In recent years, new hope for understanding the pathogenesis of Parkinson's disease (PD) and Lewy body dementia (LBD) has emerged with the discovery of mutations and duplications in the a-synuclein (a-syn) gene that are associated with rare familial forms of Parkinsonism [1][2][3]. Moreover, it has been shown that a-syn is centrally involved in the pathogenesis of both sporadic and inherited forms of PD and LBD because this molecule accumulates in Lewy bodies (LBs) [4][5][6], synapses, and axons, and its expression in transgenic (tg) mice [7][8][9] and Drosophila [10] mimics several aspects of PD.The mechanisms through which a-syn leads to neurodegeneration and the characteristic symptoms of LBD are unclear. However, recent evidence indicates that abnormal accumulation of misfolded a-syn in the Accumulation of a-synuclein resulting in the formation of oligomers and protofibrils has been linked to Parkinson's disease and Lewy body dementia. In contrast, b-synuclein (b-syn), a close homologue, does not aggregate and reduces a-synuclein (a-syn)-related pathology. Although considerable information is available about the conformation of a-syn at the initial and end stages of fibrillation, less is known about the dynamic process of a-syn conversion to oligomers and how interactions with antiaggregation chaperones such as b-synuclein might occur. Molecular modeling and molecular dynamics simulations based on the micelle-derived structure of a-syn showed that a-syn homodimers can adopt nonpropagating (head-to-tail) and propagating (head-to-head) conformations. Propagating a-syn dimers on the membrane incorporate additional a-syn molecules, leading to the formation of pentamers and hexamers forming a ring-like structure. In contrast, b-syn dimers do not propagate and block the aggregation of a-syn into ring-like oligomers. Under in vitro cell-free conditions, a-syn aggregates formed ring-like structures that were disrupted by b-syn. Similarly, cells expressing a-syn displayed increased ion current activity consistent with the formation of Zn 2+ -sensitive nonselective cation channels. These results support the contention that in Parkinson's disease and Lewy body dementia, a-syn oligomers on the membrane might form pore-like structures, and that the beneficial effects of b-synuclein might be related to its ability to block the formation of pore-like structures.Abbreviations aa, amino acid; a-syn, a-synuclein; b-syn, b-synuclein; GFP, green fluorescent protein; LBD, Lewy body disease; PD, Parkinson's disease; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; tg, transgenic.

Background-Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role in the development of idiopathic pulmonary arterial hypertension (IPAH), whereas a rise in cytosolic Ca 2ϩ concentration triggers PASMC contraction and stimulates PASMC proliferation. Recently, we demonstrated that upregulation of the TRPC6 channel contributes to proliferation of PASMCs isolated from IPAH patients. This study sought to identify single-nucleotide polymorphisms (SNPs) in the TRPC6 gene promoter that are associated with IPAH and have functional significance in regulating TRPC6 activity in PASMCs. Methods and Results-Genomic DNA was isolated from blood samples of 237 normal subjects and 268 IPAH patients.Three biallelic SNPs, Ϫ361 (A/T), Ϫ254(C/G), and Ϫ218 (C/T), were identified in the 2000-bp sequence upstream of the transcriptional start site of TRPC6. Although the allele frequencies of the Ϫ361 and Ϫ218 SNPs were not different between the groups, the allele frequency of the Ϫ254(C3 G) SNP in IPAH patients (12%) was significantly higher than in normal subjects (6%; PϽ0.01). Genotype data showed that the percentage of Ϫ254G/G homozygotes in IPAH patients was 2.85 times that of normal subjects. Moreover, the Ϫ254(C3 G) SNP creates a binding sequence for nuclear factor-B. Functional analyses revealed that the Ϫ254(C3 G) SNP enhanced nuclear factor-B-mediated promoter activity and stimulated TRPC6 expression in PASMCs. Inhibition of nuclear factor-B activity attenuated TRPC6 expression and decreased agonist-activated Ca 2ϩ influx in PASMCs of IPAH patients harboring the Ϫ254G allele. Conclusions-These results suggest that the Ϫ254(C3 G) SNP may predispose individuals to an increased risk of IPAH by linking abnormal TRPC6 transcription to nuclear factor-B, an inflammatory transcription factor. (Circulation. 2009;119:2313-2322.)Key Words: calcium Ⅲ hypertension, pulmonary Ⅲ ion channels Ⅲ muscle, smooth Ⅲ NF-kappa B P ulmonary arterial hypertension (PAH) is a fatal and progressive disease characterized by elevated pulmonary vascular resistance resulting from severe pulmonary vascular remodeling. [1][2][3] Approximately 6% of PAH patients have a family history of the condition and are referred to as having familial PAH; the rest are considered to have idiopathic PAH (IPAH). Although the cause of PAH remains unclear, elevated levels of mitogenic, angiogenic, and proinflammatory factors such as platelet-derived growth factor, endothelin-1, interleukin-1␤/-6, soluble CD40 ligand, angiopoietin-1, and serotonin have been reported to correlate with the onset of IPAH. [2][3][4] Other factors associated with IPAH include the downregulation and dysfunction of voltage-gated K ϩ channels 5 and upregulation of the serotonin receptors and transporter. 6 Moreover, mutations in the bone morphogenetic protein receptor-type II gene (BMPR2) have been demonstrated to associate with the development of familial PAH and IPAH. 7,8 However, because BMPR2 mutations are present in Received March 25, 2008; accepted Februar...

The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of ␣-␥-␣-␦-␤. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg 313 -Lys 314 sequence in the large cytoplasmic loop of the ␣-subunit to K314Q promotes the trafficking of the mutant unassembled ␣-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q ␣-subunit with ␥-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type ␣-subunits when co-expressed with the ␤-, ␦-, and ␥-subunits. This suggests that the Arg 313 -Lys 314 sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the ␣-subunit. Although unassembled K314Q ␣-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q ␣-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface.Multimeric transmembrane proteins, including complex ligand-gated ion channels represented by nicotinic acetylcholine receptors (nAchR), 1 generally require subunit assembly to be transported beyond the endoplasmic reticulum (ER) into the secretory pathway leading to the cell surface (1-3). As integral membrane components of lipid trafficking vesicles, unassembled subunits are re-localized to the ER, stabilized by chaperones and either assembled with neighboring subunits or targeted for degradation by ubiquitination and cleavage in the proteasome (4, 5). Cellular mechanisms that distinguish whether a protein is folded and/or assembled and directed to the cell surface or misfolded and/or unassembled and shuttled into a degradative pathway are poorly understood, especially for the polytypic membrane proteins represented by ligandgated ion channels. The importance for shedding light on this topic is borne out by several debilitating disorders associated with mutations that are believed to cause misfolding and inhibit the trafficking of physiologically important ion channels. Examples include inherited mutations in potassium channel subunits that increase the propensity to develop cardiac arrhythmias (6, 7) and amino acid substitutions in cystic fibrosis transmembrane conductance regulator that result in cystic fibrosis (8 -10). In this study, we employ the nAChR ␣-subunits as a model to identify fac...