Biomedical subjects

Thomas J Jentsch

Publications and source records attributed to Thomas J Jentsch.

At least 19 recordsLinked to original sources

PubMed2026

Abnormal ClC-3/TMEM9-mediated endosomal ion transport in CLCN3-associated neurodevelopmental disease.

Endolysosomal abnormalities are particularly detrimental to the nervous system and have been implicated in neuropsychiatric disorders. Key regulators of the lysosomal and endosomal luminal ion homeostasis are CLC chloride/proton exchangers. We report 15 individuals carrying variants in CLCN3, encoding a ubiquitous endosomal 2Cl-/H+ exchanger, and provide updated clinical information for 5 previously reported individuals. Subjects displayed a broad spectrum of neuropsychiatric symptoms, including developmental delay, intellectual disability, and epilepsy. To reveal the pathogenic mechanism, we investigated ClC-3 variants-mediated ion transport and its regulation by the recently discovered inhibitory beta subunit TMEM9. 12/20 missense variants exhibited altered properties and fell into two classes: those affecting the region binding inhibitory TMEM9 carboxy-termini, and those that broaden the voltage range over which ClC-3 conducts ions. Surprisingly, the latter variants also attenuated TMEM9-mediated inhibition. Both classes produced a toxic gain-of-function, as evident from endolysosomal vacuolization by mutant ClC-3/TMEM9 overexpression. Our results expand the genetic and clinical spectrum of CLCN3-related disease, provide a solid basis for genetic counseling, and uncover an unexpected link between gating-associated conformational changes and inhibition by TMEM9.

Chloride Channels↗

PubMed2006

Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters.

Several members of the CLC family of Cl- channels and transporters are expressed in vesicles of the endocytotic-lysosomal pathway, all of which are acidified by V-type proton pumps. These CLC proteins are thought to facilitate vesicular acidification by neutralizing the electric current of the H+-ATPase. Indeed, the disruption of ClC-5 impaired the acidification of endosomes, and the knock-out (KO) of ClC-3 that of endosomes and synaptic vesicles. KO mice are available for all vesicular CLCs (ClC-3 to ClC-7), and ClC-5 and ClC-7, as well as its beta-subunit Ostm1, are mutated in human disease. The associated mouse and human pathologies, ranging from impaired endocytosis and nephrolithiasis (ClC-5) to neurodegeneration (ClC-3), lysosomal storage disease (ClC-6, ClC-7/Ostm1) and osteopetrosis (ClC-7/Ostm1), were crucial in identifying the physiological roles of vesicular CLCs. Whereas the intracellular localization of ClC-6 and ClC-7/Ostm1 precluded biophysical studies, the partial expression of ClC-4 and -5 at the cell surface allowed the detection of strongly outwardly rectifying currents that depended on anions and pH. Surprisingly, ClC-4 and ClC-5 (and probably ClC-3) do not function as Cl- channels, but rather as electrogenic Cl--H+ exchangers. This hints at an important role for luminal chloride in the endosomal-lysosomal system.

Animals↗

PubMed2006

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6.

Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

Animals↗

PubMed2006

Structural determinants of M-type KCNQ (Kv7) K+ channel assembly.

The ability of KCNQ (Kv7) channels to form hetero-oligomers is of high physiological importance, because heteromers of KCNQ3 with KCNQ2 or KCNQ5 underlie the neuronal M-current, which modulates neuronal excitability. In KCNQ channels, we recently identified a C-terminal subunit interaction (si) domain that determines their subunit-specific assembly. Within this si domain, there are two motifs that comprise approximately 30 amino acid residues each and that exhibit a high probability for coiled-coil formation. Transfer of the first or the second coiled-coil (TCC) domain from KCNQ3 into the KCNQ1 scaffold resulted in chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3, both of which coimmunoprecipitated with KCNQ2. However, only KCNQ1(TCC2)Q3 enhanced KCNQ2 currents and surface expression or exerted a strong dominant-negative effect on KCNQ2. Deletion of TCC2 within KCNQ2 yielded functional homomeric channels but prevented the current augmentation measured after coexpression of KCNQ2 and KCNQ3. In contrast, deleting TCC1 within KCNQ2 did not give functional homomeric KCNQ2 or heteromeric KCNQ2/KCNQ3 channels. Mutations that disrupted the predicted coiled-coil structure of TCC1 in KCNQ2 or KCNQ3 abolished channel activity after expressing these constructs singly or in combination, whereas helix-breaking mutations in TCC2 of KCNQ2 gave functional homomeric channels but prevented the heteromerization with KCNQ3. In contrast, KCNQ3 carrying a coiled-coil disrupting mutation in TCC2 hetero-oligomerized with KCNQ2. Our data suggest that the TCC1 domains of KCNQ2 and KCNQ3 are required to form functional homomeric as well as heteromeric channels, whereas both TCC2 domains facilitate an efficient transport of heteromeric KCNQ2/KCNQ3 channels to the plasma membrane.

