Na+-coupled alanine transport in LLC-PK1 cells: the relationship between the Km for Na+ at low [Alanine] and potential dependence for the system.
Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (Km and Vmax) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na+/alanine cotransporter in LLC-PK1 cells (1:1 Na+ to substrate coupling, and an ordered binding sequence), the algebraic expressions for Km for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of Km of the first-bound substrate (Na+) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the KNam includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na+, and (ii) conformational 'translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent KNam for the LLC-PK1 alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for KNam at 5 micron alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system.
Wilson
J J
JJ
Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA.
Randles
J
J
Kimmich
G A
GA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000378:metabolism* / Q000494:pharmacology; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D007700:Kinetics; D018374:LLC-PK1 Cells; D008433:Mathematics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D009550:Nigericin / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D013552:Swine; D027981:Symporters; D014634:Valinomycin / Q000494:pharmacology
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D027981:Symporters; C052168:sodium-alanine cotransporter; D014634:Valinomycin; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000409:Alanine; D009550:Nigericin