The role of calcium-activated potassium channels and store-operated calcium channels in human macrophages
Intracellular Ca2+ is an important regulator of diverse functions of macrophages. Store-operated Ca2+ entry (SOCE) is the major Ca2+ influx pathway of human macrophages. Ca2+ activated potassium channels of the KCa3.1 subtype (IKCa channels) are expressed in human macrophages. We hypothesized that I...
Normale und Pathologische Physiologie
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|Summary:||Intracellular Ca2+ is an important regulator of diverse functions of macrophages. Store-operated Ca2+ entry (SOCE) is the major Ca2+ influx pathway of human macrophages. Ca2+ activated potassium channels of the KCa3.1 subtype (IKCa channels) are expressed in human macrophages. We hypothesized that IKCa may be activated by store-operated Ca2+ entry and that .the resulting hyperpolarization may serve to sustain the Ca2+ influx through store-operated channels (SOCs).
Human macrophages were differentiated from peripheral blood mononuclear cells and had the typical morphological properties and expressed the macrophage marker CD14. The calcium stores were depleted in Ca2+-free solution by activation of P2Y purinergic receptors with UTP or by application of the calcium pump inhibitor thapsigargin. Current-clamp experiments showed that re-addition of Ca2+ to the bath solution resulted in membrane hyperpolarization. This hyperpolarization was inhibited by IKCa blocker charybdotoxin (CHTX) and by the SOC blocker 2-APB. Whole-cell patch clamp at 0 mV showed that SOCE induced an outward current which was also blocked by CHTX and 2-APB. These data indicate that IKCa channels are the dominant KCa channels in human macrophages and can be activated by SOCE, which results in hyperpolarization of macrophages. By using cesium-based pipette solution, we recorded the inward current carried by calcium-release-activated channels (ICRAC) after depletion of the intracellular calcium stores with the calcium buffer EGTA, with the purinergic agonist UTP or the Ca2+-pump blocker thapsigargin. ICRAC had a reversal potential >+50 mV and could be blocked by 2-APB.
Fluoro¬metric measurements of intra¬cellu¬lar free Ca2+ with fluo-3 showed that UTP or thapsigargin induced a transient increase of intracellular Ca2+ in Ca2+ free solution, which was followed by a sustained Ca2+ influx after re-addition of Ca2+ to bath solution. Blockage of IKCa with CHTX resulted in accelerated decay of Ca2+ fluorescence but had no effects on initial rate of Ca2+ influx. These findings suggest that Ca2+ influx activates IKCa and hyperpolarizes the membrane potential, which will maintain the driving force for Ca2+ influx by providing counter ions for Ca2+ -influx through store-operated channels.
Very recent studies have shown that protein Orai1, 2, 3 may be the molecular candidates of ICRAC and that the protein STIM1 may represent the sensor of ER Ca2+ content. Using RT-PCR, we found that Orai1, Orai 2, Orai3 and STIM1 were expressed by human macrophages. These results suggest that one or more members of Orai protein family may form the store-operated Ca2+ entry pathway in human macrophages and that STIM1 may act as a Ca2+ sensor.
Our data indicate that IKCa channels and SOCE may provide a feedback loop for Ca2+ influx, The sustained Ca2+ influx is important for proper function of immune system.|