Amino Acid Sequence↗

PubMed2006

ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function.

Mutations in ClC-7, a late endosomal/lysosomal member of the CLC family of chloride channels and transporters, cause osteopetrosis and lysosomal storage disease in humans and mice. Severe osteopetrosis is also observed with mutations in the OSTM1 gene, which encodes a membrane protein of unknown function. Here we show that both ClC-7 and Ostm1 proteins co-localize in late endosomes and lysosomes of various tissues, as well as in the ruffled border of bone-resorbing osteoclasts. Co-immunoprecipitations show that ClC-7 and Ostm1 form a molecular complex and suggest that Ostm1 is a beta-subunit of ClC-7. ClC-7 is required for Ostm1 to reach lysosomes, where the highly glycosylated Ostm1 luminal domain is cleaved. Protein but not RNA levels of ClC-7 are greatly reduced in grey-lethal mice, which lack Ostm1, suggesting that the ClC-7-Ostm1 interaction is important for protein stability. As ClC-7 protein levels in Ostm1-deficient tissues and cells, including osteoclasts, are decreased below 10% of normal levels, Ostm1 mutations probably cause osteopetrosis by impairing the acidification of the osteoclast resorption lacuna, which depends on ClC-7 (ref. 3). The finding that grey-lethal mice, just like ClC-7-deficient mice, show lysosomal storage and neurodegeneration in addition to osteopetrosis implies a more general importance for ClC-7-Ostm1 complexes.

Animals↗

PubMed2006

Mice with altered KCNQ4 K+ channels implicate sensory outer hair cells in human progressive deafness.

KCNQ4 is an M-type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4-/- mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I(K,n) current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.

Animals↗

PubMed2006

Neurogenic mechanisms contribute to hypertension in mice with disruption of the K-Cl cotransporter KCC3.

The neurodegenerative disorder Andermann syndrome is caused by mutations of the K-Cl cotransporter KCC3. Mice with a targeted disruption of the corresponding gene, Slc12a6, reproduce neurodegeneration of the peripheral and central nervous system (CNS) and display arterial hypertension. Kcc3 is expressed in numerous tissues, including the CNS and vascular smooth muscle cells. As the intracellular chloride concentration may influence myogenic tone and hence blood pressure, we measured the chloride concentration in vascular smooth muscle cells. It was indeed increased in superficial brain arteries and saphenous arteries of Kcc3(-/-) mice. Isolated saphenous arteries and their third-order branches, however, reacted indistinguishably to changes in intravascular pressure, stimulation of alpha1-adrenoreceptors, exogenous nitric oxide, or blockade of calcium-activated chloride channels. Likewise, the responses to alpha1-adrenergic stimulation or exogenous nitric oxide in vivo were identical in both genotypes. These results argue against a major vascular-intrinsic component of arterial hypertension in Kcc3(-/-) mice. In contrast, either alpha1-adrenergic blockade or inhibition of ganglionic transmission abolished the difference in arterial blood pressure between both genotypes. This demonstrates a neurogenic component in the maintenance of this phenotype, which is further supported by an increase of urinary norepinephrine and epinephrine excretion in Kcc3(-/-) mice. Our data indicate that local control of myogenic tone does not require KCC3 and that hypertension in Kcc3(-/-) mice depends on an elevated sympathetic tone.

Aldosterone↗

PubMed2006

Mice with a targeted disruption of the Cl-/HCO3- exchanger AE3 display a reduced seizure threshold.

Neuronal activity results in significant pH shifts in neurons, glia, and interstitial space. Several transport mechanisms are involved in the fine-tuning and regulation of extra- and intracellular pH. The sodium-independent electroneutral anion exchangers (AEs) exchange intracellular bicarbonate for extracellular chloride and thereby lower the intracellular pH. Recently, a significant association was found with the variant Ala867Asp of the anion exchanger AE3, which is predominantly expressed in brain and heart, in a large cohort of patients with idiopathic generalized epilepsy. To analyze a possible involvement of AE3 dysfunction in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy, and neither displayed gross histological and behavioral abnormalities nor spontaneous seizures or spike wave complexes in electrocorticograms. However, the seizure threshold of AE3-knockout mice exposed to bicuculline, pentylenetetrazole, or pilocarpine was reduced, and seizure-induced mortality was significantly increased compared to wild-type littermates. In the pyramidal cell layer of the hippocampal CA3 region, where AE3 is strongly expressed, disruption of AE3 abolished sodium-independent chloride-bicarbonate exchange. These findings strongly support the hypothesis that AE3 modulates seizure susceptibility and, therefore, are of significance for understanding the role of intracellular pH in epilepsy.

