Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Abstract

The utility of the lipophilic anion thiocyanate (SCN-) as a probe for the indirect estimation of the cell membrane potential (Vm) in Ehrlich ascites tumor cells has been evaluated by comparison to direct electrophysiological measurements. SCN accumulation is consistent with first-order uptake into a single, kinetically-identifiable cellular compartment, achieving steady-state distribution in 20-30 min at 22 degrees C. The steady state distribution ratio ([SCN-]e/[SCN-]e) in physiological saline is 0.44 +/- 0.02. Treatment of the cells with propranolol (0.13 mM), an activator of Ca2+ dependent K+ channels, reduces the steady-state distribution ratio to 0.19 +/- 0.02. Conversely, treatment with BaCl2 (10 mM), an antagonist of the pathway, increases the SCN- distribution ratio to 0.62 +/- 0.01. The equilibrium potentials (VSCN) calculated under these conditions are virtually identical to direct electrophysiological measurements of the Vm made under the same conditions. The effect of varying extracellular [K+] ([K+]e) in the presence of constant [Na+]e = 100 mM has also been tested. In control cells, elevation of [K+]e from 6 to 60 mM reduces VSCN from -20.6 +/- 1.0 to -13.2 +/- 1.2 mV. Again, microelectrode measurements give excellent quantitative agreement. Propranolol increases the sensitivity of the cells to varying [K+]e, so that a 10-fold elevation reduces VSCN by approximately 31 mV. BaCl2 greatly reduces this response: a 10-fold elevation in [K+]e yielding only a 4-mV reduction in VSCN. It is concluded that the membrane potential of Ehrlich cells can be estimated accurately from SCN- distribution measurements.