Institute For Neurophysiologie

Hannover, Germany

Institute For Neurophysiologie

Hannover, Germany
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Cordeiro S.,Universitatsklinikum Hamburg Eppendorf | Cordeiro S.,Institute For Neurophysiologie | Strauss O.,Universitatsklinikum Hamburg Eppendorf
Graefe's Archive for Clinical and Experimental Ophthalmology | Year: 2011

Background: The retinal pigment epithelium (RPE) fulfills a large variety of tasks that are important for visual function. Many of these tasks, such as phagocytosis, growth factor secretion, or transepithelial ion transport, are regulated by increases in intracellular Ca 2+ as second-messenger. Despite the multitude of Ca 2+-dependently regulated functions, only few Ca 2+ channels have been described so far in the RPE to couple Ca 2+ conductance and Ca 2+ signaling. Methods: RT-PCR experiments with mRNA of freshly isolated RPE cells as well as from the RPE cell line ARPE-19 and measurements of the intracellular free Ca 2+ concentration were performed. Results: The RT-PCR experiments revealed the expression of the I CRAC channel proteins Orai 1, 2, and 3 and their stimulators Stim-1 and Stim-2. The classic maneuver to stimulate capacitive Ca 2+ entry (depletion of Ca 2+ stores by 1 μM thapsigargin under extracellular Ca 2+-free conditions and then re-adding extracellular Ca 2+) led to an increase in intracellular free Ca 2+, which could be blocked by application of a high concentration of 2-APB (75 μM) either before or during induction of capacitive Ca 2+ entry. On the other hand, application of a low concentration of 2-APB (2 μM) led to enhancement of the Ca 2+ increase induced by capacitive Ca 2+ entry. Depletion of cytosolic Ca 2+ stores by administration of an extracellular divalent cation-free solution led to an increase in the whole-cell conductance. Conclusions: With these data we show a new Ca 2+ entry pathway linked to the Ca 2+/inositolphosphate second-messenger system in RPE cells which help to further understand regulatory pathways of agonists. The expression of Orai channels enables the RPE cells to generate sustained or repetitive Ca 2+ signals as they are known to be induced by different stimuli like ATP, bFGF, and the stimulation with photoreceptor outer segments. © 2010 Springer-Verlag.


Ewers D.,Institute For Neurophysiologie | Becher T.,Institute For Neurophysiologie | Machtens J.-P.,Institute For Neurophysiologie | Machtens J.-P.,Jülich Research Center | And 3 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Excitatory amino acid transporters (EAATs) are a class of glutamate transporters that terminate glutamatergic synaptic transmission in the mammalian CNS. GltPh, an archeal EAAT homolog from Pyrococcus horikoshii, is currently the only member with a known 3D structure. Here, we studied the kinetics of substrate binding of a single tryptophan mutant (L130W) GltPh in detergent micelles. At low millimolar [Na+], the addition of L-aspartate resulted in complex time courses of W130 fluorescence changes over tens of seconds. With increasing [Na+], the kinetics were dominated by a fast component [kobs,fast; KD (Na+) = 22 ± 3 mM, nHill = 1.7 ± 0.3] with values of kobs,fast rising in a saturable manner to 500 s-1 (at 6 °C) with increasing [L-aspartate]. The binding kinetics of L-aspartate differed from the binding kinetics of two alternative substrates: L-cysteine sulfinic acid and D-aspartate. L-cysteine sulfinic acid bound with higher affinity than L-aspartate but involved lower saturating rates, whereas the saturating rates after D-aspartate binding were higher. Thus, after the association of two Na+ to the empty transporter, GltPh binds amino acids by induced fit. Cross-linking and proteolysis experiments suggest that the induced fit results from the closure of helical hairpin 2. This conformational change is faster for GltPh than for most mammalian homologues, whereas the amino acid association rates are similar. Our data reveal the importance of induced fit for substrate selection in EAATs and illustrate how high-affinity binding and the efficient transport of glutamate can be accomplished simultaneously by this class of transporters.


