Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen).

In most organisms, high affinity ammonium uptake is catalyzed by members of the ammonium transporter family (AMT/MEP/Rh). A single point mutation (G458D) in the cytosolic C terminus of the plasma membrane transporter LeAMT1;1 from tomato leads to loss of function, although mutant and wild type proteins show similar localization when expressed in yeast or plant protoplasts. Co-expression of LeAMT1;1 and mutant in Xenopus oocytes inhibited ammonium transport in a dominant negative manner, suggesting homo-oligomerization. In vivo interaction between LeAMT1;1 proteins was confirmed by the split ubiquitin yeast two-hybrid system. LeAMT1;1 is isolated from root membranes as a high molecular mass oligomer, converted to a ϳ35-kDa polypeptide by denaturation. To investigate interactions with the LeAMT1;2 paralog, co-localizing with LeAMT1;1 in root hairs, LeAMT1;2 was characterized as a lower affinity NH 4 ؉ uniporter. Co-expression of wild types with the respective G458D/G465D mutants inhibited ammonium transport in a dominant negative manner, supporting the formation of heteromeric complexes in oocytes. Thus, in yeast, oocytes, and plants, ammonium transporters are able to oligomerize, which may be relevant for regulation of ammonium uptake. Ammonium transporters (AMTs)1 of the AMT/MEP/Rh protein family have been identified in all domains of life, including plants, bacteria, archea, yeast, and animals (1, 2). AMT/ MEP/Rh proteins are highly hydrophobic membrane proteins with a predicted molecular mass of ϳ45-55 kDa and 11 or 12 putative transmembrane spans. Initially AMT/MEP/Rh ammonium transporters from yeast and plants were identified molecularly by functional complementation of a yeast mutant defective in ammonium uptake (3-5). Later, homologs were isolated from bacteria (6) and animals (Caenorhabditis elegans), and phylogenetic analysis showed that mammalian Rh (rhesus) blood group polypeptides belong to the same superfamily (7). Heterologously expressed RhAG and a homolog from kidney (RhGK ϭ RhCG) were also shown to function as ammonium transporters (8, 9).Plants require transporters for NH 4 ϩ acquisition from a wide range of external concentrations and are able to concentrate and transiently accumulate NH 4 ϩ in the cytosol before being metabolized or further compartmentalized (10). Ammonium transport across root plasma membranes is biphasic, consisting of a high-affinity and a low-affinity nonsaturating component (11,12). The high-affinity transport system, which operates predominantly at low external ammonium concentrations, is energized by the membrane potential. In tomato, the NH 4 ϩ -uniporter LeAMT1;1 encodes a component of the high-affinity transport system that depends on the membrane potential (13). The molecular identity of the low-affinity transport system, however, is less clear. It contributes significantly to overall NH 4 ϩ uptake at higher external ammonium concentrations (Ͼ1 mM) and may have a distinct transport mechanism, because uncharged NH 3 or charged NH 4 ϩ may be the substrate (11, 12, 14,...

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“…Ref. 48). At low amounts of cRNA injected per oocyte (Ϸ6 -12 ng), ammonium currents increased linearly with the amount of injected cRNA (Fig.…”

Section: Resultsmentioning

confidence: 84%

Homo- and Hetero-oligomerization of Ammonium Transporter-1 NH4+ Uniporters

Ludewig

Wilken

et al. 2003

Journal of Biological Chemistry

157

150

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“…In contrast to all other C1C proteins, including those from yeast and bacteria, it does not have a glutamic acid at position 208, but glutamine, and also lacks a proline at position 508 (replaced by serine). It is this highly conserved proline, which, when mutated to leucine in the muscle channel CIC-1, causes Thomsen's disease (autosomal dominant myotonia) in Dr. Thomsen's own family [19].…”

