Current Research Topics:

1. Linkage of TRPC (canonical transient receptor potential channels) to Ca²⁺-transcription coupling

Michael Poteser

TRPC channels have been identified as upstream components of Ca²⁺/calcineurin/nuclear factor of activated T cells (NFAT)-signalling and as important players in immune cell function and pathology of cardiac remodeling. The linkage between Ca²⁺ signalling processes and NFAT activity is still inclompletely understood. Calcineurin has been identified as an important regulator of TRPC channel function, mediated by its intrinsic phosphatase activity. Immunophilins, physically linking TRPC channels and calcineurin in many cellular systems, do not only provide a common scaffold for TRP channels and calcineurin, but exert in addition chaperone-like functions upon the TRPC ion channels. Thus, immunophilins, calcineurin and TRPC channels appear to form a tightly regulated signalplex, allowing for local calcium signals dedicated to NFAT triggered transcription of genes. We are investigating the relations between signalplex location, assembly and downstream signling by FRET- and TIRF- microscopy in combination with electrophysiology and fura2-calcium measurements.

2. Linkage of TRPC (canonical transient receptor potential channels) to cardiac excitability, electrical- and hypertrophic remodeling.

Bernhard Doleschal, Dieter Platzer

TRPC3 was recently suggested as a player in the development of cardiac hypertrophy. Little is known about the direct pro-arrhythmogenic role for TRPC3.  We investigate the involvement of TRPC3 in cardiac actions of pathophysiologically relevant mediators and activators, like angiotensin II, for GPCR/Gq/TRPC3 signalling, using a TRPC3 transgenic mouse model. We use hearts and heart-cells of mature and neonatal transgenic and normal mice and apply methods including fura2-Ca²⁺ imaging, isolated Langendorff perfused heart experiments, measurement of Ca²⁺-transients, measurement of sarcomere shortening and patch-clamp experiments.

3. Multimodal regulation of TRPC channel complexes by mechanical stimuli, membrane lipid metabolims and redox processes

Michaela Lichtenegger

The mammalian TRPC3 protein is a non-selective cation channel that is activated via G-protein coupled receptors and PLC-metabolism. While the important role of TRPC3 in physiological functions is well recognized, only little information is available on the molecular architecture, structure-function relations of the TRPC permeation pathway and its linkage to cellular functions. Based on a computational homology model of the pore structure, we set out to explore the permeation pathway. We aim to identify and characterize important structural elements of the TRPC3 channel, like selectivity filter, gate and lipid-binding (=activation) sites. Our computational model is tested by patch-clamp experiments in combination with site directed mutagenesis and by SCAM (substituted cysteine accessibility method)

The projects I develop are sponsored by a Marie Curie International Incoming Fellowship, which I obtained in collaboration with Professor Groschner; they are part of an ongoing effort to elucidate the fundamental mechanisms underpinning normal function in vascular smooth muscle. In particular, we will focus on the calcium signals (and their possible relation to intracellular sodium) which regulate healthy cell function.
At the core of the projects is the development of a quantitative model of two critical calcium (Ca2+) signalling steps hypothesized to be at the basis of proper physiological function of vascular smooth muscle: The generation of localized sodium (Na+) transients via transient receptor potential canonical (TRPC) channels promoting reversal of Na+/Ca2+ exchangers (NCX) for extra-cellular Ca2+ ingress and the capture of Ca2+ entering through NCX by sarco/endoplasmic reticulum ATPases (SERCA).  These are some of the main signalling pillars sustaining Ca2+ wave-like oscillations observed during physiological activation of vascular smooth muscle and understanding their mechanism will enable us to address key questions of vascular dysfunction and disease.
Experimental evidence suggests that both steps are assisted by plasma membrane (PM)-sarcoplasmic reticulum (SR) nanojunctions, regions of the cell where the PM and the SR are separated by about 20-nm for an extension of several hundred nm. Quantitative modeling is currently the only way to enhance our understanding of transport processes at this scale as the required resolution is <5 nm, which cannot be satisfactorily reached by available instrumentation. Preliminary stochastic models show a quantitative order-of-magnitude fit between the required Ca2+ flux to maintain contraction and that calculated by simulating Ca2+ transport across PM-SR junctions, from NCX to SERCA. Models of the local Na+ transients measured in cultured vascular smooth muscle cells, and presumed to be the precursors to NCX reversal in native tissue as well, led to the introduction of several assumptions, which will be investigated in the proposed studies.
Combining my expertise in quantitative modeling of Ca2+ processes in vascular smooth muscle with the knowledge and understanding of TRP(C)–NCX communication available within the Institut provides an advantageous situation to improve our comprehension of the above mentioned mechanisms as well as their link to cardiovascular disease.