Thrombin-dependent calcium signalling in single human erythroleukaemia cells

Area of Science:

• Cell Biology
• Calcium Signaling
• Hematology

Background:

• Thrombin plays a role in cellular signaling pathways.
• Calcium ions (Ca2+) are crucial intracellular messengers.
• Human erythroleukemia (HEL) cells are a model for platelet-megakaryocyte lineage.

Purpose of the Study:

• To investigate the mechanisms of thrombin-evoked calcium (Ca2+) signals in HEL cells.
• To determine the specific calcium channels involved in thrombin-induced Ca2+ influx.
• To elucidate the role of intracellular Ca2+ stores in regulating Ca2+ signaling.

Main Methods:

• Single-cell fluorescence microscopy to measure intracellular Ca2+ ([Ca2+]i).
• Patch clamp electrophysiology to record Ca2+ currents.
• Use of thrombin and thapsigargin to stimulate Ca2+ release and influx.
• Manganese (Mn2+) quench studies to assess channel permeability.

Main Results:

• Thrombin induced a transient increase in [Ca2+]i via intracellular store release and extracellular Ca2+ influx.
• The Ca2+ influx pathway was activated during store depletion.
• Thapsigargin mimicked thrombin's effects on store release and Ca2+ influx, revealing single-cell heterogeneity.
• Patch clamp data identified an inwardly rectifying Ca2+ current (ICRAC) activated by both stimuli, blocked by Zn2+.
• Thrombin did not activate additional current after thapsigargin-induced current developed.

Conclusions:

• Thrombin activates Ca2+ influx in HEL cells exclusively through a store-operated Ca2+ current (ICRAC).
• ICRAC, not receptor-operated or second messenger-dependent channels, mediates thrombin-evoked Ca2+ influx.
• The level of ICRAC expression is a key determinant of the duration of thrombin-induced [Ca2+]i responses.