Biomedical subjects

Christoph Fahlke

Publications and source records attributed to Christoph Fahlke.

12 recordsLinked to original sources

PubMed2006

Barttin modulates trafficking and function of ClC-K channels.

Barttin is an accessory subunit of a subgroup of ClC-type chloride channels expressed in renal and inner ear epithelia. In this study, we examined the effects of barttin on two ClC-K channel isoforms, rat ClC-K1 and human ClC-Kb, using heterologous expression, patch clamping, confocal imaging, and flow cytometry. In the absence of barttin, only a small percentage of rClC-K1 and hClC-Kb channels are inserted into the plasma membrane. Coexpression of barttin enhances surface membrane insertion and furthermore modifies permeation and gating of ClC-K channels. hClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. We mapped the separate functions of barttin to structural domains by a deletion analysis. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin might be necessary for a tight adjustment of epithelial Cl(-) conductances to ensure a precise regulation of body salt content and endocochlear potential.

Cell Line↗

PubMed2006

Intersubunit interactions in EAAT4 glutamate transporters.

Excitatory amino acid transporters (EAATs) play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian CNS. A functional transporter is assembled as oligomer consisting of three subunits, each of which appears to transport glutamate independently from the neighboring subunits. EAATs do not only sustain a secondary-active glutamate transport but also function as anion channel. We here address the question whether intersubunit interactions play a role in pore-mediated anion conduction. We expressed a neuronal isoform, EAAT4, heterologously in Xenopus oocytes and mammalian cells and measured glutamate flux and anion currents under various concentrations of Na+ and glutamate. EAAT4 anion channels are active in the absence of both substrates, and increasing concentrations activate EAAT4 anion currents with a sigmoidal concentration dependence. Because only one glutamate molecule is cotransported per uptake cycle, the cooperativity between glutamate binding sites most likely arises from an interaction between different carrier domains. This interaction is modified by two point mutations close to the putative glutamate binding site, G464S and Q467S. Both mutations alter the dissociation constants and Hill coefficient of the substrate dependence of anion currents, leaving the concentration dependence of glutamate uptake unaffected. Our results demonstrate that glutamate carriers cooperatively interact during anion channel activation.

Animals↗

PubMed2005

A dynamic switch between inhibitory and excitatory currents in a neuronal glutamate transporter.

Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission and maintain extracellular glutamate concentrations in the central nervous system below excitotoxic levels. In addition to sustaining a secondary-active glutamate transport, EAAT glutamate transporters also function as anion-selective channels. Here, we report a gating process that makes anion channels associated with a neuronal glutamate transporter, EAAT4, permeable to cations and causes a selective increase of the open probability at voltages negative to the actual current reversal potential. The activation process depends on both membrane potential and extracellular glutamate concentration and causes an accumulation of EAAT4 anion channels in a state favoring cation influx and anion efflux. Gating of EAAT4 anion channels thus allows a switch between inhibitory currents in resting cells and excitatory currents in electrically active cells. This transporter-mediated conductance could modify the excitability of Purkinje neurons, providing them with an unprecedented mechanism for adaptation.

Amino Acid Transport Systems↗

PubMed2005

Carboxy-terminal truncations modify the outer pore vestibule of muscle chloride channels.

Mammalian ClC-type chloride channels have large cytoplasmic carboxy-terminal domains whose function is still insufficiently understood. We investigated the role of the distal part of the carboxy-terminus of the muscle isoform ClC-1 by constructing and functionally evaluating two truncation mutants, R894X and K875X. Truncated channels exhibit normal unitary conductances and anion selectivities but altered apparent anion binding affinities in the open and in the closed state. Since voltage-dependent gating is strictly coupled to ion permeation in ClC-1 channels, the changed pore properties result in different fast and slow gating. Full length and truncated channels also differed in methanethiosulphonate (MTS) modification rate constants of an engineered cysteine at position 231 near the selectivity filter. Our data demonstrate that the carboxy-terminus of ClC channels modifies the conformation of the outer pore vestibule.

Animals↗

PubMed2004

A trimeric quaternary structure is conserved in bacterial and human glutamate transporters.

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.

Amino Acid Sequence↗

PubMed2004

The role of the carboxyl terminus in ClC chloride channel function.

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-beta-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxyl-terminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.

Animals↗

PubMed2003

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels.

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.

Amino Acid Transport System X-AG↗

PubMed2003

Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies.

Idiopathic generalized epilepsy (IGE) is an inherited neurological disorder affecting about 0.4% of the world's population. Mutations in ten genes causing distinct forms of idiopathic epilepsy have been identified so far, but the genetic basis of many IGE subtypes is still unknown. Here we report a gene associated with the four most common IGE subtypes: childhood and juvenile absence epilepsy (CAE and JAE), juvenile myoclonic epilepsy (JME), and epilepsy with grand mal seizures on awakening (EGMA; ref. 8). We identified three different heterozygous mutations in the chloride-channel gene CLCN2 in three unrelated families with IGE. These mutations result in (i) a premature stop codon (M200fsX231), (ii) an atypical splicing (del74-117) and (iii) a single amino-acid substitution (G715E). All mutations produce functional alterations that provide distinct explanations for their pathogenic phenotypes. M200fsX231 and del74-117 cause a loss of function of ClC-2 channels and are expected to lower the transmembrane chloride gradient essential for GABAergic inhibition. G715E alters voltage-dependent gating, which may cause membrane depolarization and hyperexcitability.

