Research Interests

Cation channels regulate the influx of calcium ions (Ca2+) as messengers that determine the functionality of individual cells and, subsequently, complex tasks and processes in whole organs as wells as systemic body functions. Our main interest is currently directed at TRP channels and voltage gated Ca2+ channels (VDDC) as well as structurally and functionally related membrane proteins. In the past we have contributed to the identifica-tion of constituents of these channels by elucidation of their biological relevance by generation and analysis of mouse models with targeted mutations (gene deletion, Knock in of pore mutation, Knock-Add-On of fluorescent proteins), e.g. for processes in the cardiovascular system including its development and its regulation by cells of the nervous system, for release of inflammatory mediators from mast cells and for male fertility.

 

TRP channels: polymodal sensors in numerous cell signaling processes

The mammalian 28 TRP proteins are classified according to structural homology into 6 subfamilies: TRPC (canonical, 7 members), TRPV (vanilloid, 6 members), TRPM (melastatin, 8 members), TRPA (ankyrin, 1 member), TRPML (mucolipin, 3 members), and TRPP (polycystin, 3 members). All TRP channels are assumed to have six-transmembrane (6TM) polypeptide subunits that assemble as tetramers to channel com-plexes that mediate the transmembrane flux of cations down their electrochemical gradi-ents, thereby raising intracellular Ca2+ and Na+ concentrations, respectively, and depolarizing the cell. TRP proteins form cation channels that are regulated through strikingly diverse mechanisms including mechanostimulation, changes in temperature, in pH and osmolarity and in cytosolic free Ca2+ concentration ([Ca2+]i) and they are molecular players in signal transduction cascades downstream of metabotropic receptors which makes them polymodal sensors for fine tuning of many cellular and systemic processes in the body (e.g. (Freichel et al., 2012). As such TRP channels are involved in manifold physiological functions, ranging from pure sensory functions, such as pheromone signaling, taste transduction, nociception, neurotransmitter release and temperature sensation, over homeostatic functions, such as Ca2+ and Mg2+ reabsorption and osmoregulation, to many other motile functions, such as muscle contraction, vasomotor control and flagellar beating in sperm.  Thus, it is not a surprise that more than 14 hereditary human diseases in areas as diverse as neurology, cardiology, pulmonology, nephrology, dermatology, urology and other genetic disorders are caused by defects in TRP channels.

Although the search for natural ligands and chemical modulators of TRP channels as therapeutics has been intensified in the last years, specific agonists or blockers are still lacking for the vast majority of TRP channels until now.

 

Information processing by TRP channels in non-exitable cells is defined by their ion per-meability profile

We showed that individual members of the TRPC subfamily  proteins are constituents of Ca2+ permeable channels and, as such,   directly contribute to Ca2+ entry across the plas-ma membrane. They are activated by agonists of G protein-coupled receptors activating phospolipase C (PLC) such as acetylcholine and thrombin in endothelial cells regulating endothelial-dependent vasorelaxation and endothelial permeability (Freichel et al., 2001; Tiruppathi et al., 2002). A role for TRPC-mediated Ca2+-entry could be demonstrated in platetets and cardiomyocytes that is associated with platelet aggregation and myocyte hy-pertrophy (ms in preparation). The focus of our future experiments is to address their con-tribution to Ca2+ signaling in cardiac fibroblast and subsequent release of hypertrophic factors that stimulate cardiomyocyte growth in a paracrine fashion (see section A) „Outlook and future aims).

TRPM4 (and TRPM5), two members of the TRPM subfamily, have distinct properties with-in the TRP class of ion channels: we showed that TRPM4 proteins form cation channels in mast cells stimulated by antigens, that do not conduct Ca2+ but Na+ under physiological conditions and are activated by elevation of cytoplasmic Ca2+ levels. The depolarization evoked by TRPM4 channels reduces the driving for Ca2+ entry carried by Ca2+-conducting channels such as the inwardly rectifying CRAC channels that are concommentantly acti-vated by IP3-mediated release of intracellular Ca2+ stores following anitgen-induced phospolipase C activation. Lack of TRPM4 in mast cells on the other hand leads to an in-crease in antigen-evoked Ca2+-rise, release of inflammatory mediators and anaphylaxis  (Vennekens et al., 2007).

