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Membrane Transport Mechanisms Probed by Capacitance Measurements With Megahertz Voltage Clamp

Lu, Chin-Chih and Kabakov, Anatoli and Markin, Vladislav S. and Mager, Sela and Frazier, Gary A. and Hilgemann, Donald W. (1995) Membrane Transport Mechanisms Probed by Capacitance Measurements With Megahertz Voltage Clamp. Proceedings of the National Academy of Sciences of the United States of America, 92 (24). pp. 11220-11224. ISSN 0027-8424. PMCID PMC40603. doi:10.1073/pnas.92.24.11220.

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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 E_1-E_2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions, For the GAT1 (y-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.

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Additional Information:© 1995 National Academy of Sciences. Communicated by A. J. Hudspeth, University of Texas Southwestern Medical Center, Dallas, TX, August 10, 1995 (received for review March 8, 1995). We thank Dr. H. Lester (California Institute of Technology) for helpful discussions, Dr. W. L. Hubbel (University of California, Los Angeles) for the generous gift of DPA, Dr. Rich Lobdill (Axon Instruments) for modifying the patch clamps employed, and Mr. Steve Callaway for technical assistance. This work was supported by Grant RO1 HL51323-02 from the National Institutes of Health and by a Grant-in-Aid from the American Heart Association.
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NIHRO1 HL51323-02
American Heart AssociationUNSPECIFIED
Issue or Number:24
PubMed Central ID:PMC40603
Record Number:CaltechAUTHORS:20150123-111051558
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Official Citation:Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp C C Lu, A Kabakov, V S Markin, S Mager, G A Frazier, and D W Hilgemann PNAS 1995 92 (24) 11220-11224
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54020
Deposited By: Ruth Sustaita
Deposited On:23 Jan 2015 19:35
Last Modified:10 Nov 2021 20:26

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