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Institut für Physiologie II - Herz-Kreislauf-Physiologie
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Physiology / Collaborative Funding / DynIon

Research Unit DynIon

Functional dynamics of ion channels and transporters

The DFG-Research Unit initiative ‘Functional dynamics of ion channels and transporters’ combines experimental and computational strategies to study ion channels and transporters. We focus on ligand activation of ion channels, including ion channels that require ligand binding for channel opening (ionotropic glutamate receptors), ion channels that are modulated by ligands (K2P channels, voltage-gated K+ channels, cyclic nucleotide modulated ion channels) and transporters that can also adopt channel-like conformations (EAAT glutamate transporters and CLC anion channels/anion-proton exchangers). A variety of experimental approaches, ranging from electrophysiology, biochemistry and fluorometry to structural biology, is integrated with computational techniques including all-atom molecular dynamics (MD), hybrid quantum mechanical/molecular mechanics simulations, as well as free energy calculations and Markov state modeling. Our strategy fosters and exploits an otherwise unattainable synergy between computer simulations and experiments. We expect that our research program advances theoretical and experimental approaches in ion channel research in general and provides novel insights into selected ion channels and transporters specifically.

Within this research unit, Klaus Benndorf and Jana Kusch lead project P2 and Indra Schröder project P10.

P2 Scrutinizing the function of the cyclic-nucleotide binding domain in the activation of HCN channels

Schematic illustrations of CN-modulated ion channels. (A) Scheme of a whole channel composed of four subunits. The four subunits are surrounding a central cation-selective pore. Each homo- and heteromeric channel carries four binding sites for cyclic nucleotides (CNBD). The CNBD is connected to the transmembrane region via a so-called C-linker. (B) Scheme of a single subunit composed of four transmembrane domains S1 to S6. The ion-conducting pore with the selectivity filter is formed by S5 and S6 and the loop between them. The illustration follows the structural information published by Lee and MacKinnon, 2017.
Schematic illustrations of CN-modulated ion channels. (A) Scheme of a whole channel composed of four subunits. The four subunits are surrounding a central cation-selective pore. Each homo- and heteromeric channel carries four binding sites for cyclic nucleotides (CNBD). The CNBD is connected to the transmembrane region via a so-called C-linker. (B) Scheme of a single subunit composed of four transmembrane domains S1 to S6. The ion-conducting pore with the selectivity filter is formed by S5 and S6 and the loop between them. The illustration follows the structural information published by Lee and MacKinnon, 2017.

In project P2, we study the function of the C-linker and the cyclic nucleotide binding domain (CNBD) in mammalian HCN (hyperpolarization-activated and cyclic nucleotide modulated) channels. They are composed of four subunits, with the C-terminus of each subunit containing a cyclic nucleotide binding domain (CNBD) that is linked to the last transmembrane S6 helix via the C-linker (CL). Confocal patch-clamp fluorometry (cPCF), a technique which combines electrophysiological approaches and confocal fluorescence microscopy will be applied in two variations:

  • to monitor activation state-dependent binding of the fluorescent cAMP derivative fcAMP and
  • to analyze activation-induced conformational changes of the CL and CNBD employing the intrinsically fluorescent unnatural amino acid Anap and fluorescent dyes coupled to the channel via click chemistry.

The data will be interpreted kinetically by complex Markovian models and by MD simulations in collaboration with Holger Gohlke (University Düsseldorf). Frank Noé (Freie Universität Berlin) will provide novel strategies to fit Markovian models to the data.

 

Dr. Jana Kusch

Phone: 03641 - 9 397668

Prof. Dr. Klaus Benndorf

Phone: 03641 - 9 397651

P10 Novel approaches to study the signal chain of the voltage-dependent gating in the selectivity filter of viral K+ channels

Schematic representation of the regulation of gating in the selectivity filter.
Schematic representation of the regulation of gating in the selectivity filter.

The subject of this proposal is a voltage-dependent gating process in the selectivity filter (SF) of a viral prototype K+ channel (Kcv) with sub-millisecond kinetics. By a combination of cell-free single-channel recordings and computational studies, we will identify a) how ion occupation shapes the flexibility of the SF, b) how the signal is transmitted from the sensor binding site to the gate and c) the location and mechanism of the gate.

Single channels will be characterized by high-resolution recordings in planar lipid bilayers. Modified ion-filter interactions will be induced by different permeant ions and point mutations. The role of the hydrogen bond network between the pore helix and the SF will also be addressed by point mutations. Anisotropic network model (ANM) calculations will be used to screen mutations and different ion occupations. This will provide estimates for the related changes in flexibility and the principal components of structural fluctuations. This will allow to identify molecular mechanisms, which will then be quantitatively tested by global fits of Hidden Markov Models. 

 

Prof. Dr. Indra Schröder

Phone: 03641 - 9 397686