Abstract
ClC-7
is a ubiquitously expressed chloride channel whose function was unknown
so far. Mice deficient for ClC-7 were generated using two different gene
targeting strategies. Whereas targeting construct C7A abolished expression
of ClC-7 completely, the LacZ gene was fused in frame into exon
7 of Clcn7 as a reporter gene in construct C7B. Using the reporter
gene to assess the expression pattern of ClC-7, we detected strong signals
in osteoclasts, dorsal root ganglia, trigeminal ganglion, retina, spinal
cord and brain beside a ubiquitous basal expression level. These findings
were corroborated by in situ hybridization.
Clcn7-/- mice of both knockout-strains
show the same severe osteopetrosis (marble bone disease) that becomes apparent
shortly after birth, leading to an increased early postnatal lethality.
Life expectancy of the surviving animals was reduced to maximal 45 days.
Those mice were anemic and much smaller than wildtype mice. Additionally,
there was progressive neurodegeneration in the retina and the brain. It
could be excluded that this degeneration was secondary to osteopetrotic
changes in Clcn7-/- mice. An accumulation of a lipofuscine-like
material was detected in cerebral neurons, whereas no such material could
be seen in the retina. Although osteoclasts are present in normal numbers,
they fail to resorb bone because they cannot acidify the extracellular
resorption lacuna. In osteoclasts, ClC-7 is highly expressed in the ruffled
membrane that is formed by the fusion of H+-ATPase containing
vesicles and that secretes protons into the lacuna. In the proximal tubule
of the kidney ClC-7 probably resides in late endosomes and lysosomes.
The murine phenotype closely resembles human
infantile malignant osteopetrosis. Therefore, in 12 patients suffering
from this disease a screening for mutations in the human gene, CLCN7,
was performed. One patient was compound heterozygous for a nonsense and
a missense mutation. These mutations lead to a complete loss of the ClC-7
protein from cultured fibroblasts of the patient. It is concluded that
ClC-7 provides the chloride conductance required for an efficient proton
pumping by the H+-ATPase of the osteoclast ruffled membrane.
The insufficient acidification in Clcn7-/- mice therefore
prevents bone resorption. A similar acidification defect in lysosomal compartments
may cause neuronal storage because of decreased degradation.
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