Paroxysmal kinesigenic dyskinesia (PKD) is a heterogeneous movement disorder characterized by recurrent dyskinesia attacks triggered by sudden movement. PRRT2 has been identified as the first causative gene of PKD. However, it is only responsible for approximately half of affected individuals, indicating that other loci are most likely involved in the etiology of this disorder. To explore the underlying causative gene of PRRT2-negative PKD, we used a combination strategy including linkage analysis, whole-exome sequencing and copy number variations analysis to detect the genetic variants within a family with PKD. We identified a linkage locus on chromosome 12 (12p13.32-12p12.3) and detected a novel heterozygous mutation c.956 T>G (p.319 L>R) in the potassium voltage-gated channel subfamily A member 1, KCNA1. Whole-exome sequencing in another 58 Chinese patients with PKD who lacked mutations in PRRT2 revealed another novel mutation in the KCNA1 gene [c.765 C>A (p.255 N>K)] within another family. Biochemical analysis revealed that the L319R mutant accelerated protein degradation via the proteasome pathway and disrupted membrane expression of the Kv1.1 channel. Electrophysiological examinations in transfected HEK293 cells showed that both the L319R and N255K mutants resulted in reduced potassium currents and respective altered gating properties, with a dominant negative effect on the Kv1.1 wild-type channel. Our study suggests that these mutations in KCNA1 cause the Kv1.1 channel dysfunction, which leads to familial PKD. The current study further extended the genotypic spectrum of this disorder, indicating that Kv1.1 channel dysfunction maybe one of the underlying defects in PKD.
Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metalloproteinase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (10, 100 or 200 μmol/L) or an MMP-2 inhibitor (10(-6) or 10(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, bone morphogenetic protein (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification proteins, and decreased anti-calcification protein levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification.
Transient receptor potential melastatin 7 (TRPM7) plays a key role in the pathophysiological response of multiple cell types. However, the role of TRPM7 channels in ox-LDL-induced proliferation and migration of VSMC remains unclear. This study used the thoracic aorta VSMCs to explore the effects of ox-LDL on cell proliferation and migration and to investigate the underlying molecular mechanisms and signaling pathways. Data demonstrated that ox-LDL significantly increased TRPM7 activity, and induced VSMC proliferation and migration. VSMC proliferation and migration were inhibited by nonspecific TRPM7 blocker 2-APB or synthetic siRNA targeting TRPM7. Furthermore, the phosphorylation of ERK1/2 and MEK1/2 associated with cell proliferation and migration decreased in TRPM7-deficient VSMC. Therefore, TRPM7 may constitute a useful target for the treatment of atherosclerosis.Keywords: MEK-ERK pathways; migration; proliferation; TRPM7; VSMCs Migration and excess proliferation of vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of various vascular diseases, such as atherosclerosis [1]. Oxidized low-density lipoprotein (Ox-LDL) contributes to the atherosclerotic plaque formation and progression by several mechanisms, including the induction of endothelial cell activation and dysfunction, macrophage foam cell formation, and smooth muscle cell migration and proliferation [2]. Ox-LDL significantly increases VSMC migration and proliferation through the ERK1/2 signaling pathway [3]. However, the mechanisms underlying the effect of ox-LDL on atherosclerosis are not fully understood.Ion channels participate in cell proliferation in diverse types of cells, including vascular endothelial cells [4], cancer cells [5], smooth muscle cells [6], and mesenchymal stem cells [7]. Transient receptor potential melastatin 7 (TRPM7) channel, a member of TRP channel superfamily [8], is a widely expressed nonselective divalent cation (Ca 2+ and Mg 2+ ) channel with protein serine/threonine kinase activity that regulates diverse physiological/pathological processes, such as Mg 2+ homeostasis [9,10], hypoxic neuronal injury [11], and tumor cell growth/proliferation [12]. Activating TRPM7 channels may also contribute to the physiology/pathophysiology of cardiovascular and cerebrovascular diseases, including arterial calcification [13], atrial fibrillation [14], and hypertension [6,15]. However, little is known about the role of TRPM7 channels in atherosclerosis-related vasculopathy.Studies have shown that TRPM7 channels greatly participate in growth/proliferation and nitric oxide production of vascular endothelial cells [4], angiotensin IIinduced phenotype switching, and VSMC proliferation through the ERK pathway [6]. Although the presence and potential function of TRPM7 channels have been Abbreviations 2-APB, 2-aminoethoxydiphenyl borate; ang II, angiotensin II; DMSO, dimethyl sulfoxide; MAPK, mitogen-activated protein kinase; Ox-LDL, oxidized low-density lipoprotein; PDGF, platelet-derived growth factor; PLC, phospholipase C;...
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