Thus, by conducting Ca2+ ions to various degrees most TRP channels directly contribute to Ca2+ influx via the plasma membrane, but individual TRP channels - by conducting Na+ - mediate electrogenic effects through plasma membrane depolarization which can have opposite consequence in non-excitable cells.

 

TRP channels convert chemical to electrical signalings by regulating voltage gated chan-nels

Based on above properties it was proposed that TRP channels play also an important role in electrogenesis in electrically excitable cells to fine tune the excitability of voltage gated channels.

In ileal smooth muscle cells TRPC4 and TRPC6 are essential components of cation channels gated by muscarinic receptors which underlie a current referred to as mICAT. In TRPC4-deficient cells this current and carbachol-induced membrane depolarizations are greatly diminished. Consequently, atropine sensitive neurogenic contraction elicited by electrical field stimulation, which critically depends on activation of voltage gated L-type Ca2+ channels, is largely reduced demonstrating a decisive role for TRPC4 and TRPC6 channels in mediating the signaling cascade evoked by muscarinic receptors to activation of voltage-activated Ca2+-influx and smooth muscle contraction (Tsvilovskyy et al., 2009).

In neurons of the lateral septum, a similar mechanism was found (collaborative effort with Zhang Feng, University of Arkansas, USA). Here, metabotropic glutamate receptor ago-nists evoke large depolarizing plateau potentials that underlie epileptiform burst firing which was completely abolished in neurons lacking TRPC1 and TRPC4. Furthermore, neuronal cell death in the lateral septum and the CA1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in these mice demon-strating a role of TRPC1/TRPC4 proteins in neuronal excitotoxicity (Phelan et al., 2012).

TRPC1 and TRPC4 were also identified in neurons of the granule cell layer of the mouse olfactory bulb where they contribute at reciprocal dendrodendritic synapses to slow synaptic transmission including the calcium dynamics required for asynchronous release (collaboration with Dr. Veronica Egger, LMU Munich). However, the mode of activation is somewhat different in these cells as TRPC1 and TRPC4 are activated downstream of NMDA receptor activation (Stroh et al., 2012).

 

Cation channel functions in multicellular networks and compartments

 

Epididymal TRPV6 channels regulate functionality of neighboring sperm

TRPV6 (and its closest structural homologue TRPV5) form channels that are highly Ca2+-selective in heterologous expression systems, which gives them a unique property within the TRP channel family. We generated mouse models (i) carrying a single point mutation (Trpv6D541A) in the channel pore or (ii) with deletion of the TRPV6 C-terminus including the channel pore to specifically block TRPV6-mediated Ca2+ permeation. We found that both mutations led to a marked reduction of the fertility of male, but not female mice. The motility, the fertilization capacity and the viability of sperms from the cauda epididymis were drastically reduced. Surprisingly, we found TRPV6 protein expression in the apical membranes of epididymal epithelial cells but not in sperm or germinal epithelium. Further analysis revealed abnormally high Ca2+ concentrations in the intraluminal fluid of the cauda epididymis of Trpv6D541A mice and a drastically reduced Ca2+ uptake from the in-traluminal fluid by epididymal epithelial cells. These experiments show that a decrease in luminal Ca2+ concentration along the epididymal segments is required for the posttesticular gain of functional competence of sperm and that TRPV6 proteins, as indispensable constituents of the underlying Ca2+-uptake channels in the epididymal epithelium, regulate the function of vicinal spermatozoa (Weissgerber et al., 2012; Weissgerber et al., 2011).

 

 

Ca2+ channel CaVβ2 subunits in cardiomyocytes determine vascular plexus formation dur-ing embryonic development

CaVβ2 represent one of four auxiliary subunits of voltage gated Ca2+ channels that affect trafficking and assembly of the calcium channel heteromultimer, and CaVβ2 is the most abundant CaVβ in the murine heart. Mice with ubiquitous inactivation of the CaVβ2 gene die during embryonic development due to impaired heart formation with abnormalities in the progression of looping architecture. We found diminished L-type Ca2+ currents in embryonic cardiomyocytes leading to a functionally compromised heart function but also to defective remodeling of intra- and extraembryonic blood vessels. The defects in vascular remodeling were also observed when the CaVβ2 gene was selectively targeted in cardiomyocytes demonstrating that they are secondary to cardiac failure rather than due to the lack of CaVβ2 proteins in the vasculature. These data show that CaVβ2 proteins are essential for L-type Ca2+ channel function in cardiomyocytes and thereby determines proper maturation of the vasculature (Weissgerber et al., 2006).

