| 研究実績の概要 |
Exome-wide analysis in a family with inherited Brugada syndrome reveal heterozygous point mutation in TMEM168, a gene with unknown function. Cloning of the human variant and expression in HL-1 cardiomyocytes found that TMEM168 is localized in the nuclear membrane and endogenous Na+ current of HL-1 cells transfected with TMEM168 variant had reduced density and Nav1.5 protein (cardiac Na+ channel alpha subunit). We generated knock-in mice (CRISPR/Cas9 genome editing) that incorporated similar to the studied human family point mutation. Like in HL-1 cells, TMEM168 is localized in the nuclear membrane and co-localized with lamin A (nuclear membrane protein). Nav1.5 protein and Na+ current density were reduced in a similar pattern. ECG recordings in the presence of Ajmaline (Na+ channel blocker) detected ST segment elevation in right precordial leads, conduction disturbances and ventricular arrhythmias selectively in the knock-in animals. There was no transcriptional dysregulation of Na+ channel subunits. We identified increased ubiquitination of Nav1.5 in the knock-in hearts. There was also increased interaction between mutated TMEM168 and alphaB-crystallin, a chaperon protein that is part of Nav1.5 molecular complex and is physiological suppressor of Nedd4 - ubiquitine ligase also associated with Nav1.5. We propose the following mechanism of Nav1.5 regulation: increased affinity of variant TMEM168 protein to alphaB-crystallin deplete its association with Nav1.5 and release inhibitory effect on Nedd4. This results in enhanced ubiquitination and degradation of Nav1.5.
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