Molecular insight into coordination sites for substrates and their coupling kinetics in Na+/HCO3- cotransporter NBCe1
Molecular insight into coordination sites for substrates and their coupling kinetics in Na+/HCO3- cotransporter NBCe1
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Na /HCO3-协同转运蛋白 NBCe1 中底物配位位点及其偶联动力学的分子洞察
DOI：
10.1113/jp282034
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发表时间：
2022-02-26
影响因子：
5.5
通讯作者：
Chen，Li-Ming
中科院分区：
文献类型：
--
作者：
Wu，Han;Liu，Shiyong;Chen，Li-Ming
第二主动转运蛋白NBCe 1将Na+和碳酸盐物质的跨膜运动偶联，表观化学计量为1 Na+：2 HCO 3 −（“流入”模式）或1 Na+：3 HCO 3 −（“流出”模式）。在这里，我们采用分子生物学，电生理学和结构生物学方法来研究NBCe 1中Na+和HCO 3 −的转运耦合的分子机制。在异种卵母细胞中，将细胞外[HCO 3 −]从66降至4 mm会逐渐降低NBCe 1的Na+亲和力。然而，将[Na+]从96降至35 mm对HCO 3 −亲和力几乎没有影响。通过突变和分子模拟研究分别确定了NBCe 1底物口袋中负责Na+和HCO 3 −配位的残基。HCO 3 −配位残基的突变降低了NBCe 1对Na+和HCO 3 −的亲和力。然而，对Na+配位的残基的突变降低了对Na+的亲和力，但对HCO 3 −的亲和力几乎没有影响。分子模拟表明，NBCe 1只能配位HCO 3 −或CO 32 −两种离子。我们提出：（1）NBCe 1具有有序的底物结合动力学，HCO 3 −的结合先于Na+的结合;（2）NBCe 1在流入模式下移动1 Na ++2HCO 3 −，而在流出模式下移动1 Na ++1HCO 3 − +1CO 32 −。NBCe 1的底物结合动力学与许多其他Na+偶联转运蛋白的已知动力学模型不同，其中Na+结合先于驱动溶质。关键点在生理条件下，次级主动转运蛋白NBCe 1可以以表观化学计量比为1 Na+：2 HCO 3 −的“流入”模式或表观化学计量比为1 Na+：3 HCO 3 −的“流出"模式运行。NBCe 1在Na+结合之前与HCO 3 −具有有序的底物结合动力学。NBCe 1的动力学与许多其他Na+驱动的协同转运蛋白的已知动力学不同，后者的Na+结合通常先于驱动的底物。NBCe 1的底物结合口袋中负责Na+配位和碳酸盐物种配位的残基通过突变和分子模拟研究确定。NBCe 1的底物结合口袋仅包含两个HCO 3 −或CO 32 −配位位点。据推测，NBCe 1在流入模式下移动1 Na ++2 HCO 3 −穿过质膜，而在流出模式下移动1 Na ++1HCO 3 − +1CO 32 −。
AbstractThe secondary active transporter NBCe1 couples the transmembrane movement of Na+and carbonate species with an apparent stoichiometry of 1Na+:2HCO3−(the ‘influx’ mode) or 1Na+:3HCO3−(the ‘efflux’ mode). Here, we employed molecular biology, electrophysiology and structural biology approaches to investigate the molecular mechanism for the transport coupling of Na+and HCO3−in NBCe1. InXenopusoocytes, decreasing extracellular [HCO3−] from 66 to 4 mmprogressively decreases the Na+affinity of NBCe1. However, decreasing [Na+] from 96 to 35 mmhas little effect on the HCO3−affinity. The residues responsible for the coordination of Na+and HCO3−in the substrate pocket of NBCe1 were respectively determined by mutational and molecular simulation studies. Mutation to the residues for HCO3−coordination decreased the affinities of NBCe1 for both Na+and HCO3−. However, mutation to the residues for Na+coordination decreased the affinity for Na+but had little effect on the affinity for HCO3−. Molecular simulation showed that NBCe1 has the capacity to coordinate only two ions of HCO3−or CO32−. We propose that (1) NBCe1 has an ordered substrate‐binding kinetics with the binding of HCO3−preceding that of Na+; (2) NBCe1 operating in the influx mode moves 1Na++ 2HCO3−, whereas NBCe1 in the efflux mode moves 1Na++ 1HCO3−+ 1CO32−. The substrate‐binding kinetics of NBCe1 is distinct from the known kinetics models of many other Na+‐coupled transporters with Na+binding preceding the driven solute.Key pointsUnder physiological conditions, the secondary active transporter NBCe1 can operate in the ‘influx’ mode with an apparent stoichiometry of 1Na+:2HCO3−or in the ‘efflux’ mode with an apparent stoichiometry of 1Na+:3HCO3−.NBCe1 has an ordered substrate‐binding kinetics with HCO3−preceding the binding of Na+. The kinetics of NBCe1 is distinct from the known kinetics of many other Na+‐driven cotransporters for which the binding of Na+usually precedes the driven substrate.The residues responsible for the coordination of Na+and those for carbonate species in the substrate‐binding pocket of NBCe1 were determined by mutation and molecular simulation studies.The substrate‐binding pocket of NBCe1 contains just two coordination sites for HCO3−or CO32−. It is proposed that NBCe1 in the influx mode moves 1Na++ 2HCO3−across the plasma membrane, whereas NBCe1 in the efflux mode moves 1Na++1HCO3−+1CO32−.