The ''CLCN5''
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
encodes the
chloride channel
Chloride channels are a superfamily of poorly understood ion channels specific for chloride. These channels may conduct many different ions, but are named for chloride because its concentration ''in vivo'' is much higher than other anions. Several ...
Cl-/H+ exchanger ClC-5. ClC-5 is mainly expressed in the
kidney
The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blo ...
, in particular in
proximal tubule
The proximal tubule is the segment of the nephron in kidneys which begins from the renal pole of the Bowman's capsule to the beginning of loop of Henle. It can be further classified into the proximal convoluted tubule (PCT) and the proximal strai ...
s where it participates to the uptake of
albumin
Albumin is a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water- soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Album ...
and low-molecular-weight proteins, which is one of the principal physiological role of proximal tubular cells.
Mutation
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, m ...
s in the ''CLCN5'' gene cause an
X-linked recessive
X-linked recessive inheritance is a mode of inheritance in which a mutation in a gene on the X chromosome causes the phenotype to be always expressed in males (who are necessarily homozygous for the gene mutation because they have one X and one Y ...
nephropathy named
Dent disease (Dent disease 1 MIM#300009) characterized by excessive urinary loss of low-molecular-weight proteins and of calcium (
hypercalciuria
Hypercalciuria is the condition of elevated calcium in the urine. Chronic hypercalciuria may lead to impairment of renal function, nephrocalcinosis, and chronic kidney disease. Patients with hypercalciuria have kidneys that put out higher levels o ...
),
nephrocalcinosis (presence of calcium phosphate aggregates in the tubular lumen and/or interstitium) and
nephrolithiasis (kidney stones).
The CLCN5 gene
Structure
The human ''CLCN5'' gene (MIM#300008, reference sequence NG_007159.2) is localized in the pericentromeric region on
chromosome
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins ar ...
Xp11.23. It extends over about 170 Kb of
genomic DNA, has a coding region of 2,238 bp and consists of 17
exon
An exon is any part of a gene that will form a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term ''exon'' refers to both the DNA sequence within a gene and to the corresponding sequen ...
s including 11 coding exons (from 2 to 12).
The ''CLCN5'' gene has 8
paralogues
Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spec ...
(''
CLCN1
The CLCN family of voltage-dependent chloride channel genes comprises nine members (CLCN1-7, Ka and Kb) which demonstrate quite diverse functional characteristics while sharing significant sequence homology. The protein encoded by this gene regula ...
'', ''
CLCN2
Chloride channel protein 2 is a protein that in humans is encoded by the ''CLCN2'' gene. Mutations of this gene have been found to cause leukoencephalopathy and Idiopathic generalised epilepsy (), although the latter claim has been disputed. CLCN ...
'', ''
CLCN3
H+/Cl− exchange transporter 3 is a protein that in humans is encoded by the ''CLCN3'' gene.
Interactions
CLCN3 has been shown to interact with PDZK1.
See also
* Chloride channel
Chloride channels are a superfamily of poorly understood ...
'',
''CLCN''4, ''
CLCN6'', ''
CLCN7
Chloride channel 7 alpha subunit also known as H+/Cl− exchange transporter 7 is a protein that in humans is encoded by the CLCN7 gene. In melanocytic cells this gene is regulated by the Microphthalmia-associated transcription factor.
Clinical ...
'', ''
CLCNKA'', ''
CLCNKB'') and 201
orthologues
Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spec ...
among jawed vertebrates (''
Gnathostomata
Gnathostomata (; from Greek: (') "jaw" + (') "mouth") are the jawed vertebrates. Gnathostome diversity comprises roughly 60,000 species, which accounts for 99% of all living vertebrates, including humans. In addition to opposing jaws, livi ...
'').
