KCTD7 expression hyperpolarizes the cell membrane and reduces the excitability of transfected neurons in patch clamp experiments. KCTD7 mRNA and protein are expressed in hippocampal neurons, deep layers of the cerebral cortex and Purkinje cells of the murine brain as shown by in situ hybridization and immunohistochemistry experiments. Immunoprecipitation assays demonstrates that KCTD7 is able to prudhommerie and directly interacts with cullin-3, a component of the ubiquitin ligase complex. These interactions are thought to be mediated via the BTB/POZ domain of KCTD7. However, KCTD7 does not show any interaction cullin-1. Immunoprecipitation assays also shows that KCTD7 does not interact with Ubiquitin-flag, suggesting a potential role of KCTD7 in the ubiquitin ligase complex without being itself subject to uiquitination. Immunofluorescence microscopy shows a cytosolic expression of the recombinant GFP-KCTD7 protein in transfected COS-7 cells. One possible hypothesis is that KCTD7 regulates indirectly the membrane expression level of a potassium channel. By conjugating with cullin-3 ubiquitin ligase complex, KCTD7 may modulate the expression level of a negative regulator of potassium channel. Therefore, the overexpression of KCTD7 in neurons would increase the degradation of that regulatory molecule leading to the increase of potassium current through the cell membrane as observed in patch clamp experiments. In cultured mouse hippocampal cells, expression is found in the cell soma, in neuritic varicosities along the developing neuronal extensions, and in neurite growth cones, but not in the nucleus. Kctd7 is widely expressed in neurons throughout the intact mouse brain, including in cortical neurons, in granular and pyramidal cell layers of the hippocampus, and in cerebellar Purkinje cells. However, not all neuronal cells are immunopositive for Kctd7, and expression is not seen in astrocytes or microglial cells. Expression is constant from P5 to 2 months in cerebellar lysates. Overexpression of KCTD7 in HeLa and COS-1 cells, which do not express endogenous KCTD7, shows diffuse cytosolic localization, with no colocalization with markers for endosomes, ER, Golgi, lysosomes, or the cytoskeleton. Beside the BTB/POZ domain of KCTD7, other residues are critical for its proper interaction with cullin-3. Furthermore, a full-length 31-kD Kctd7 isoform is expressed in mouse brain. Other major immunoreactive bands included a 28-kD species in the spleen, liver, and kidneys, a 37-kD species in the kidneys, and a 62-kD form most likely corresponding to a stable dimer. The presence of multiple bands was consistent with alternative splicing and tissue-specific regulation.
Clinical significance
In 3 affected members of a large consanguineous Moroccan family with progressive myoclonic epilepsy-3, a homozygous nonsense mutation in the KCTD7 gene has been identified. In 2 Mexican siblings with infantile onset of progressive myoclonic epilepsy and pathologic findings of neuronal ceroid lipofuscinosis in multiple cell types, a homozygous mutation in the KCTD7 gene has been identified. The mutation was identified by whole-exome sequencing and confirmed by Sanger sequencing. This phenotype has been identified as CLN14. KCTD7 mutations were not found in 32 additional CLN samples.
