to the melastatin-related subfamily of TRP ion channels. TRPM6 has been identified as the magnesium (Mg2+) entry pathway in the distal convoluted tubule (DCT) of the kidney, where it functions as gatekeeper for controlling the bodys Mg2+ balance. The tight control of blood Mg2+ levels (0.7-1.1 mmol/L) is of central importance for various physiological processes and a low Mg2+ status (hypomagnesemia) has been found to be involved in the pathogenesis of diabetes mellitus type 2, osteoporosis, asthma, and heart and vascular diseases. TRPM6 is, together with its homologue TRPM7, the only known protein that combines a channel domain with an alpha-kinase domain. Kinases are key players in numerous cellular processes. They act as enzymes to phosphorylate target proteins and subsequently modulate their function. Importantly, the function of this carboxyl-terminally fused alpha-kinase domain is still poorly understood. The following key objectives will, therefore, be addressed: I) Phosphorylation-dependent regulation of TRPM6: phosphomapping by mass spectrometry will reveal phosphorylated residues in TRPM6 and their functional implications will be analyzed by electrophysiological and fluorescence microscopy methods. II) Identification of renal substrates of TRPM6: detect new kidney-specific proteins by mass spectrometry and study their effect on TRPM6 channel activity, kinase function and renal Mg2+ handling. III) Development and characterization of a cytosolic fluorescent Mg2+ probe: a new technically advanced fluorescent indicator for measuring intracellular Mg2+ concentrations ([Mg2+]i) will boost in-depth understanding of TRPM6 regulation. Taken together, research on the regulation of TRPM6 is still in its infancy. By using a complementary array of novel tools and state-of-the-art techniques, this project will disclose the molecular regulation of TRPM6 specifically involving Mg2+-dependent regulation, identification of renal substrates of the remarkable alpha-kinase and the functional implications of channel autophosphorylation.