Animals↗

PubMed2005

Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins.

Eukaryotic members of the CLC gene family function as plasma membrane chloride channels, or may provide neutralizing anion currents for V-type H(+)-ATPases that acidify compartments of the endosomal/lysosomal pathway. Loss-of-function mutations in the endosomal protein ClC-5 impair renal endocytosis and lead to kidney stones, whereas loss of function of the endosomal/lysosomal protein ClC-7 entails osteopetrosis and lysosomal storage disease. Vesicular CLCs have been thought to be Cl- channels, in particular because ClC-4 and ClC-5 mediate plasma membrane Cl- currents upon heterologous expression. Here we show that these two mainly endosomal CLC proteins instead function as electrogenic Cl-/H+ exchangers (also called antiporters), resembling the transport activity of the bacterial protein ClC-e1, the crystal structure of which has already been determined. Neutralization of a critical glutamate residue not only abolished the steep voltage-dependence of transport, but also eliminated the coupling of anion flux to proton counter-transport. ClC-4 and ClC-5 may still compensate the charge accumulation by endosomal proton pumps, but are expected to couple directly vesicular pH gradients to Cl- gradients.

Animals↗

PubMed2005

Molecular determinants of KCNQ (Kv7) K+ channel sensitivity to the anticonvulsant retigabine.

Epilepsy is caused by an electrical hyperexcitability in the CNS. Because K+ channels are critical for establishing and stabilizing the resting potential of neurons, a loss of K+ channels could support neuronal hyperexcitability. Indeed, benign familial neonatal convulsions, an autosomal dominant epilepsy of infancy, is caused by mutations in KCNQ2 or KCNQ3 K+ channel genes. Because these channels contribute to the native muscarinic-sensitive K+ current (M current) that regulates excitability of numerous types of neurons, KCNQ (Kv7) channel activators would be effective in epilepsy treatment. A compound exhibiting anticonvulsant activity in animal seizure models is retigabine. It specifically acts on the neuronally expressed KCNQ2-KCNQ5 (Kv7.2-Kv7.5) channels, whereas KCNQ1 (Kv7.1) is not affected. Using the differential sensitivity of KCNQ3 and KCNQ1 to retigabine, we constructed chimeras to identify minimal segments required for sensitivity to the drug. We identified a single tryptophan residue within the S5 segment of KCNQ3 and also KCNQ2, KCNQ4, and KCNQ5 as crucial for the effect of retigabine. Furthermore, heteromeric KCNQ channels comprising KCNQ2 and KCNQ1 transmembrane domains (attributable to transfer of assembly properties from KCNQ3 to KCNQ1) are retigabine insensitive. Transfer of the tryptophan into the KCNQ1 scaffold resulted in retigabine-sensitive heteromers, suggesting that the tryptophan is necessary in all KCNQ subunits forming a functional tetramer to confer drug sensitivity.

Animals↗

PubMed2005

Chloride transport in the kidney: lessons from human disease and knockout mice.

Knockout mouse models and human inherited diseases have provided important new insights into the physiologic role of chloride transport by CLC Cl(-) channels and KCC K-Cl co-transporters. ClC-K/barrtin Cl(-) channels are important for renal salt reabsorption and possibly for acid secretion by intercalated cells. The endosomal ClC-5 protein is crucial for proximal tubular endocytosis. Its disruption in mice and patients with Dent's disease leads to hypercalciuria and kidney stones through a pathologic cascade that may be entirely explained by an impairment of endocytosis. KCC4 is important for recycling Cl(-) for the basolateral anion exchanger in intercalated cells, as is evident from the renal tubular acidosis resulting from its knockout. Finally, both KCC3 and KCC4 are crucial for proximal tubular cell volume regulation.

Animals↗

PubMed2005

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration.

ClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis.

Animals↗

PubMed2005

CLC chloride channels and transporters.

CLC proteins are found in cells from prokaryotes to mammals and perform functions in plasma membranes and intracellular vesicles. Several genetic human diseases and mouse models underscore their broad physiological functions in mammals. These functions range from the control of excitability to transepithelial transport, endocytotic trafficking and acidification of synaptic vesicles. The recent crystallization of bacterial CLC proteins gave surprising insights into CLC Cl(-)-channel permeation and gating and provides an excellent basis for structure-function studies. Surprisingly, the CLC from Escherichia coli functions as a Cl-/H+ exchanger, thus demonstrating the thin line separating transporters and channels.

