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.

Heart failure poses a large burden on patients and healthcare, largely because heart failure patients have low fitness and require frequent hospitalisation for close monitoring. In CardiacCare@Home, researchers work together with patients, doctors, industry, and others to develop technology for home-based monitoring of cardiac function and rehabilitation. This approach facilitates early detection of worsening of cardiac function, which allows doctors to rapidly alter treatment and prevent hospitalisation. Moreover, home-based rehabilitation will improve patients’ fitness levels. Technological innovations will facilitate a new care path that improves patients’ quality of life and lower socio-economic costs, and lower burden for hospital staff.

Het lopen van een marathon wordt steeds populairder. Naast de vele positieve gezondheidseffecten van duurinspanning, kan duurinspanning ook gepaard gaan met maagdarmklachten. Zo’n 30-90% van de hardlopers heeft last van maagdarmklachten tijdens of in de uren na het hardlopen. Het ontstaan van maagdarmklachten heeft waarschijnlijk te maken met de herverdeling van het bloedvolume, resulterend in minder bloedtoevoer naar het spijsverteringskanaal en een minder goed functionerende darmbarrière. Doordat de darmbarrière minder goed functioneert kunnen er ongewenste stoffen (endotoxinen) de bloedbaan intreden en voor ontstekingsreacties zorgen. De vele micro-organismen in onze darm, gezamenlijk onze darmmicrobiota genoemd, zijn van invloed op de voedselvertering, maar ook op het functioneren van de cellen die de darmwand bekleden en de verbindingen tussen deze cellen. Mogelijk hebben hardlopers met maagdarmklachten tijdens duurinspanning te maken met een afwijkende samenstelling van de darmmicrobiota en/of metabolieten ten opzichte van hardlopers zonder klachten, waardoor de darmbarrière minder goed functioneert en er problemen kunnen optreden. Vandaar dat het voornaamste doel van ons onderzoeksproject is om te onderzoeken of er een relatie bestaat tussen de samenstelling van de darmmicrobiota en/of metabolieten en het ontstaan van maagdarmklachten tijdens duurinspanning. De onderzoeksvragen die zullen worden bestudeerd zijn: 1) Verschilt de samenstelling van de darmmicrobiota en/of metabolieten van hardlopers die wel en niet last krijgen van maagdarmklachten tijdens het lopen van een marathon? En zo ja, hoe? 2) Kan de samenstelling van de darmmicrobiota en/of metabolieten van getrainde sporters die maagdarmklachten ervaren tijdens duurinspanning positief beïnvloed worden door probiotica-suppletie, zodat de kans op en/of intensiteit van maagdarmklachten tijdens duurinspanning wordt verminderd en de sportprestatie verbeterd? Het onderzoeksproject richt zich op de identificatie van sporters die last hebben van maagdarmklachten tijdens duurinspanning. We hopen met de beoogde resultaten bij te kunnen dragen aan op de persoon gerichte preventie van maagdarmklachten door het aanpassen van de darmmicrobiota.

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.

The impact of climate change on health related to heat stress (indoor and outdoor), water quality, and plant diversity is increasing. Blue (lakes, canals) and green infrastructure (trees, herbs) may significantly contribute to reduce heat stress and the warming up of built-up areas. BENIGN aims to investigate how blue and green infrastructure can be employed in urban areas to create healthy living conditions. To do so, 3 living labs in Dutch municipalities will be set up. A key outcome of BENIGN will be a decision support system for municipalities to guide them in creating healthier living conditions.