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Principal Investigators

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P10  Klaus Fendler
Professor

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Max Planck Institute of Biophysics
Max-von-Laue-Str. 3
60438 Frankfurt am Main, Germany

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P10  Ernst Bamberg
Professor and Director

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Max Planck Institute of Biophysics
Max-von-Laue-Str. 3
60438 Frankfurt am Main, Germany

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P10 Light gated ion channels and secondary active transporters: mechanism and application

The proteins of interest are from the domains of ion channels and transporters. Using electrophysiological and spectroscopic methods the transport properties of these membrane proteins are investigated. More specifically, the light-gated ion channel Channelrhodopsin and secondary active transporters of the cation/proton antiporter (CPA) and the major facilitator superfamilies (MFS) are studied. The applied methods allow the elucidation of their transport and gating mechanism as well as the development of cell biological applications.

The molecular mechanism of Channelrhodopsin-2 (ChR2) is investigated in detail by electrophysiological and spectroscopic methods and this approach is complemented by structural analysis. Because the field of optogenetics has made an enormous progress, an urgent need exists for improved tools especially in basic neuroscience and for biomedical applications. Based on recent results, a light-gated channel with a higher Ca++ selectivity and a ChR tandem cassette, we will prepare constructs with drastically increased light sensitivity and with a higher selectivity for K+ or Na+ ions for biomedical applications such as recovery of vision. The localized expression of ChR2 in mitochondria for light dependent uncoupling experiments will be continued.

To cover different aspects of the mechanism of secondary active transport, antiporters (Na+/H+ exchangers) as well as symporters (cation/sugar cotransporters) are studied. We investigate the dynamics of these transporters using electrophysiological techniques allowing identification of intermediates and isolation of partial reactions in the transport cycle. This part of the project aims at a molecular description of the antiport/cotransport process with special emphasis on the dynamics of the involved conformational transitions. Furthermore, we will use our expertise in Na+/H+ exchangers to study a eukaryotic member of the CPA2 subfamily, hsNHA1/2. This transporter implicated in essential hypertension is still poorly characterized.

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Fig. 1: Fast firing of hippocampal cells induced by the Ca++ permeable ChR2 variant CatCh(L132C) (A,C)). B) shows the Ca++ induced change of the surface potential on the inner side of hippocampal neurons and indicates the interaction with the Ca++ activated BK channels. D) Time to peak in absence and presence of Ca++

Fig. 2: Kinetic models for the Na+/H+ exchanger NhaA and the lactose permease LacY from E. coli. Identification of individual partial reactions, their electrogenicity and rate constants. NhaA: The conformational transitions are associated with a displacement of the two negatively charged aspartate residues (D163, D164) and one Na+ or two H+ ions, respectively. In the case of Na+ translocation this leads to a net charge displacement as indicated by red arrows. LacY: A rapid and weakly electrogenic reaction (green, 6%) follows sugar binding. H+ release is slow and strongly electrogenic (red, 94%).

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Publications

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Gushchin I, Shevchenko V, Polovinkin V, Kovalev K, Alekseev A, Round E, Borshchevskiy V, Balandin T, Popov A, Gensch T, Fahlke C, Bamann C, Willbold D, Büldt G, Bamberg E, Gordeliy V (2015) Crystal structure of a light-driven sodium pump. Nat Struct Mol Biol 22, 390-5. 


Mace E, Caplette R, Marre O, Sengupta A, Chaffiol A, Barbe P, Desrosiers M, Bamberg E, Sahel JA, Picaud S, Duebel J, Dalkara D (2015) Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice. Mol Ther 23, 7-16.


Müller M, Bamann C, Bamberg E, Kühlbrandt W (2015) Light-induced helix movements in channelrhodopsin-2. J Mol Biol 427, 341-9.


Lorenz-Fonfria VA, Bamann C, Resler T, Schlesinger R, Bamberg E, Heberle J (2015a) Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance. Proc Natl Acad Sci USA 112, E5796-804.


Calinescu O, Danner E, Bohm M, Hunte C, Fendler K (2014a) Species differences in bacterial NhaA Na+/H+ exchangers. FEBS Lett 588, 3111-6 .


Calinescu O, Paulino C, Kuhlbrandt W, Fendler K (2014b) Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers. J Biol Chem 289, 13168-76. 


Lorenz-Fonfria VA, Resler T, Krause N, Nack M, Gossing M, Fischer von Mollard G, Bamann C, Bamberg E, Schlesinger R, Heberle J (2013) Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating. Proc Natl Acad Sci USA 110, E1273-81. 


Sattig T, Rickert C, Bamberg E, Steinhoff HJ, Bamann C (2013) Light-induced movement of the transmembrane helix B in channelrhodopsin-2. Angew Chem Int Ed Engl 52, 9705-8. 


Grewer C, Gameiro A, Mager T, Fendler K (2013) Electrophysiological characterization of membrane transport proteins. Annu Rev Biophys 42, 95-120.


Mager T, Braner M, Kubsch B, Hatahet L, Alkoby D, Rimon A, Padan E, Fendler K (2013) Differential effects of mutations on the transport properties of the Na+/H+-antiporter NhaA from Escherichia coli. J Biol Chem 288, 24666-75.

Geibel S, Lorinczi E, Bamberg E, Friedrich T (2013). Voltage dependence of proton pumping by bacteriorhodopsin mutants with altered lifetime of the M intermediate. PloS One 8, e73338.

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Collaborations

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Wachtveitl (P12), Gottschalk (P11), Glaubitz (P6), Kühlbrandt (P1), Michel (P3),
Schwalbe (P13), Dötsch/Bernhard (P2), Ziegler (P4)