TRP channels with diverse permeability profiles: regulation of blood pressure and fertility

Published on February 4, 2014   35 min

Other Talks in the Series: Calcium Signaling I

0:00
I am professor Marc Friechel. I'm working at the Pharmacological Institute, and I'm heading the Department of General Pharmacology. Welcome to my talk with the title, TRP Channels with Diverse Permeability Profiles: Regulation of Blood Pressure and Fertility.
0:20
The TRP class of ion channels consists of 6 sub-families, which cover altogether 28 members, at least in mice. Each TRP protein consists of six putative transmembrane domains, and it is generally believed that four of such TRP proteins together form hetero or homomultimeric channel complexes.
0:44
Most TRP channels are non selective with a permeability for calcium of a sodium below 10. Therefore TRP channels are not only important for calcium entry across the plasma membrane, but play also an important role in electrogenesis, regulating the driving force for calcium entry via other calcium permeable channels. This table gives you an overview about the permeability of channels formed by individual TRP proteins of the TRPC, TRPV, and TRPM sub-family. It should be emphasized that most of this information is based on studies of heterologously expressed channel proteins. And that characteristics of those channels may differ significantly from native channel complexes that exist in primary cells, since the ectopically expressed channel proteins do not necessarily act in accordance with the native cellular context as in primary sets. Within the TRP family of cation channels the highlighted channel proteins TRPV5 and TRPV6, as well as TRPM4 and TRPM5, have distinct characteristic features. TRPV6 proteins for instance, are able to form channels that conduct calcium ions with the selectivity of calcium over sodium of about more than 100. Whereas TRPM4 protein similar like TRPM5 from cation channels that selectively conduct monovalent cations.
2:15
In my talk, I will show you a recent results regarding the function of these two proteins-- TRPM4 and TRPV6-- that we have obtained using corresponding TRP deficient mouse models. I will highlight our findings about TRPM4-- which we found to regulate blood pressure-- and the role of TRPV6 for regulation of fertility in mice.
2:38
The initial cell types in which we characterize the function of TRPM4 were bone marrow derived mast cells. You can see in this northern blot that TRPM4 transcripts are abundantly expressed in these mast cells. In the upper right part, you can see a face contrast image of these cells that can be cultured from bone marrow in the presence of interleukin 3 and stem cell factor. FACs analysis with mast cell specific antibodies against FC-epsilon receptor and C-Kit reveal that more than 99% of this cell population does express these mast cell markers.
3:16
Using electrophysiological recordings that were performed by Rudi Vennekens, who worked with me in that study, were used to characterize TRPM4 mediated currents. Here you see wholesale measurements with 10 micro molar calcium in a pipette solution. And in the upper part you see a characteristic time course of the developing currents. Inward but not outward currents are blocked by the large cation, NMDG, and in the lower left part, you see a typical current voltage relationship derived from these recordings. Experiments in which the composition of the recording solutions were systematically substituted, revealed that a relative permeability sequence of sodium over cesium, over lithium, whereas, the channel was found to be virtually impermeable to the monovalent NMDG, as well as for the divalent calcium ions. In mast cells from TRPM4 deficient cells as shown on the right side of that slide, it's current is almost completely lacking.
4:19
To activate the mast cells with a physiological stimulation, the cells were incubated overnight with IGE molecules directed against DNP. On the next day, they were then challenged with the multivalent antigen, DNP, which was coupled to serum albumin. Resting calcium was not significantly different between WT and TRPM4 efficient mast cells over several preps. But both the peak and the plateau of calcium increase that is observed following stimulation of the FC-epsilon receptor is largely increased in TRPM4-deficient mast cells.
4:55
So how can this increased calcium entry in TRPM4 deficient mast cells be explained? We could show that following stimulation of the FC-epsilon receptors and subsequent activation of the phospholipase C signaling pathway, the calcium entry through cracked channels is largely inhibited by TRPM4 channels. TRPM4 channels form calcium activated cation channels that carry sodium ions on the physiological conditions leading to depolarization of the cell membrane. And as you can see in these membrane potential measurements below, you find that in the absence of TRPM4, this repolarization-- which follows the hyperpolarization induced by FC-epsilon receptor stimulation-- is either completely absent as shown in the left cell, or the cell oscillates around a more negative membrane potential. In average, the membrane potential is shifted by about 25 millivolts towards hyperpolarized membrane potentials.
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TRP channels with diverse permeability profiles: regulation of blood pressure and fertility

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