Thrombin-dependent calcium signalling in single human erythroleukaemia cells

1. A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin-evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet-megakaryocyte lineage. 2. Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+](i)), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin-evoked divalent cation-permeable pathway was activated during, but not prior to, release from internal stores. 3. Thapsigargin (1 mu M) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca2+-influx mechanism. The amplitude of the thrombin- and thapsigargin-induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient. 4. In whole-cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range -110 to +6O mV and was blocked by 1 mM Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+ >> Ba2+ > Mn2+, Mg2+. The thapsigargin-evoked current density at -100 mV varied between 0.42 and 2.1 pA pF-(1) in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin-induced inward current had fully developed. 5. These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca2+ store-release-activated Ca2+ current (I-crac) rather than via receptor-operated or second messenger-dependent Ca2+ channels. The level of expression of I-crac appears to be a major factor in determining the duration of the thrombin-evoked [Ca2+](1) response and therefore represents a means by which cells can exert control over [Ca2+](i)-dependent events.