Patch-clamp and Optogenetics
Dr. Ulrich Terpitz
|+49 931 31-84226|
Patch-clamp and Optogenetics
Membrane transport proteins allow rather membrane-impermeable molecules and ions for crossing membranes and entering/leaving cells and organelles. In order to understand the dynamics, interaction, and gating of electrogenic membrane transport proteins, we combine super-resolution microscopy and electrophysiology (Patch-clamp technique). We focus on light-gated membrane proteins, especially rhodopsins. Furthermore we use optogenetical tools to investigate physiological downstream processes upon light-trigger.
We are especially interested in light-triggered microbial rhodopsins, with focus on fungal rhodopsins (Fig. 1). Although these green-light sensing proteins are widespread in the fungal kingdom, little is known about their physiological function and distribution in the hyphae. Microbial rhodopsins consist of seven transmembrane helices (TM) forming an interior pocket for the chromophore, the all-transretinal, which is covalently bound via a protonated Schiff’base to a lysine located in the TM7. In light-driven ion pumps, a single ion is translocated per photocycle that is triggered by light-induced retinal isomerization and characterized by a sequence of photointermediates responsible for ion-uptake, -transfer, -release, and chromophore-recycling. Fungal rhodopsins are eukaryotic proteins thus they are promising candidates for new optical switches in optogenetical applications.
DFG project Interdisziplinäre Analyse pilzlicher Rhodopsine und ihrer physiologischen Funktion in Myzelien
Fig. 1: Electrophysiological Measurements of the fungal rhodopsin CarO from the filamentous fungus Fusarium fujikuroi expressed in HEK293 cells. a. Typical current trace of the ion pump recorded in 140 mM NaCl pH 7.4 at membrane potentials of 0 mV. Cell was illuminated by a 561 nm diode pumped laser. The green bar represents illumination time. b. Influence of external pH on pump-activity. The relative pump activity is shown, which was normalized to the pump current at pH 7.4 and 0 mV. pH of bath solution varied from pH 5 (black squares, 10 cells), to pH 7.4 (red circles, 25 cells) and pH 9 (blue triangles, 13 cells); intracellular pH was kept at 7.4.
Fluorescence Microscopy in Fungi
Filamentous fungi exhibit tiny tubular cells often not extending diameters of 3-5 µm. Thus using conventional fluorescence microscopy it is often challenging if not impossible to resolve subcellular information in fungi. In collaboration with Prof. J. Avalos in Sevilla (Spain), we aim in unravelling the subcellular location of rhodopsins as well as enzymes involved in the carotenoid synthesis applying dSTORM technology.
Fig. 2. Fluorescence micrograph showing the distribution ot the rhodopsin CarO::YFP in hyphae of Fusarium fujikuroi
Ca2+ is a key signal in cell regulation, modulating the activity of a plenitude of sensitive proteins. Prerequisite for accurate signalling is the exact buffering of Ca2+ concentrations within the cell. The steep Ca2+ gradients between the cytosol (100 nM), endosomes (4-40 µM), lysosomes (~500 µM), and the extracellular lumen (~1 mM) allow for the generation of fast, spatially, and temporally modulated Ca2+ signals.
We developed an optochemokine tandem to control the release of Ca2+ from endosomes into the cytosol by light and to analyse the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology (Fig. 2). The light-gated Ca2+-permeable cation channel ChR2(L132C), CatCh was combined with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signalling pathway. The kinetics of internalization was followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca2+ by tandem endosomes into the cytosol via CatCh was visualized using the Ca2+- sensitive dye rhod2(AM) showing an increase of intracellular Ca2+ in response to light.
Fig. 3. The optochemokine tandem tCXCR4/CatCh. a. The CXCR4-protein was placed at the N-terminus, followed C-terminally by eYFP and the ß-subunit of the rat H+/K+-ATPase, to combine the intracellular C-terminus of eYFP with the extracellular N-terminus of ChR2 (aa 1-309). b. CLSM micrographs of NG108-15 expressing the tCXCR4/CatCh(D156C) (cyan) for visualisation of intracellular, light-induced Ca2+-signalling . White arrows highlight such endosomes that express the respective membrane protein and are loaded with rhod2. All rhod2-experiments were performed in absence of extracellular Ca2+. Scale bars represent 10 µm
Application of Patch-clamp techniques is often limited when membrane proteins are expressed in a low level. Therefore, we enlarge yeast protoplasts by multi-cell electrofusion (MCE). Within a Patch-clamp experiment a slight extension of the cell diameter is accompanied by a marked enhancement of the electrically accessible membrane area. At the same time also the number of electrogenic membrane proteins increases thus leading to strong signal amplification (Fig. 3).
Fig. 4: Patch-clamp-experiments with baker yeast protoplasts expressing Channelrhodopsin-2 from Chlamydomonas rheinhardtii (figure modified from Terpitz et al., 2008).
Feldbauer, K., Schlegel, J., Weissbecker, J., Sauer, F., Wood, P.G., Bamberg, E., Terpitz, U., Optochemokine Tandem for Light-Control of Intracellular Ca2+. PLoS ONE 11(10): e0165344 (2016)
García-Martínez, J., Brunk, M., Avalos, J., and Terpitz, U., The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination. Sci. Rep. 5, 7798 (2015)
Terpitz, U., Letschert, S., Bonda, U., Spahn, C., Guan, C., Sauer, M., Zimmermann, U., Bamberg, E., Zimmermann, D., and Sukhorukov, V.L., Dielectric analysis and multi-cell electrofusion of the yeast Pichia pastoris for electrophysiological studies. J. Membr. Biol. 245, 815-826 (2012)
Kleinlogel, S., Terpitz, U., Legrum, B., Gökbuget, D., Boyden, E. S., Bamann, C., Wood, P. G., and Bamberg, E., A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins. Nature Meth. 8, 1083-1088 (2011)