Five different ''CLCN5''
gene transcripts have been discovered, two of which (transcript variants 3
M_000084.5and 4
M_001282163.1 encode for the canonical 746 amino acid
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
, two (transcript variants 1
M_001127899.3and 2
M_001127898.3 for the NH
2-terminal extended 816 amino acid protein
and one does not encode for any protein (Transcript variant 5,
M_001272102.2.
The
5’ untranslated region (5’UTR) of ''CLCN5'' is complex and not entirely clarified. Two strong and one weak
promoters were predicted to be present in the ''CLCN5'' gene.
Several different 5’ alternatively used exons have been recognized in the human kidney.
The three promoters drive with varying degree of efficiency 11 different
mRNAs
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
, with transcription initiating from at least three different start sites.
The chloride channel H+/Cl− exchanger ClC-5
Like all ClC channels, ClC-5 needs to dimerize to create the pore through which the ions pass.
ClC-5 can form both homo- and hetero-
dimers due to its marked sequence homology with
ClC-3 and
ClC-4.
The canonical 746-
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...
ClC-5 protein has 18
membrane
A membrane is a selective barrier; it allows some things to pass through but stops others. Such things may be molecules, ions, or other small particles. Membranes can be generally classified into synthetic membranes and biological membranes. ...
spanning
α-helices
The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
(named A to R), an intracellular N- terminal
domain
Domain may refer to:
Mathematics
*Domain of a function, the set of input values for which the (total) function is defined
** Domain of definition of a partial function
** Natural domain of a partial function
**Domain of holomorphy of a function
* ...
and a cytoplasmic C-terminus containing two
cystathionine beta-synthase (CBS) domains which are known to be involved in the regulation of ClC-5 activity.
Helices B, H, I, O, P, and Q are the six major helices involved in the formation of dimer’s interface and are crucial for proper pore configuration.
The Cl
− selectivity filter is principally driven by helices D, F, N, and R, which are conveyed together near the channel center.
Two important amino acids for the proper ClC-5 function are the
glutamic acid
Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can synt ...
s at position 211 and 268 called respectively “gating glutamate” and “proton glutamate”.
The gating glutamate is necessary for both H
+ transport and ClC-5 voltage dependence.
The proton glutamate is crucial to the H
+ transport acting as an H
+ transfer site.
Localization and function
ClC-5 belongs to the family of voltage gated chloride channel that are regulators of membrane excitability, transepithelial transport and cell volume in different
tissues. Based on sequence homology, the nine mammalian ClC proteins can be grouped into three classes, of which the first (
ClC-1,
ClC-2,
ClC-Ka and
ClC-Kb) is expressed primarily in plasma membranes, whereas the other two (
ClC-3,
ClC-4, and ClC-5 and
ClC-6 and
ClC-7) are expressed primarily in
organellar
In cell biology, an organelle is a specialized subunit, usually within a cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as organs are to the body, hence ''organelle,'' the ...
membranes.
ClC-5 is expressed in minor to moderate level in
brain
A brain is an organ (biology), organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as Visual perception, vision. I ...
,
muscle
Skeletal muscles (commonly referred to as muscles) are organs of the vertebrate muscular system and typically are attached by tendons to bones of a skeleton. The muscle cells of skeletal muscles are much longer than in the other types of mus ...
,
intestine
The gastrointestinal tract (GI tract, digestive tract, alimentary canal) is the tract or passageway of the digestive system that leads from the mouth to the anus. The GI tract contains all the major organs of the digestive system, in humans an ...
but highly in the kidney, primarily in proximal tubular cells of S3 segment, in alfa intercalated cells of cortical
collecting duct
The collecting duct system of the kidney consists of a series of tubules and ducts that physically connect nephrons to a minor calyx or directly to the renal pelvis. The collecting duct system is the last part of nephron and participates in elect ...
of and in cortical and medullary thick ascending limb of
Henle’s loop.