 

 

TRP channels in sympathetic neurons determine arterial pressure by regulation of neuro-transmitter release

TRPM4 proteins are expressed in cells of the cardiovascular system including heart, endothelial cells, kidney and in arterial smooth muscle cells. We found that inactivation of TRPM4 channels leads to increased blood pressure without evidence for impairment of endothelium- or smooth muscle-dependent regulation of vascular tone, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. In contrast, TRPM4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines associated with elevated levels of epinephrine in the plasma and its metabolites in the urine. We therefore conclude that TRPM4 limits catecholamine release from chromaffin cells and that lack of this inhibitory action contributes to increased sympathetic and vascular tone and, eventually, hypertension (Mathar et al., 2010). Whether catecholamine release is also regulated by TRPM4 in other neurons of the sympathetic nervous system still needs to be clarified .

 

 

Outlook

We plan to extend our current analyses to gain a better understanding of the composition and characteristics of TRP channel complexes as signaling molecules regulating activation of primary cell types. In the long run, this knowledge will be the basis for our aim to identify and validate compounds that can regulate these channels with sufficient specifity and potency and may serve for pharmacotherapy. 

 

 

 

References

Freichel, M., Almering, J., and Tsvilovskyy, V. (2012). The Role of TRP Proteins in Mast Cells. Frontiers in immunology 3, 150.

Freichel, M., Suh, S.H., Pfeifer, A., Schweig, U., Trost, C., Weissgerber, P., Biel, M., Philipp, S., Freise, D., Droogmans, G., et al. (2001). Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice. Nat Cell Biol 3, 121-127.

Phelan, K.D., Mock, M.M., Kretz, O., Shwe, U.T., Kozhemyakin, M., Greenfield, L.J., Dietrich, A., Birnbaumer, L., Freichel, M., Flockerzi, V., et al. (2012). Heteromeric canonical transient receptor potential 1 and 4 channels play a critical role in epileptiform burst firing and seizure-induced neurodegeneration. Mol Pharmacol 81, 384-392.

Stroh, O., Freichel, M., Kretz, O., Birnbaumer, L., Hartmann, J., and Egger, V. (2012). NMDA receptor-dependent synaptic activation of TRPC channels in olfactory bulb granule cells. J Neurosci 32, 5737-5746.

Tiruppathi, C., Freichel, M., Vogel, S.M., Paria, B.C., Mehta, D., Flockerzi, V., and Malik, A.B. (2002). Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability. CircRes 91, 70-76.

Tsvilovskyy, V.V., Zholos, A.V., Aberle, T., Philipp, S.E., Dietrich, A., Zhu, M.X., Birnbaumer, L., Freichel, M., and Flockerzi, V. (2009). Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo. Gastroenterology 137, 1415-1424.

Vennekens, R., Olausson, J., Meissner, M., Bloch, W., Mathar, I., Philipp, S.E., Schmitz, F., Weissgerber, P., Nilius, B., Flockerzi, V., et al. (2007). Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4. Nat Immunol 8, 312-320.

Weissgerber, P., Held, B., Bloch, W., Kaestner, L., Chien, K.R., Fleischmann, B.K., Lipp, P., Flockerzi, V., and Freichel, M. (2006). Reduced cardiac L-type Ca2+ current in Ca(V)beta2-/- embryos impairs cardiac development and contraction with secondary defects in vascular maturation. Circ Res 99, 749-757.

Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Mannebach, S., Wissenbach, U., Vennekens, R., Middendorff, R., et al. (2012). Excision of Trpv6 Gene Leads to Severe Defects in Epididymal Ca2+ Absorption and Male Fertility Much Like Single D541A Pore Mutation. J Biol Chem 287, 17930-17941.

Weissgerber, P., Kriebs, U., Tsvilovskyy, V., Olausson, J., Kretz, O., Stoerger, C., Vennekens, R., Wissenbach, U., Middendorff, R., Flockerzi, V., et al. (2011). Male fertility depends on Ca(2)+ absorption by TRPV6 in epididymal epithelia. Sci Signal 4, ra27.