Mechanisms of ion channel and transporter function

The lipid bilayer is a perfect electrical insulator and provides the basis for molecular information processing at the cell membrane. Active transport proteins establish electrochemical potential gradients across the membrane, providing the energy for passive ion flux through channels. Electrical signaling in cells is mediated by the opening and closing of voltage-gated ion channels in response to electrical stimuli and the consequent transmembrane voltage changes.
Patch-clamp electrophysiology enables direct recordings of electrogenic transport processes mediated by ion channels and transporters. State-of-the-art biomolecular dynamics simulations provide detailed in-sight into their transport mechanisms at atomic resolution-including accurate estimates of ion conductance, selectivity, and voltage dependence. Together, experiments and molecular simulations provide validated insights into transport mechanisms, thereby establishing a direct link between molecular structure and physiological function.
We use all-atom MD simulations, combined with patch-clamp electrophysiology and time-resolved fluorescence spectroscopy, to investigate functional dynamics of neurotransmitter transporters and Cl^- channels. We developed kinetic state models to explain the functional coupling of secondary active glutamate transport and channel-like anion conduction in EAAT glutamate transporters (1-3), and advanced noise analysis techniques to measure unitary properties of transporter-associated channels (4). Using stopped-flow fluorescence recordings, we identified an induced-fit substrate binding mechanism in EAATs (4). The prokaryotic EAAT homolog Glt_Ph is the founding member of the group of transporters with an elevator transport mechanism, and we used essential dynamics sampling to simulate the inward–outward transition path (5). We identified the Cl^- permeation pathway and Cl^- conduction mechanism in EAATs (5,6) using Computational Electrophysiology, a simulation technique for all-atom MD simulations of membrane proteins under sustained electrochemical gradients (7). To investigate the voltage dependence of membrane proteins, we developed a novel method for gating charge calculations using Computational Electrophysiology (8), and proposed a novel voltage-sensing mechanism in G protein-coupled receptors (9,10).
Further research projects include ion transport mechanisms in vesicular glutamate transporters, Ca²⁺-activated Cl^- channels and lipid scramblases of the TMEM16 (anoctamin) family and Cl^- channels and Cl^-/H⁺ exchangers of the ClC family.

1. Leinenweber, A., Machtens, J. P., Begemann, B. & Fahlke, C. Regulation of glial glutamate transporters by C-terminal domains. The Journal of biological chemistry 286, 1927-1937 (2011).
   
2. Kovermann, P., Machtens, J. P., Ewers, D. & Fahlke, C. A conserved aspartate determines pore properties of anion channels associated with excitatory amino acid transporter 4 (EAAT4). The Journal of biological chemistry 285, 23676-23686, doi:10.1074/jbc.M110.126557 (2010).
3. Machtens, J. P., Kovermann, P. & Fahlke, C. Substrate-dependent gating of anion channels associated with excitatory amino acid transporter 4. The Journal of biological chemistry 286, 23780-23788, doi:10.1074/jbc.M110.207514 (2011).
4. Machtens, J. P., Fahlke, C. & Kovermann, P. Noise analysis to study unitary properties of transporter-associated ion channels. Channels (Austin) 5, 468-474, doi:10.4161/chan.5.6.17453 (2011).
5. Machtens, J. P. et al. Mechanisms of anion conduction by coupled glutamate transporters. Cell 160, 542-553, doi:10.1016/j.cell.2014.12.035 (2015).
6. Fahlke, C., Kortzak, D. & Machtens, J. P. Molecular physiology of EAAT anion channels. Pflugers Arch 468, 491-502, doi:10.1007/s00424-015-1768-3 (2016).
7. Kutzner, C. et al. Insights into the function of ion channels by computational electrophysiology simulations. Biochim Biophys Acta 1858, 1741-1752, doi:10.1016/j.bbamem.2016.02.006 (2016).
8. Machtens, J. P., Briones, R., Alleva, C., de Groot, B. L. & Fahlke, C. Gating Charge Calculations by Computational Electrophysiology Simulations. Biophysical journal 112, 1396-1405, doi:10.1016/j.bpj.2017.02.016 (2017).
9. Vickery, O. N., Machtens, J. P., Tamburrino, G., Seeliger, D. & Zachariae, U. Structural Mechanisms of Voltage Sensing in G Protein-Coupled Receptors. Structure 24, 997-1007, doi:10.1016/j.str.2016.04.007 (2016).
10. Vickery, O. N., Machtens, J. P. & Zachariae, U. Membrane potentials regulating GPCRs: insights from experiments and molecular dynamics simulations. Curr Opin Pharmacol 30, 44-50, doi:10.1016/j.coph.2016.06.011 (2016).

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