Proximal tubular cells (PTCs) are the main site of ClC-5 expression. By means of the
receptor-mediated endocytosis
Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins – and in some cases viruses – by the inward budding of the plasma membrane ( invagination). Th ...
process, they uptake albumin and low-molecular-weight proteins freely passed through the
glomerular filter. ClC-5 is located in early
endosome
Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membrane can ...
s of PTCs where it co-localizes with the electrogenic vacuolar H
+‐ATPase (
V‐ATPase).
ClC-5 in this compartment contributes to the maintenance of intra-endosomal acidic
pH. Environment acidification is necessary for the dissociation of
ligand
In coordination chemistry, a ligand is an ion or molecule (functional group) that binds to a central metal atom to form a coordination complex. The bonding with the metal generally involves formal donation of one or more of the ligand's elect ...
from its
receptor
Receptor may refer to:
*Sensory receptor, in physiology, any structure which, on receiving environmental stimuli, produces an informative nerve impulse
*Receptor (biochemistry), in biochemistry, a protein molecule that receives and responds to a n ...
. The receptor is then recycled to the apical membrane, while ligand is transported to the late endosome and
lysosome
A lysosome () is a membrane-bound organelle found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane p ...
where it is degraded. ClC-5 supports efficient acidification of endosomes either by providing a Cl
− conductance to counterbalance the accumulation of positively charged H
+ pumped in by V-ATPase or by directly acidifying endosome in parallel with V-ATPase.
Experimental evidence indicates that endosomal Cl
− concentration, which is raised by ClC-5 in exchange for protons accumulated by the V-ATPase, may play a role in endocytosis independently from endosomal acidification, thus pointing to another possible mechanism by which ClC-5 dysfunction may impair endocytosis.
ClC-5 is located also at the cell surface of PTCs where probably it plays a role in the formation/function of the endocytic complex that also involves
megalin
Low density lipoprotein receptor-related protein 2 also known as LRP-2 or megalin is a protein which in humans is encoded by the ''LRP2'' gene.
Function
LRP2 was identified as the antigen of rat experimental membranous nephropathy (Heyman neph ...
and
cubilin
Cubilin is a protein that in humans is encoded by the ''CUBN'' gene.
Function
Cubilin (CUBN) acts as a receptor for intrinsic factor-vitamin B12 complexes. The role of receptor is supported by the presence of 27 CUB domains. Cubilin shows a re ...
/
amnionless receptors, the sodium-hydrogen antiporter 3 (
NHE3), and the V-ATPase.
It was demonstrated at the C-terminus of ClC-5 binds the
actin
Actin is a family of globular multi-functional proteins that form microfilaments in the cytoskeleton, and the thin filaments in muscle fibrils. It is found in essentially all eukaryotic cells, where it may be present at a concentration of ov ...
-depolymerizing protein
cofilin. When the nascent endosome forms, the recruitment of cofilin by ClC-5 is a prerequisite for the localized dissolution of the actin
cytoskeleton
The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is co ...
, thus permitting the endosome to pass into the
cytoplasm
In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. ...
. It is conceivable that at the cell surface, the large intracellular C-terminus of ClC-5 has a crucial function in mediating the assembly, stabilization and disassembly of the endocytic complex via protein–protein interactions. Therefore, ClC-5 may accomplish two roles in the receptor-mediated endocytosis: i) vesicular acidification and receptor recycling; ii) participation to the non-selective megalin–cubilin-amnionless low-molecular-weight protein uptake at the apical membrane.
Clinical significance
Dent disease is mainly caused by
loss-of-function
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosi ...
mutations in the ''CLCN5'' gene (Dent disease 1; MIM#300009).
Dent disease 1 shows a marked
allelic heterogeneity
Allelic heterogeneity is the phenomenon in which different mutations at the same locus lead to the same or very similar phenotypes. These allelic variations can arise as a result of natural selection processes, as a result of exogenous mutagens ...
. To date, 265 different ''CLCN5'' pathogenic variants have been described.
A small number of pathogenic variants were found in more than one family.
