MEMBRANE-TRANSPORT MECHANISMS PROBED BY CAPACITANCE MEASUREMENTS WITH MEGAHERTZ VOLTAGE-CLAMP
MEMBRANE-TRANSPORT MECHANISMS PROBED BY CAPACITANCE MEASUREMENTS WITH MEGAHERTZ VOLTAGE-CLAMP
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DOI：
10.1073/pnas.92.24.11220
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发表时间：
1995-11-21
影响因子：
11.1
通讯作者：
HILGEMANN, DW
中科院分区：
文献类型：
--
作者：
LU, CC;KABAKOV, A;HILGEMANN, DW
我们使用了1亩S电压钳技术的电容测量技术来探测巨型离体膜贴片中产生的离子-转运体之间的相互作用，疏水离子二苦胺被用来检验一个简单的电荷移动反应的模型预测。由1-mV正弦微扰监测到的二苯甲胺诱导的表观电容的电压和频率依赖关系，对应于在0 mV时单电荷以9500 S(-1)的速率移动通过76%的膜场。对于心脏Na，K泵，细胞质中的三磷酸腺苷和钠的联合存在引起表观膜电容的增加，这需要细胞外钠的存在。电容变化对频率、电压、三磷酸腺苷和钠的依赖关系证实，磷酸化使缓慢的300-900-S(-1)泵转变(E(1)-E(2)构象变化)，进而使快速、电产生的细胞外钠结合反应。对于非洲爪哇卵母细胞膜表达的GAT1(γ-氨基丁酸，钠，氯)共转运体，我们发现氯结合来自细胞质一侧，而且可能是来自细胞外侧的钠结合，导致以1到50 kHz的扰动频率监测的膜电容的下降。显然，GAT1转运体的离子结合抑制了内在的快速电荷运动，这种运动可能源于转运体结合位点的带电残基的移动。结果表明，快速电容测量可以为研究与膜转运体离子结合密切相关的电生过程提供新的视角。
We have used capacitance measurements with a 1-mu s voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches, The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s(-1) at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP-and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium, The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow 300- to 900-s(-1), pump transition (the E(1)-E(2) conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions, For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which mag originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.