Adolescent↗

PubMed2003

Anion permeation in human ClC-4 channels.

ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.

Anions↗

PubMed2002

A novel alteration of muscle chloride channel gating in myotonia levior.

Mutations in the voltage-dependent skeletal muscle chloride channel, ClC-1, result in dominant or recessive myotonia congenita. The Q552R mutation causes a variant of dominant myotonia with a milder phenotype, myotonia levior. To characterise the functional properties of this mutation, homodimeric mutant and heterodimeric wild-type (WT) mutant channels were expressed in tsA201 cells and studied using the whole-cell recording technique. Q552R ClC-1 mutants formed functional channels with normal ion conduction but altered gating properties. Mutant channels were activated by membrane depolarisation, with a voltage dependence of activation that was shifted by more than +90 mV compared to WT channels. Q552R channels were also activated by hyperpolarisation, and this process was dependent upon the intracellular chloride concentration ([Cl(-)](i)). Together, these alterations resulted in a substantial reduction in the open probability at -85 mV at a physiological [Cl(-)](i). Heterodimeric WT-Q552R channels did not exhibit hyperpolarisation-activated gating transitions. As was the case for WT channels, activation occurred upon depolarisation, but the activation curve was shifted by 28 mV to more positive potentials. The functional properties of heterodimeric channels suggest a weakly dominant effect, a finding that correlates with the inheritance pattern and symptom profile of myotonia levior.

Algorithms↗

PubMed2002

The myotonia congenita mutation A331T confers a novel hyperpolarization-activated gate to the muscle chloride channel ClC-1.

Mutations in the muscle chloride channel gene CLCN1 cause myotonia congenita, an inherited disorder of skeletal muscle excitability leading to a delayed relaxation after muscle contraction. Here, we examine the functional consequences of a novel disease-causing mutation that predicts the substitution of alanine by threonine at position 331 (A331T) by whole-cell patch-clamp recording of recombinant mutant channels. A331T hClC-1 channels exhibit a novel slow gate that activates during membrane hyperpolarization and closes at positive potentials. This novel gate acts in series with fast opening and closing transitions that are common to wild-type (WT) and mutant channels. Under conditions at which this novel gate is not activated, i.e., a holding potential of 0 mV, the typical depolarization-induced activation gating of WT hClC-1 was only slightly affected by the mutation. In contrast, A331T hClC-1 channels with an open slow gate display an altered voltage dependence of open probability. These novel gating features of mutant channels produce a decreased open probability at -85 mV, the normal muscle resting potential, leading to a reduced resting chloride conductance of affected muscle fibers. The A331T mutation causes an unprecedented alteration of ClC-1 gating and reveals novel processes defining transitions between open and closed states in ClC chloride channels.

Adult↗

PubMed2002

Novel CLCN1 mutations with unique clinical and electrophysiological consequences.

Myotonia is a condition characterized by impaired relaxation of muscle following sudden forceful contraction. We systematically screened all 23 exons of the CLCN1 gene in 88 unrelated patients with myotonia and identified mutations in 14 patients. Six novel mutations were discovered: five were missense (S132C, L283F, T310M, F428S and T550M) found in heterozygous patients, and one was a nonsense mutation (E193X) in a homozygous patient. While five patients had a clinical diagnosis of myotonia congenita, the patient with the F428S mutation exhibited symptoms characteristic of paramyotonia congenita--a condition usually thought to be caused by mutations in the sodium channel gene SCN4A. Nevertheless, no mutations in SCN4A were identified in this patient. The functional consequences of the novel CLCN1 sequence variants were explored by recording chloride currents from human embryonic kidney cells transiently expressing homo- or heterodimeric mutant channels. The five tested mutations caused distinct functional alterations of the homodimeric human muscle chloride ion channel hClC-1. S132C and T550M conferred novel hyperpolarization-induced gating steps, L283F and T310M caused a shift of the activation curve to more positive potentials and F428S reduced the expression level of hClC-1 channels. All showed a dominant-negative effect. For S132C, L283F, T310M and T550M, heterodimeric channels consisting of one wild-type (WT) and one mutant subunit exhibited a shifted activation curve at low intracellular [Cl(-)]. WT-F428S channels displayed properties similar to WT hClC-1, but expressed at significantly lower levels. The novel mutations exhibit a broad variety of functional defects that, by distinct mechanisms, cause a significant reduction of the resting chloride conductance in muscle of heterozygous patients. Our results provide novel insights into functional alterations and clinical symptoms caused by mutations in CLCN1.

Adult↗