The 48% are truncating mutations (
nonsense
Nonsense is a communication, via speech, writing, or any other symbolic system, that lacks any coherent meaning. Sometimes in ordinary usage, nonsense is synonymous with absurdity or the ridiculous. Many poets, novelists and songwriters have u ...
,
frameshift or complex), 37% non-truncating (
missense or in-frame
insertions/
deletions), 10%
splice site mutation
A splice site mutation is a genetic mutation that inserts, deletes or changes a number of nucleotides in the specific site at which splicing takes place during the processing of precursor messenger RNA into mature messenger RNA. Splice site c ...
s, and 5% other type (large deletions,
Alu insertions or 5’UTR mutations). Functional investigations in ''
Xenopus laevis
The African clawed frog (''Xenopus laevis'', also known as the xenopus, African clawed toad, African claw-toed frog or the ''platanna'') is a species of African aquatic frog of the family Pipidae. Its name is derived from the three short claws o ...
''
oocyte
An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female ...
s and mammalian cells
enabled these ''CLCN5'' mutations to be classified according to their functional consequences.
The most common mutations lead to a defective
protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduc ...
and
processing
Processing is a free graphical library and integrated development environment (IDE) built for the electronic arts, new media art, and visual design communities with the purpose of teaching non-programmers the fundamentals of computer programming ...
, resulting in
endoplasmic reticulum
The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum ...
retention of the mutant protein for further degradation by the
proteasome
Proteasomes are protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases.
Proteasomes are part of a major mechanism by whi ...
.
Animal models
Two independent ClC-5
knock-out mice
A knockout mouse, or knock-out mouse, is a genetically modified mouse (''Mus musculus'') in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are important ...
, the so called Jentsch
and Guggino models,
provided critical insights into the mechanisms of proximal tubular dysfunction in Dent disease 1. These two murine models recapitulated the major features of Dent disease (low-molecular-weight
proteinuria
Proteinuria is the presence of excess proteins in the urine. In healthy persons, urine contains very little protein; an excess is suggestive of illness. Excess protein in the urine often causes the urine to become foamy (although this symptom ma ...
,
hypercalciuria
Hypercalciuria is the condition of elevated calcium in the urine. Chronic hypercalciuria may lead to impairment of renal function, nephrocalcinosis, and chronic kidney disease. Patients with hypercalciuria have kidneys that put out higher levels o ...
and
nephrocalcinosis/
nephrolithiasis) and demonstrated that ClC-5 inactivation is associated with severe impairment of both fluid phase and
receptor-mediated endocytosis
Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins – and in some cases viruses – by the inward budding of the plasma membrane ( invagination). Th ...
, as well as
trafficking
Smuggling is the illegal transportation of objects, substances, information or people, such as out of a house or buildings, into a prison, or across an international border, in violation of applicable laws or other regulations.
There are various ...
defects leading to the loss of
megalin
Low density lipoprotein receptor-related protein 2 also known as LRP-2 or megalin is a protein which in humans is encoded by the ''LRP2'' gene.
Function
LRP2 was identified as the antigen of rat experimental membranous nephropathy (Heyman neph ...
and
cubilin
Cubilin is a protein that in humans is encoded by the ''CUBN'' gene.
Function
Cubilin (CUBN) acts as a receptor for intrinsic factor-vitamin B12 complexes. The role of receptor is supported by the presence of 27 CUB domains. Cubilin shows a re ...
at the
brush border
A brush border (striated border or brush border membrane) is the microvilli-covered surface of simple cuboidal and simple columnar epithelium found in different parts of the body. Microvilli are approximately 100 nanometers in diameter and the ...
of proximal tubules. However, targeted disruption of ClC-5 in the Jentsch model did not lead to
hypercalciuria
Hypercalciuria is the condition of elevated calcium in the urine. Chronic hypercalciuria may lead to impairment of renal function, nephrocalcinosis, and chronic kidney disease. Patients with hypercalciuria have kidneys that put out higher levels o ...
