Quantitative use of weak bases for estimation of cellular pH gradients

An improvement in the usual procedure for estimating cellular H+ gradients by distribution of weak bases is described, which involves evaluation of and correction for the permeability of the ionic form of the sensor. In the case of methylamine, unidirectional uptake (influx) of the methylammonium ion is calculated by comparing the total influx of [14C]methylamine with the influx of the highly permeant ion [14C]tetraphenyl-phosphonium ([14C]TPP+) for two experimental situations in which the membrane potential differs. Comparison of the potential-dependent changes in unidirectional influx of methylamine and TPP+ allows calculation of the magnitude of influx for the methylammonium ion. This value can then be used to determine the error in the H+ gradient as estimated from the steady-state distribution of methylamine across the plasma membrane. By using ATP-depleted isolated small intestine cells from the chick as the test system, and imposed H+ gradients of defined magnitude, it can be shown that the observed error matches the calculated error.