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Hannover, Germany

Stolting G.,Julich Research Center | Fischer M.,Institute For Neurophysiologie | Fahlke C.,Julich 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.,Julich Research Center | Fischer M.,Institute For Neurophysiologie | Fahlke C.,Julich 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.


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.


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.


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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