,
kidney stones
Kidney stone disease, also known as nephrolithiasis or urolithiasis, is a crystallopathy where a solid piece of material (kidney stone) develops in the urinary tract. Kidney stones typically form in the kidney and leave the body in the urine s ...
or
nephrocalcinosis, while the Guggino model did.
The Jentsch murine model produced slightly more acidic urines. Urinary phosphate excretion was increased in both models by about 50%. Hyperphosphaturia in the Jentsch model was associated with decreased apical expression of the sodium/phosphate cotransporter
NaPi2a that is the predominant phosphate transporter in the proximal tubule. However, NaPi2a expression is ClC-5-independent since apical NaPi2a was normally expressed in any proximal tubules of chimeric female mice, while it was decreased in all male proximal tubular knock-out cells. Serum parathormone (PTH) is normal in knock-out mice while urinary PTH is increased of about 1.7 fold. Megalin usually mediates the endocytosis and degradation of PTH in proximal tubular cells. In knock-out mice, the downregulation of megalin leads to PTH defective endocytosis and progressively increases luminal PTH levels that enhance the internalization of NaPi2a.
DNA testing and genetic counselling
A clinical diagnosis of Dent disease can be confirmed through molecular genetic testing that can detect mutations in specific genes known to cause Dent disease. However, about 20-25% of Dent disease patients remain genetically unresolved.
Genetic testing is useful to determine the status of
healthy carrier in the mother of an affected male. In fact, being Dent disease an
X-linked recessive disorder, males are more frequently affected than females, and females may be
heterozygous
Zygosity (the noun, zygote, is from the Greek "yoked," from "yoke") () is the degree to which both copies of a chromosome or gene have the same genetic sequence. In other words, it is the degree of similarity of the alleles in an organism.
Mo ...
healthy carrier. Due to
skewed X-inactivation
Skewed X-chromosome inactivation (skewed X-inactivation) occurs when the X-inactivation of one X chromosome is favored over the other, leading to an uneven number of cells with each chromosome inactivated. It is usually defined as one allele being ...
, female carriers may present some mild symptoms of Dent disease such as low-molecular-weight
proteinuria
Proteinuria is the presence of excess proteins in the urine. In healthy persons, urine contains very little protein; an excess is suggestive of illness. Excess protein in the urine often causes the urine to become foamy (although this symptom ma ...
or
hypercalciuria
Hypercalciuria is the condition of elevated calcium in the urine. Chronic hypercalciuria may lead to impairment of renal function, nephrocalcinosis, and chronic kidney disease. Patients with hypercalciuria have kidneys that put out higher levels o ...
. Carriers will transmit the disease to half of their sons whereas half of their daughters will be carriers. Affected males do not transmit the disease to their sons since they pass
Y chromosome
The Y chromosome is one of two sex chromosomes (allosomes) in therian mammals, including humans, and many other animals. The other is the X chromosome. Y is normally the sex-determining chromosome in many species, since it is the presence or abse ...
to males, but all their daughters will inherited mutated
X chromosome
The X chromosome is one of the two sex-determining chromosomes (allosomes) in many organisms, including mammals (the other is the Y chromosome), and is found in both males and females. It is a part of the XY sex-determination system and XO sex-d ...
.
Preimplant and
prenatal genetic testing is not advised for Dent disease 1 since the prognosis for the majority of the patients is good and a clear correlation between
genotype
The genotype of an organism is its complete set of genetic material. Genotype can also be used to refer to the alleles or variants an individual carries in a particular gene or genetic location. The number of alleles an individual can have in a ...
and
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
is lacking.
See also
*
Chloride channel
Chloride channels are a superfamily of poorly understood ion channels specific for chloride. These channels may conduct many different ions, but are named for chloride because its concentration ''in vivo'' is much higher than other anions. Severa ...
Notes
References
Further reading
*
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External links
*
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{{Ion channels, g4
Ion channels