Functional expression of P-glycoproteins in secretory vesicles.
Functional expression of P-glycoproteins in secretory vesicles.
复制标题
分泌囊泡中 P-糖蛋白的功能表达。
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
P. Gros
中科院分区：
文献类型：
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作者：
S. Ruetz;P. Gros
我们表达的P-糖蛋白（P-GPS）编码的三个小鼠MDR基因的分泌囊泡（SV）积累在酵母突变株秒6-4的膜。Mdr 1和Mdr 3亚型在SV膜中的表达导致药物长春碱（VBL）的蓄积显著增加，超过对照SV中测量的背景水平。P-gp调节剂维拉帕米可完全消除Mdr 1/Mdr 3介导的药物蓄积增加。相比之下，Mdr 2在这些囊泡中的过表达未能增加囊泡内VBL积累超过背景水平。Mdr 3介导的VBL转运不受膜电位变化的影响，因为在存在或不存在内源性质子转运PMA 1 H（+）-ATP酶的情况下测量了相同的VBL摄取速率，该质子转运PMA 1 H（+）-ATP酶负责跨SV膜的强电化学膜电位。此外，在几乎90 mV的穿过SV膜（内部为阳性）的Δ微H+存在下，我们在表达Mdr 3的SV中检测到亲脂性阳离子和P-gp底物四苯基磷的积累增强，表明P-gp介导的该阳离子的摄取发生在囊内去极化膜上。同样，VBL运输Mdr 3表达SV不受存在或不存在陡峭的质子梯度（酸内），是独立的任何质子运动，不包括质子synport或反向端口机制的P-gp介导的药物转运。最后，我们可以证明，秋水仙素积累在Mdr 3表达SV发生对显着的底物浓度梯度，达到7倍以上的囊泡外介质药物浓度的囊泡内秋水仙素浓度增加。我们的研究表明，SV分离的温度敏感的酵母sec 6-4突变体是一个理想的工具，表达和功能特性异源膜蛋白，一般和P-GPS，特别是。
We expressed P-glycoproteins (P-gps) encoded by the three mouse mdr genes in the membranes of secretory vesicles (SV) accumulating in the yeast mutant strain sec 6-4. Expression of the Mdr1 and Mdr3 isoforms in SV membranes caused a significant increased accumulation of the drug vinblastine (VBL) over background levels measured in control SV. The Mdr1/Mdr3-mediated increased drug accumulation could be completely abolished by the P-gp modulator verapamil. By contrast, overexpression of Mdr2 in these vesicles failed to increase intravesicular VBL accumulation over background levels. Mdr3-mediated VBL transport was not affected by changes in the membrane potential, since identical rates of VBL uptake were measured in the presence or absence of the endogenous proton-translocating PMA1 H(+)-ATPase responsible for the strong electrochemical membrane potential across SV membranes. Moreover, in the presence of a delta micro-H+ across the SV membranes (inside positive) of almost 90 mV, we detected in Mdr3-expressing SV an enhanced accumulation of the lipophilic cation and P-gp substrate tetraphenylphosphonium, suggesting that P-gp-mediated uptake of this cation occurs against an intravesicular depolarized membrane. Likewise, VBL transport in Mdr3-expressing SV was not affected by the presence or absence of a steep proton gradient (inside acid) and was independent of any proton movements, excluding a proton synport or antiport mechanism for P-gp-mediated drug transport. Finally, we could demonstrate that colchicine accumulation in Mdr3-expressing SV occurred against a significant substrate concentration gradient, reaching a 7-fold increase in intravesicular colchicine concentration above the extravesicular medium drug concentration. Our studies show that SV isolated from the temperature-sensitive yeast sec 6-4 mutants are an ideal tool to express and to functionally characterize heterologous membrane proteins, in general and P-gps, in particular.