Section: Clc-6 Clc-7mentioning

confidence: 99%

“…Since mutations in C1C-1 and C1C-5 cause inherited disease in humans [14,17,19], we localized C1C-6 and C1C-7 in the human genome to see whether they could be candidate genes for human disease. FISH to human metaphase chromosomes localized CIC-6 on chromosome lp36, while C1C-7 is encoded on 16p13.…”

Section: Clc-6 Clc-7mentioning

confidence: 99%

“…Injecting large amounts of cDNA, in contrast, yielded anion currents with an iodide > chloride conductivity sequence which activated slowly upon strong hyperpolarization (in excess of-100 mV) (data not shown). However, similar currents are endogenous to Xenopus oocytes [25] and can also be provoked by overexpression of phospholemman [26], Mat-8 [27], minK (also named IsK [28]) [29] and mutants of C1C-1 which are closed in that voltagerange [19]. Thus, it seems that these currents are just an artifact of overexpressing membrane proteins in Xenopus oocytes [30].…”

Section: Clc-6 Clc-7mentioning

confidence: 99%

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ClC‐6 and ClC‐7 are two novel broadly expressed members of the CLC chloride channel family

1995

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We cloned two novel members of the CLC chloride channel family from rat and human brain. CIC-6 is a 97-kDa protein, and CIC-7 a 89-kDa protein roughly 45% identical with CIC-6. Together they define a new branch of this gene family. Both genes are very broadly expressed, e.g. in brain, testes, muscle and kidney. In mouse embryos, both genes are expressed as early as day 7. While the human gene for CIC-6 is located on human chromosome lp36 and shares this region with hCIC-Ka and hCIC-Kb, CIC-7 is on 16p13. CIC-6 has a highly conserved glycosylation site between transmembrane domains D8 and D9, while CIC-7 is the only known eukaryotic CIC protein which lacks this site. Hydropathy analysis indicates that domain D4 cannot serve as a transmembrane domain. Both CIC-6 and CIC-7 cannot be expressed as chloride channels in Xenopus oocytes, either singly or in combination.

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“…3 -5 The vast majority of these are found in recessive pedigrees, whereas approximately only 10 mutations exclusively result in dominant MC presumably through a dominant-negative effect. 6 More that 10 mutations have been associated with both recessive and dominant MC, 3 making a clear distinction between the two modes of inheritance of the disease difficult. This peculiar phenomenon has been explained by reduced penetrance of dominant-negative mutations, incomplete dominance, founder effect and incomplete mutation detection.…”

Section: Introductionmentioning

confidence: 99%

Difference in allelic expression of the CLCN1 gene and the possible influence on the myotonia congenita phenotype

et al. 2004

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Mutations in the CLCN1 gene, encoding a muscle-specific chloride channel, can cause either recessive or dominant myotonia congenita (MC). The recessive form, Becker's myotonia, is believed to be caused by two loss-of-function mutations, whereas the dominant form, Thomsen's myotonia, is assumed to be a consequence of a dominant-negative effect. However, a subset of CLCN1 mutations can cause both recessive and dominant MC. We have identified two recessive and two dominant MC families segregating the common R894X mutation. Real-time quantitative RT-PCR did not reveal any obvious association between the total CLCN1 mRNA level in muscle and the mode of inheritance, but the dominant family with the most severe phenotype expressed twice the expected amount of the R894X mRNA allele. Variation in allelic expression has not previously been described for CLCN1, and our finding suggests that allelic variation may be an important modifier of disease progression in myotonia congenita.

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Multimeric structure of ClC-1 chloride channel revealed by mutations in dominant myotonia congenita (Thomsen).

Cited by 209 publications

References 44 publications

Homo- and Hetero-oligomerization of Ammonium Transporter-1 NH4+ Uniporters

Homo- and Hetero-oligomerization of Ammonium Transporter-1 NH4+ Uniporters

ClC‐6 and ClC‐7 are two novel broadly expressed members of the CLC chloride channel family

Difference in allelic expression of the CLCN1 gene and the possible influence on the myotonia congenita phenotype

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