Stolting G.,Jülich Research Center | Fischer M.,Institute For Neurophysiologie | Fahlke C.,Jülich Research Center
Frontiers in Physiology | Year: 2014

CLC channels and transporters are expressed in most tissues and fulfill diverse functions. There are four human CLC channels, ClC-1, ClC-2, ClC-Ka, and ClC-Kb, and five CLC transporters, ClC-3 through -7. Some of the CLC channels additionally associate with accessory subunits. Whereas barttin is mandatory for the functional expression of ClC-K, GlialCam is a facultative subunit of ClC-2 which modifies gating and thus increases the functional variability within the CLC family. Isoform-specific ion conduction and gating properties optimize distinct CLC channels for their cellular tasks. ClC-1 preferentially conducts at negative voltages, and the resulting inward rectification provides a large resting chloride conductance without interference with the muscle action potential. Exclusive opening at voltages negative to the chloride reversal potential allows for ClC-2 to regulate intracellular chloride concentrations. ClC-Ka and ClC-Kb are equally suited for inward and outward currents to support transcellular chloride fluxes. Every human CLC channel gene has been linked to a genetic disease, and studying these mutations has provided much information about the physiological roles and the molecular basis of CLC channel function. Mutations in the gene encoding ClC-1 cause myotonia congenita, a disease characterized by sarcolemmal hyperexcitability and muscle stiffness. Loss-of-function of ClC-Kb/barttin channels impairs NaCl resorption in the limb of Henle and causes hyponatriaemia, hypovolemia and hypotension in patients suffering from Bartter syndrome. Mutations in CLCN2 were found in patients with CNS disorders but the functional role of this isoform is still not understood. Recent links between ClC-1 and epilepsy and ClC-Ka and heart failure suggested novel cellular functions of these proteins. This review aims to survey the knowledge about physiological and pathophysiological functions of human CLC channels in the light of recent discoveries from biophysical, physiological, and genetic studies. © 2014 Stölting.


Stolting G.,Jülich Research Center | Fischer M.,Institute For Neurophysiologie | Fahlke C.,Jülich Research Center
Pflugers Archiv European Journal of Physiology | Year: 2014

CLC-type chloride channels exhibit a unique double-barreled architecture with two independently functioning ion conduction pathways, the so-called protopores. There exist gating processes that open and close individual protopores as well as common processes that jointly mediate slow opening and closing of both protopores. Different isoforms exhibit distinct voltage dependences and kinetics of gating. Whereas opening of the individual and common gate of homo-dimeric ClC-1 is promoted by membrane depolarization, ClC-2 is closed at positive potentials and opens only at negative voltages. To characterize the functional interaction of protopores we engineered a concatameric construct linking the coding regions of ClC-1 and ClC-2 in an open reading frame, expressed it in mammalian cells and measured anion currents through whole-cell and single channel patch clamping. In the hetero-dimeric assembly, each protopore displayed two kinetically distinct gating processes. Fast gating of the ClC-1 protopore closely resembled fast protopore gating of homo-dimeric channels. The voltage dependence of ClC-2 fast gating was shifted to more positive potentials by the adjacent ClC-1 protopore, resulting in open ClC-2 protopores at positive voltages. We observed two slow gating processes individually acting on ClC-1 and ClC-2 protopores, with distinct time and voltage dependences. Single channel recordings demonstrated that hetero-dimerization additionally modified the unitary conductance of ClC-2 protopores. Our findings suggest that inter-subunit interactions do not only affect common gating, but also ion permeation and gating of individual protopores in hetero-dimeric ClC channels. © 2014, Springer-Verlag Berlin Heidelberg.


Machtens J.-P.,Jülich Research Center | Machtens J.-P.,Institute For Neurophysiologie | Machtens J.-P.,Max Planck Institute for Biophysical Chemistry | Kortzak D.,Jülich Research Center | And 9 more authors.
Cell | Year: 2015

Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na+/H+/K+ transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward-and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels. © 2015 Elsevier Inc.


Alekov A.K.,Institute For Neurophysiologie
Frontiers in Physiology | Year: 2015

Dent's disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5; however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent's disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 toward more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function. © 2015 Alekov.


Grieschat M.,Institute For Neurophysiologie | Alekov A.K.,Institute For Neurophysiologie
Biophysical Journal | Year: 2014

Most mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl-] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates. © 2014 by the Biophysical Society.


