Isolation and characterization of membrane potential changes associated with release of calcium from intracellular stores in rat thymic lymphocytes

Abstract

Membrane potential changes accompanying Ca²⁺ influx stimulated by release of Ca²⁺ from intracellular stores (store-regulated Ca²⁺ uptake) were monitored in BAPTA-loaded rat thymic lymphocytes using the fluorescent indicator bis(1,3-diethylthiobarbituric acid)trimethine oxonol. Depletion of [Ca²⁺] _i stores by the application of thapsigargin, ionomycin or cyclopiazonic acid induced a depolarization which was (i) dependent upon BAPTA-loading, (ii) dependent upon extracellular Ca²⁺, (iii) independent of extracellular Na⁺ and (iv) abolished by 5 mm extracellular Ni²⁺. This depolarization was followed by a charybdotoxin-sensitive repolarization and subsequent hyperpolarization to values approximating the K⁺ equilibrium potential, consistent with secondary activation of a K⁺ conductance. These membrane potential changes temporally correlated with Ca²⁺ influx from the extracellular medium as measured fluorimetrically with indo-1. The divalent cation permeability sequence was investigated by monitoring the magnitude of the depolarization observed following the addition of 4 mm Ca²⁺, Mn²⁺, Ba²⁺ or Sr²⁺ to cells pretreated with doses of thapsigargin or ionomycin known to activate the store-regulated calcium uptake pathway. On the basis of these experiments, we conclude that the store-regulated Ca²⁺ uptake pathway has the following permeability sequence: Ca²⁺ > Mn²⁺ ≫ Ba²⁺, Sr²⁺ with Mn²⁺ displaying significant permeability relative to Ca²⁺. This pathway is distinguishable from other divalent cation uptake pathways reported in other cells types on the basis of its activation by thapsigargin and its high Mn²⁺ permeability.