Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant p-glycoprotein with deleted Phe335
Loss of cyclosporin and azidopine binding are associated with altered ATPase activity by a mutant p-glycoprotein with deleted Phe335
复制标题
DOI：
10.1124/mol.57.4.769
复制
发表时间：
2000-04-01
影响因子：
3.6
通讯作者：
Sikic, BI
中科院分区：
文献类型：
--
作者：
Chen, GK;Lacayo, NJ;Sikic, BI
在这项研究中，我们进一步表征了一种突变的p -糖蛋白（P-gp），它具有Phe（335）的缺失，并且对环孢素的抑制具有抗性。与野生型P-gp相比，[H-3]环孢素和[H-3]氮氮吡啶的光亲和标记显示与突变型P-gp的结合明显减少。P-gp突变体在多药耐药细胞系MES-SA/ DxP （DxP）细胞中的表达与具有野生型P-gp的多药耐药细胞系MES-SA/ Dx5 （Dx5）细胞的2倍高的基础atp酶活性相关。环孢素抑制两种细胞类型的atp酶活性，而环孢素D类似物valspodar （PSC 833）、长春花碱和放线菌素在Dx5细胞中刺激atp酶活性，但在突变型DxP细胞中没有。此外，不同细胞系对维拉帕米的反应不同，维拉帕米对Dx5细胞的atp酶的刺激比DxP细胞更大。维拉帕米显著逆转了野生型Dx5细胞中[H-3]柔红霉素积累缺陷，但对突变型DxP细胞中[H-3]柔红霉素积累无显著影响。表达突变型或野生型P-gp的细胞都不能运输维拉帕米。突变体P-gp对叠氮- atp的钒酸捕获明显受损。综上所述，我们的数据表明跨膜6的Phe（335）是环孢素和叠氮吡啶药物结合位点形成的重要氨基酸残基。在野生型P-gp中，Phe（335）还在维拉帕米结合的偶联和柔红霉素细胞内积累的调节中发挥作用。此外，跨膜6中的Phe（335）可能在药物结合与atp酶活性的偶联中发挥作用。Phe（335）的缺失导致基础ATP酶活性显著增加，同时其捕获ATP和运输一些P-gp底物的能力下降。
In this study, we further characterize a mutant P-glycoprotein (P-gp) that has a deletion of Phe(335) and is resistant to inhibition by cyclosporins. Photoaffinity labeling with [H-3] cyclosporine and [H-3] azidopine revealed markedly decreased binding to the mutant P-gp compared with wild-type P-gp. Expression of the mutant P-gp in multidrug-resistant variant cell line MES-SA/ DxP (DxP) cells was associated with a 2-fold higher basal ATPase activity relative to multidrug-resistant cell line MES-SA/ Dx5 (Dx5) cells with wild-type P-gp. Cyclosporine inhibited ATPase activity in both cell types, whereas the cyclosporin D analog valspodar (PSC 833), vinblastine, and dactinomycin stimulated ATPase activity in Dx5 but not in mutant DxP cells. Moreover, the cell lines differed in their responses to verapamil, which produced greater stimulation of ATPase in Dx5 than DxP cells. Verapamil significantly reversed the [H-3] daunorubicin accumulation defect in wild-type Dx5 cells, but it had no significant effect on [H-3] daunorubicin accumulation in the mutant DxP cells. Verapamil was not transported by cells expressing either mutant or wild-type P-gp. Vanadate trapping of azido-ATP was markedly impaired in mutant P-gp. In conclusion, our data demonstrate that Phe(335) of transmembrane 6 is an important amino acid residue for the formation of cyclosporine and azidopine drug-binding site(s). Phe(335) also plays a role in the coupling of verapamil binding and modulation of daunorubicin intracellular accumulation in wild-type P-gp. In addition, Phe(335) in transmembrane 6 may play a role in coupling drug binding to ATPase activity. The deletion of Phe(335) results in a significant increase in the basal ATPase activity with a concomitant decrease in its ability to trap ATP and transport some P-gp substrates.