Transepithelial transport in cell culture: stoichiometry of Na/phlorizin binding and Na/D-glucose cotransport. A two-step, two sodium model of binding and translocation

Abstract

The renal cell line LLC-PK1 cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependent D-glucose coupled transport. The short-circuit current (Isc) was accounted for by net apical-to-basolateral D-glucose coupled Na flux, which was 0.53 +/- 0.09(8) mueq cm-2hr-1, and Isc, 0.50 +/- 0.50(8) mueq cm-2hr-1. A linear plot of concurrent net Na vs. net D-glucose apical-to-basolateral fluxes a gave a regression coefficient of 2.08. As support for a 2:1 transepithelial stoichiometry, sodium was added in the presence of D-glucose and the response of Isc analyzed by a Hill plot. A slope of 2.08 +/- 0.06(5) was obtained confirming a requirement of 2 Na for 1 D-glucose coupled transport. A Hill plot of Isc increase to added D-glucose in the presence of Na gave a slope of 1.02 +/- 0.02(5). A direct determination of the initial rates of Na and D-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2. A coupling ratio of 2 for Na, D-glucose uptake, doubles the potential energy available for Na-gradient coupled D-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependent-phlorizin binding was 1.1 +/- 0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na].(ABSTRACT TRUNCATED AT 250 WORDS)