Animals↗

PubMed2005

Unique structure and function of chloride transporting CLC proteins.

CLC proteins are a large structurally defined family of Cl- ion channels and H+/Cl- antiporters with nine distinct genes in mammals. The membrane-embedded part of CLC proteins bears no obvious similarity to any other class of membrane proteins, while the cytoplasmic C-terminus of most eukaryotic and some prokaryotic CLCs contains two regions with homology to cystathionine beta synthase (CBS) domains that are found in other proteins as well. Different members serve a broad range of physiological roles, including stabilization of the membrane potential, transepithelial ion transport, and vesicular acidification. Their physiological importance is underscored by the causative involvement in at least four different human genetic diseases. From functional studies of the Torpedo homologue ClC-0, a homodimeric architecture with two physically separate ion conduction pathways was anticipated and fully confirmed by solving the crystal structure of prokaryotic CLC homologues. The structure revealed a complex fold of 18 alpha-helices per subunit with at least two Cl- ions bound in the center of each protopore. A critical glutamic acid residue was identified whose side-chain seems to occupy a third Cl- ion binding site in the closed state and that moves away to allow Cl- binding. While the overall architecture and pore structure is certainly conserved from bacteria to humans, the bacterial proteins that were crystallized are actually not Cl- ion channels, but coupled H+/Cl- antiporters. These recent breakthroughs will allow us to study in further detail the structure, function, and the physiological and pathophysiological role of CLC proteins.

Animals↗

PubMed2005

Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models.

The CLC gene family encodes nine different Cl() channels in mammals. These channels perform their functions in the plasma membrane or in intracellular organelles such as vesicles of the endosomal/lysosomal pathway or in synaptic vesicles. The elucidation of their cellular roles and their importance for the organism were greatly facilitated by mouse models and by human diseases caused by mutations in their respective genes. Human mutations in CLC channels are known to cause diseases as diverse as myotonia (muscle stiffness), Bartter syndrome (renal salt loss) with or without deafness, Dent's disease (proteinuria and kidney stones), osteopetrosis and neurodegeneration, and possibly epilepsy. Mouse models revealed blindness and infertility as further consequences of CLC gene disruptions. These phenotypes firmly established the roles CLC channels play in stabilizing the plasma membrane voltage in muscle and possibly in neurons, in the transport of salt and fluid across epithelia, in the acidification of endosomes and synaptic vesicles, and in the degradation of bone by osteoclasts.

Animals↗

PubMed2005

Chloride channel diseases resulting from impaired transepithelial transport or vesicular function.

The transport of anions across cellular membranes is crucial for various functions, including the control of electrical excitability of muscle and nerve, transport of salt and water across epithelia, and the regulation of cell volume or the acidification and ionic homeostasis of intracellular organelles. Given this broad range of functions, it is perhaps not surprising that mutations in Cl- channels lead to a large spectrum of diseases. These diverse pathologies include the muscle disorder myotonia, cystic fibrosis, renal salt loss in Bartter syndrome, kidney stones, deafness, and the bone disease osteopetrosis. This review will focus on diseases related to transepithelial transport and on disorders involving vesicular Cl- channels.

Animals↗

PubMed2004

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte.

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Animals↗

PubMed2004

Additional disruption of the ClC-2 Cl(-) channel does not exacerbate the cystic fibrosis phenotype of cystic fibrosis transmembrane conductance regulator mouse models.

Cystic fibrosis is a fatal inherited disease that is caused by mutations in the gene encoding a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). It has been suggested that the cystic fibrosis phenotype might be modulated by the presence of other Cl(-) channels that are coexpressed with CFTR in some epithelial cells. Because the broadly expressed plasma membrane Cl(-) channel, ClC-2, is present in the tissues whose function is compromised in cystic fibrosis, we generated mice with a disruption of both Cl(-) channel genes. No morphological changes in their intestine, lung, or pancreas, tissues affected by cystic fibrosis, were observed in these mice. The mortality was not increased over that observed with a complete lack of functional CFTR. Surprisingly, mice expressing mutant CFTR (deletion of phenylalanine 508), survived longer when ClC-2 was disrupted additionally. Currents across colonic epithelia were investigated in Ussing chamber experiments. The disruption of ClC-2, in addition to CFTR, did not decrease Cl(-) secretion. Colon expressing wild-type CFTR even secreted more Cl(-) when ClC-2 was disrupted, although CFTR transcript levels were unchanged. It is concluded that ClC-2 is unlikely to be a candidate rescue channel in cystic fibrosis. Our data are consistent with a model in which ClC-2 is located in the basolateral membrane.

Animals↗