The use of HgCl2 to evaluate the cosubstrate: Amino acid transport stoichiometry in ehrlich ascites tumor cells

W. David Dawson, Susan C. Robinson, Thomas C. Smith

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and α‐aminoisobutyric acid (α‐AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+ ‐free HEPES‐buffered medium (pH 7.2) containing 160 mM K+ and 2.5 μM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H‐AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, −7.0 ± 0.1 mV (SEM), does not change with the addition of AIB, −7.3 ± 0.6 mV (SEM). HgCl2 (10 m̈M) added to the medium inhibited AIB flux and AIB‐stimulated Na+ flux by 45–50% but did not change the coupling ratio. HgCl2 (10 m̈M) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is −22.3 ± 0.8 mV (SEM) and depolarizes to −16.7 ± 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 ± 0.02 (SEM) in the presence of HgCl2 (10 m̈M). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of α‐AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+ :AIB stoichiometry approaches 1:1.

Original languageEnglish (US)
Pages (from-to)131-136
Number of pages6
JournalJournal of Cellular Physiology
Volume115
Issue number2
DOIs
StatePublished - May 1983
Externally publishedYes

ASJC Scopus subject areas

  • Physiology
  • Clinical Biochemistry
  • Cell Biology

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