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首页> 外文期刊>Cellular Physiology and Biochemistry >K-Cl cotransport: Immunohistochemical and ion flux studies in human embryonic kidney (HEK293) cells transfected with full-length and C-terminal-domain-truncated KCC1 cDNAs
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K-Cl cotransport: Immunohistochemical and ion flux studies in human embryonic kidney (HEK293) cells transfected with full-length and C-terminal-domain-truncated KCC1 cDNAs

机译：K-Cl共转运：全长和C端域截断的KCC1 cDNAs转染的人胚肾（HEK293）细胞中的免疫组织化学和离子通量研究

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摘要

Coupled K and CI movements are mediated by several isoforms of the K-CI cotransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, important for cell volume and ion homeostasis, has 12 transmembrane domains (Tmds), and cytoplasmic N- and C- terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, activation of K-CI COT by various non-osmotic and osmotic interventions with net unidirectional K and Rb fluxes at 37 degreesC, and the effect of Ctd deletion on K-CI COT regulation. Human embryonic kidney (HEK293) cells were transfected with full-length (fl) rabbit (rb)KCC1 and -CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal anti-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a.943-1020) revealed granular membrane and cytoplasmic immunostaining, presumably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunofluorescence in flKCC1-transfected cells was absent in -CtdKCC1 -transfected cells. Monoclonal antibodies against a c-myc epitope at the protein Ntd showed both membrane and cytosolic fluoresence. Basal and N-ethylmaleimide (NEM)-stimulated Rb influxes through K-CI GOT, calculated as Cl-dependent Rb fluxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significantly lower in stably and abrogated in transiently -CtdKCC1-transfected cells. Furosemide, calyculin and genistein inhibited basal and NEM-stimulated K-Cl COT in normal and transfected cells. Staurosporine and hydroxylamine were ineffective stimulators. No effect of pH(o) changes (16.3-8.4) was observed in basal or NEM-stimulated K-Cl GOT, in both normal and transfected cells. However, inhibition by NEM occurred at pH(o) 8.4. Furthermore, in a Cl-independent manner, NEM lowered cell K content by > 30% and hypotonicity (210-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cultured normal and KCC1-transfected cells, K-C1 COT shows significant differences from erythrocytes, and NEM and cell swelling open furosemide-sensitive and Cl-independent K/Rb channels. Failure of K-CI COT in cells transfected with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction. Copyright (C) 2001 S. Karger AG, Basel. [References: 38]
机译：K和CI的耦合运动是由KCC基因编码的K-CI共转运蛋白（COT）的几种同工型介导的。普遍存在的KCC1亚型对细胞体积和离子稳态具有重要作用，具有12个跨膜结构域（Tmds），以及胞质N和C末端结构域（Ntd和Ctd）。本研究通过共聚焦显微镜研究了KCC1的细胞定位，各种非渗透和渗透干预（在37°C时具有净单向K和Rb通量）激活K-CI COT以及Ctd缺失对K-CI COT调节的影响。在2011年核苷酸截短后获得的全长（fl）兔（rb）KCC1和-CtdKCC1 cDNA转染人胚肾（HEK293）细胞。正常细胞暴露于77个氨基酸序列内的抗Ctd表位的多克隆抗Ctd抗体（aa943-1020）发现颗粒状膜和胞质免疫染色，推测是内源性KCC1。在-CtdKCC1转染的细胞中不存在flKCC1转染的细胞中的其他弥漫膜和细胞质免疫荧光。针对蛋白质Ntd处的c-myc表位的单克隆抗体同时显示了膜和胞质荧光。基础和N-乙基马来酰亚胺（NEM）刺激的通过K-CI GOT刺激的Rb流入（计算为Cl依赖的Rb通量）在经flKCC1转染的细胞中比正常细胞高2至3倍。在flKCC1转染的细胞中，NEM对K-Cl COT的刺激最高，而在瞬时-CtdKCC1转染的细胞中，NEM刺激的稳定性显着降低，而废除。速尿，calyculin和染料木黄酮抑制正常和转染细胞中基础和NEM刺激的K-Cl COT。星形孢菌素和羟胺是无效的刺激物。在正常细胞和转染细胞中，在基础或NEM刺激的K-Cl GOT中均未观察到pH（o）变化的影响（16.3-8.4）。但是，NEM的抑制作用发生在pH（o）8.4。此外，以不依赖Cl的方式，NEM将细胞K含量降低了> 30％，并且低渗（210-70mOsM）刺激了呋塞米敏感的Rb涌入和K丢失。因此，在培养的正常细胞和KCC1转染的细胞中，K-C1 COT与红细胞，NEM和细胞膨胀开放了速尿敏感和不依赖Cl的K / Rb通道，显示出显着差异。用Ctd截短的KCC1转染的细胞中的K-CI COT对NEM的反应失败表明Ctd在信号转导中的作用。版权所有（C）2001 S.Karger AG，巴塞尔。 [参考：38]

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