利用荧光指示剂对肾刷状缘氯离子转运机制进行表征。

Renal brush-border chloride transport mechanisms characterized using a fluorescent indicator.

作者信息

Chen P Y, Illsley N P, Verkman A S

机构信息

Division of Nephrology and Cardiovascular Research Institute, University of California, San Francisco 94143.

出版信息

Am J Physiol. 1988 Jan;254(1 Pt 2):F114-20. doi: 10.1152/ajprenal.1988.254.1.F114.

DOI:10.1152/ajprenal.1988.254.1.F114

PMID:3337241

Abstract

Cl transport mechanisms in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex were characterized using the Cl-sensitive fluorescent indicator 6-methoxy-N-[3-sulfopropyl]quinolinium (SPQ). In control experiments using 36Cl, SPQ did not itself alter rates for Cl transport. Cl fluxes (JCl) in nanomoles per second per milligram vesicle protein in response to gradients of Cl and other ions were calculated from the SPQ fluorescence time course, fluorescence vs. [Cl] calibration, and BBMV glucose space (microliter/mg protein). For a 50 mM inwardly directed Cl gradient in BBMV (voltage and pH clamped) JCl was 0.26 +/- 0.01 nmol.s-1.mg-1 (mean +/- SD, 4 vesicle preparations). JCl was not altered by a 50 mM inwardly directed Na gradient (0.25 +/- 0.01 nmol.s-1.mg-1) but increased to 0.52 +/- 0.04 nmol.s-1.mg-1 when driven by a 1.5 pH unit inwardly directed proton gradient. The pH driven increase in JCl was blocked by H2DIDS (100 microM, JCl = 0.38 +/- 0.02 nmol.s-1.mg-1). In the absence of a gradient, JCl increased by 64 +/- 4% in the presence of 1 mM formate on both sides of the membrane; the increase was blocked by H2DIDS. JCl increased to 0.38 +/- 0.02 nmol.s-1.mg-1 for a 60-mV K valinomycin diffusion potential (internal positive); the increase was inhibited by the Cl channel blocker diphenylamine-2-carboxylate (DPC, 100 microM, JCl = 0.26 +/- 0.03 nmol.s-1.mg-1). Br and I transport was also blocked by DPC.(ABSTRACT TRUNCATED AT 250 WORDS)

摘要

利用对氯离子敏感的荧光指示剂6-甲氧基-N-[3-磺丙基]喹啉鎓（SPQ），对从兔肾皮质分离出的刷状缘膜囊泡（BBMV）中的氯离子转运机制进行了表征。在使用³⁶Cl的对照实验中，SPQ本身不会改变氯离子的转运速率。根据SPQ荧光时间进程、荧光与[Cl]校准以及BBMV葡萄糖空间（微升/毫克蛋白质），计算出每毫克囊泡蛋白每秒纳摩尔数的氯离子通量（JCl），以响应氯离子和其他离子的梯度。对于BBMV中50 mM内向的氯离子梯度（电压和pH钳制），JCl为0.26±0.01 nmol·s⁻¹·mg⁻¹（平均值±标准差，4个囊泡制剂）。50 mM内向的钠离子梯度不会改变JCl（0.25±0.01 nmol·s⁻¹·mg⁻¹），但当由1.5个pH单位的内向质子梯度驱动时，JCl增加到0.52±0.04 nmol·s⁻¹·mg⁻¹。pH驱动的JCl增加被H2DIDS（1 mM，JCl = 0.38±0.02 nmol·s⁻¹·mg⁻¹）阻断。在没有梯度的情况下，膜两侧存在1 mM甲酸盐时，JCl增加64±4%；该增加被H2DIDS阻断。对于60 mV的钾缬氨霉素扩散电位（内部为正），JCl增加到0.38±0.02 nmol·s⁻¹·mg⁻¹；该增加被氯离子通道阻滞剂二苯胺-2-羧酸盐（DPC，1 mM，JCl = 0.26±0.03 nmol·s⁻¹·mg⁻¹）抑制。溴离子和碘离子的转运也被DPC阻断。（摘要截断于250字）