Tetraphenylphosphonium cation (TPP⁺) is not a reliable membrane potential probe in the cyanobacterium Synechococcus R-2 PCC 7942

Abstract

Uptake of the lipophilic cation, tetraphenylphosphonium (TPP⁺), which is often used as a membrane potential probe, was studied in the cyanobacterium Synechococcus R-2 (PCC 7942) to determine if the technique was valid for these cells. Lipophilic cation probes are notorious for giving inconsistent estimates of membrane potential (ΔΨ) using the Nernst equation (E_TPP⁺=ΔΨ). It is necessary to correct for the large amounts of TPP⁺ bound to the surface of the cells. The rate of equilibration of intracellular TPP⁺ is very fast (t_½≈1–3 min), hence the permeability of the plasmalemma to TPP⁺ (P_TPP⁺≈10–50 nm s⁻¹) is very high. There appears to be an active uptake of TPP⁺ with a pH optimum at about pH_o 7.5. TPP⁺ does not readily uncouple Synechococcus cells. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and dinitrophenol depolarized E_TPP⁺, but monensin made E_TPP⁺ more negative. Accumulation of TPP⁺ is not affected by [K⁺] in the medium in a way consistent with it acting as a reporter of the membrane potential. Addition of K⁺, NH₄⁺ and HCO₃⁻ altered the uptake rate of TPP⁺ in a way consistent with electrogenic uptake of these ions but the effects were smaller than those found using the ⁸⁶Rb⁺/valinomycin and ²⁰¹TI⁺ probes. A comparison has been made with ΔΨ determinations using other membrane potential probes. E_TPP⁺ was often close to the value of the membrane potential obtained using other methods under control conditions but effects of experimental treatments are sometimes inconsistent with those found using other ΔΨ probes. The TPP⁺ method is therefore unreliable and could be seriously misleading.