The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with [3H]bumetanide

Abstract

Simultaneous measurements were made of net Cl influxes and [3H]bumetanide binding to Ehrlich ascites tumor cells in which the chloride-cation cotransport pathway had been activated by hypertonic challenge. There was a good linear correlation between inhibition of Cl influx during regulatory volume increase and numbers of bumetanide molecules bound per cell, consistent with high specificity of bumetanide binding to cotransport sites. The extrapolation to the number of bumetanide binding sites per cell at maximal inhibition of Cl transport thus gives the number of cotransport sites per cell as 2.0 X 10(6). From this, and the fluxes measured (not necessarily maximum fluxes), the turnover number is calculated at 50 Cl ions per site per second. Unstimulated cells in isotonic medium, with negligible bumetanide-inhibitable fluxes, have the same number of bumetanide binding sites as the activated cells undergoing volume regulation.