Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
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DOI：
10.1371/journal.pgen.1004376
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发表时间：
2014-07
期刊：
影响因子：
4.5
通讯作者：
North American Pancreatitis Study Group
中科院分区：
文献类型：
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作者：
LaRusch J;Jung J;General IJ;Lewis MD;Park HW;Brand RE;Gelrud A;Anderson MA;Banks PA;Conwell D;Lawrence C;Romagnuolo J;Baillie J;Alkaade S;Cote G;Gardner TB;Amann ST;Slivka A;Sandhu B;Aloe A;Kienholz ML;Yadav D;Barmada MM;Bahar I;Lee MG;Whitcomb DC;North American Pancreatitis Study Group
CFTR是一个动态调节的阴离子通道。细胞内WNK1-SPAK激活导致CFTR通过未知的机制改变从氯离子偏好通道到碳酸氢盐偏好通道的渗透性和电导特性。两种严重的CFTR突变（CFTRsev）导致CFTR功能完全丧失，并导致囊性纤维化（CF），这是一种影响汗腺、鼻窦、肺、胰腺、肝脏、肠道和男性生殖系统的严重遗传性疾病。我们假设，那些破坏WNK1-SPAK激活机制的CFTR突变会导致通道功能的选择性碳酸氢盐缺陷（CFTRBD），影响利用CFTR分泌碳酸氢盐的器官（如胰腺、鼻窦、输精管），但不会导致典型的CF。为了了解CFTR偏好碳酸氢盐通道的结构和功能要求，我们(a)筛选了984例表型良好的胰腺炎病例，从81个先前描述的CFTR变体中筛选候选CFTRBD突变；(b)对在胰腺炎而非CF中发现的变异克隆进行电生理学研究；(c)通过计算构建了新的完整的CFTR结构模型，用于野生型和突变型变异体的分子动力学模拟；(d)利用CFTR检测碳酸氢盐分泌，检测新定义的CFTRBD变异对非胰腺器官疾病的影响。与典型CF无关的9个变异（CFTR R74Q、R75Q、R117H、R170H、L967S、L997F、D1152H、S1235R和D1270N）与胰腺炎相关（OR 1.5, p = 0.002）。在WNK1-SPAK活化的HEK 293T细胞中表达的克隆具有正常的氯化物渗透性和电导率，而不是碳酸氢盐。分子动力学模拟表明CFTR通道的物理限制和动态通道调节的改变。比较胰腺炎患者和对照组，CFTRBD增加了鼻窦炎（OR 2.3, p<0.005）和男性不育（OR 395, p<<0.0001）的风险。WNK1-SPAK通路激活的CFTR碳酸氢盐渗透性增加通过多种机制被CFTRBD变异所改变。CFTRBD变异与胰腺、鼻窦和男性生殖系统的临床显著疾病相关。离子通道的遗传紊乱会在许多方面影响身体正常运作的能力。CFTR是一种离子通道，调节氯离子和碳酸氢盐在细胞膜上的运动，对吸收和分泌液体很重要。如果负责CFTR通道的基因发生严重突变，就会导致囊性纤维化，这是一种遗传性疾病，患者会出现粘稠的粘液，尤其是在肺部，同时胰腺也会形成疤痕（纤维化）。囊性纤维化还会影响汗腺、鼻窦、肠道、肝脏和男性生殖系统。不引起囊性纤维化的CFTR基因突变被认为是良性的。然而，我们发现9个CFTR突变不会引起囊性纤维化，但会引起胰腺炎症和瘢痕形成（慢性胰腺炎）。这些突变的CFTR通道分泌氯化物，这在汗腺、肺和肠中很重要，但不分泌碳酸氢盐，而碳酸氢盐在胰腺、鼻窦和男性生殖道中很重要。我们发现患有这9种突变中的任何一种的患者都有慢性胰腺炎，经常有鼻窦感染和男性不育，但没有其他囊性纤维化的症状。我们的计算机模型和数据将帮助研究人员开发更好的药物，并帮助医生治疗慢性胰腺炎患者。
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system. Genetic disorders of ion channels can affect the body's ability to function properly in many ways. CFTR, an ion channel regulating movement of chloride and bicarbonate across cell membranes, is important for absorbing and secreting fluids. If the gene responsible for the CFTR channel is mutated severely, the result is cystic fibrosis, a hereditary disorder in which the patient develops thick mucus, especially in the lungs, as well as scarring (fibrosis) in the pancreas. Cystic fibrosis also affects the sweat glands, nasal sinuses, intestines, liver, and male reproductive system. Mutations to the CFTR gene that do not cause cystic fibrosis have been considered benign. However, we discovered 9 CFTR mutations that do not cause cystic fibrosis but do cause inflammation and scarring of the pancreas (chronic pancreatitis). These mutant CFTR channels secrete chloride, which is important in the sweat glands, lungs, and intestines, but not bicarbonate, which is important in the pancreas, sinuses, and male reproductive tract. We found patients with any of these 9 mutations had chronic pancreatitis, and often sinus infections, and male infertility, but not other symptoms of cystic fibrosis. Our computer models and data will help researchers develop better drugs and help physicians treating patients with chronic pancreatitis.