Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions

Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [(3)H]tetraphenylphosphonium (TPP(+)) against a concentration gradient. The steady-state level of TPP(+) accumulation is about twice as great in physiological media of low K(+) concentration (i.e., 5 mM K(+)/135 mM Na(+)) than in a medium of high K(+) concentration (i.e., 121 mM K(+)/13.5 mM Na(+)). The latter manipulation depolarizes the NG108-15 plasma membrane and indicates that the resting membrane potential (DeltaPsi) is due primarily to a K(+) diffusion gradient (K(in) (+) --> K(out) (+)). TPP(+) accumulation is time and temperature dependent, achieving a steady state in 15-20 min at 37 degrees C, and is a linear function of cell number and TPP(+) concentration (i.e., the concentration gradient is constant). The difference in TPP(+) accumulation in low and high K(+) media under various conditions has been used to calculate mean (+/-SD) DeltaPsi values of -56 +/- 3, -63 +/- 4, and -66 +/- 5 mV at 26, 33, and 37 degrees C, respectively. Importantly, these values are virtually identical to those obtained by direct electrophysiological measurements made under the same conditions. TPP(+) accumulation is abolished by the protonophore carbonylcyanide-m-chlorophenylhydrazone, whereas the neurotoxic alkaloid veratridine diminishes uptake to the same level as that observed in high K(+) media. In addition, the effect of veratridine is dependent upon the presence of external Na(+) and is blocked by tetrodotoxin. The steady-state level of TPP(+) accumulation is enhanced by monensin, indicating that this ionophore induces hyperpolarization under appropriate conditions. Finally, ouabain has essentially no effect on the steady-state level of TPP(+) accumulation in short-term experiments, suggesting that Na(+),K(+)-ATPase activity makes little contribution to the resting potential in these cells. Because many of these observations are corroborated by intracellular recording techniques, it is concluded that TPP(+) distribution measurements can provide a biochemical method for determining membrane potentials in populations of cultured neuronal cells.