Lymphocyte membrane potential and Ca2+‐sensitive potassium channels described by oxonol dye fluorescence measurements

Abstract

A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (Ψ) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of Ψ than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte Ψ was calculated to be −70 mV. T and B lymphocytes maintain a constant Ψ as extracellular K⁺ is varied from 2 to 10 mM and the deviation from K⁺ equilibrium potentials (E_k) is shown to result from Na⁺ permeability. At [K⁺]_o values greater than 10 mM, lymphocytes behave as K⁺ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca²⁺ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca²⁺‐sensitive K⁺ channel by pharmacologic manipulations. Hence, T cells depolarized by 4‐aminopyridine (4‐AP, 10 mM) were observed to return to resting Ψ by A23187‐induced elevation of [Ca²⁺]_i. Cells depolarized by quinine (100 μM) were unaffected by A23187. The Ca²⁺‐activated channel does not contribute to resting Ψ in T cells since it may be selectively blocked by quinine (20 μM) or modulated by calmodulin antagonists (5 μM trifluperazine) without affecting resting Ψ.