Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp

Chin Chih Lu, Anatolii Kabakov, Vladislav S. Markin, Sela Mager, Gary A. Frazier, Donald W. Hilgemann

Research output: Contribution to journalArticlepeer-review

65 Scopus citations

Abstract

We have used capacitance measurements with a 1-μs 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 E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (γ- amonibutyric 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 may 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.

Original languageEnglish (US)
Pages (from-to)11220-11224
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number24
DOIs
StatePublished - Nov 21 1995

ASJC Scopus subject areas

  • General

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