ISOLATION AND CHARACTERIZATION OF MEMBRANE-POTENTIAL CHANGES ASSOCIATED WITH RELEASE OF CALCIUM FROM INTRACELLULAR STORES IN RAT THYMIC LYMPHOCYTES

Membrane potential changes accompanying Ca2+ influx stimulated by rele ase of Ca2+ from intracellular stores (store-regulated Ca2+ uptake) we re monitored in BAPTA-loaded rat thymic lymphocytes using the fluoresc ent indicator bis(1,3-diethylthiobarbituric acid)trimethine oxonol. De pletion of [Ca2+](i) stores by the application of thapsigargin, ionomy cin or cyclopiazonic acid induced a depolarization which was (i) depen dent upon BAPTA-loading, (ii) dependent upon extracellular Ca2+, (iii) independent of extracellular Na+ and (iv) abolished by 5 mM extracell ular Ni2+. This depolarization was followed by a charybdotoxin-sensiti ve repolarization and subsequent hyperpolarization to values approxima ting the K+ equilibrium potential, consistent with secondary activatio n of a K+ conductance. These membrane potential changes temporally cor related with Ca2+ influx from the extracellular medium as measured flu orimetrically with indo-l. The divalent cation permeability sequence w as investigated by monitoring the magnitude of the depolarization obse rved following the addition of 4 mM Ca2+, Mn2+, Ba2+ or Sr2+ to cells pretreated with doses of thapsigargin or ionomycin known to activate t he store-regulated calcium uptake pathway. On the basis of these exper iments, we conclude that the store-regulated Ca2+ uptake pathway has t he following permeability sequence: Ca2+ > Mn2+ >> Ba2+, Sr2+ with Mn2 + displaying significant permeability relative to Ca2+. This pathway i s distinguishable from other divalent cation uptake pathways reported in other cells types on the basis of its activation by thapsigargin an d its high Mn2+ permeability.