Fischer M.,Institute For Neurophysiologie | Janssen A.G.H.,Institute For Neurophysiologie | Fahlke C.,Institute For Neurophysiologie
Journal of the American Society of Nephrology | Year: 2010

Barttin is an accessory subunit that modifies protein stability, subcellular distribution, and voltage-dependent gating of ClC-K chloride channels expressed in renal and inner ear epithelia. ClC-K channels are double-barreled channels with two identical protopores that may be opened by individual or common gating processes. Using heterologous expression in mammalian cells and patch-clamp recordings, we studied the effects of barttin on gating of rat ClC-K1 and human ClC-Ka. In the absence of barttin, rClC-K1 channels displayed two gating processes with distinct kinetics and voltage dependence. A fast gating process, activated by membrane hyperpolarization, opens and closes individual rClC-K1 protopores. In addition, slow common gating steps, stimulated by membrane depolarization, act on both protopores together. Coexpression of barttin results in voltage-independent open probabilities of the common gate, causing increased channel activity at physiologic potentials. In contrast to rClC-K1, human ClC-Ka is functional only when coexpressed with barttin. Single-channel recordings of hClC-Ka/barttin show double-barreled channels with fast protopore gating without apparent cooperative gating steps. These findings demonstrate that barttin stimulates chloride flux through ClC-K channels by modifying cooperative gating of the double-barreled channels and highlight a physiologic role for gating of epithelial ClC chloride channels. Copyright © 2010 by the American Society of Nephrology.


Schanzler M.,Institute For Neurophysiologie | Fahlke C.,Institute For Neurophysiologie
Journal of Physiology | Year: 2012

Prestin is a member of the SLC26 solute carrier family and functions as a motor protein in cochlear outer hair cells. While other SLC26 homologues were demonstrated to transport a wide variety of anions, no electrogenic transport activity has been assigned so far to mammalian prestin. We here use heterologous expression in mammalian cells, patch clamp recordings and measurements of expression levels of individual cells to study anion transport by rat prestin. We demonstrated that cells expressing rat prestin exhibit SCN - currents that are proportional to the number of prestin molecules. Variation of the SCN - concentration resulted in changes of the current reversal potential that obey the Nernst equation indicating that SCN - transport is not stoichiometrically coupled to other anions. Application of external SCN - causes large increases of anion currents, but only minor changes in non-linear charge movements suggesting that only a very small percentage of prestin molecules function as SCN - transporters under these conditions. Unitary current amplitudes are below the resolution limit of noise analysis and thus much smaller than expected for pore-mediated anion transport. A comparison with a non-mammalian prestin fromD. rerio- recently shown to function as Cl -/SO 4 2- antiporter - and an SLC26 anion channel, human SLC26A7, revealed that SCN - transport is conserved in these distinct members of the SLC26 family. We conclude that mammalian prestin is capable of mediating electrogenic anion transport and suggest that SLC26 proteins converting membrane voltage oscillations into conformational changes and those functioning as channels or transporters share certain transport capabilities. © 2012 The Authors. Journal compilation © 2012 The Physiological Society.


Calcium entry through voltage-gated calcium channels (VGCC) initiates diverse cellular functions. VGCC pore-forming subunit (Ca(V)alpha(1)) contains four homology repeats, each encompassing a voltage sensor and a pore domain. Three main classes of Ca(V)alpha(1) subunits have been described, Ca(V)1, Ca(V)2 and Ca(V)3 that differ in their voltage-dependence of activation and in the extent in which this process is modulated by the auxiliary beta-subunit (Ca(V)beta). Association of Ca(V)beta induces a coil-to-helix conformation of the I-II intracellular linker joining the first and second repeat of Ca(V)alpha(1) that is thought to be crucial for modulation of channel function. When expressed in Xenopus laevis oocytes in the absence of Ca(V)beta, the voltage to reach 50% activation (V(0.5)) for Ca(V)1.2 and Ca(V)2.3 differs by more than 60 mV and the channel current-carrying capacity by more than thirty-fold. Here we report that the difference in V(0.5) is reduced to about 30 mV and the current-carrying capacity becomes virtually identical when the I-II linkers of Ca(V)1.2 and Ca(V)2.3 are swapped. Co-expression with Ca(V)beta increases the current-carrying capacity of chimeric channels by the same extent, while the difference in V(0.5) with respect to their corresponding parental channels vanishes. Our findings indicate that Ca(V)beta modulatory potency is determined by both, the nature of the I-II linker and the pore-forming subunit background. Moreover, they demonstrate that the I-II linker encodes self-reliant molecular determinants for channel activation and suggest that besides the secondary structure adopted by this segment upon Ca(V)beta association, its chemical nature is as well relevant.

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