ELEVATION IN INTRACELLULAR CALCIUM ACTIVATES BOTH CHLORIDE AND PROTON CURRENTS IN HUMAN MACROPHAGES

The transition of a resting macrophage into the activated state is accompanied by changes in membrane potential, cytoplasmic pH, and intracellular calcium (Ca-i). Activation of Cl- as well as H+-selective currents may give rise to stimulus-induced changes in membrane potential and counteract changes in intracellular pH (pH(i).) which have been observed to be closely associated with respiratory burst activation and superoxide production in macrophages. We carried out whole-cell voltage clamp experiments on human monocyte-derived macrophages (HMDMs) and characterized currents activated following an elevation in Ca-i using isosmotic pipette and bath solutions in which Cl- was the major permeant species. Ca-i was elevated by exposing cells to the Ca2+ ionophore A23187 (1-10 mu M) in the presence of extracellular Ca2+ or by internally exchanging the patch-electrode solution with ones buffered to free Ca2+ concentrations between 40 and 2,000 nM. We have identified two Ca2+-dependent ion conductances based on differences in their characteristic time-dependent kinetics: a rapidly activating Cl- conductance that shouted variable inactivation at depolarized potentials and a H+ conductance with delayed activation kinetics. Both conductances were inhibited by the disulfonic acid stilbene DIDS (100 mu M). Current activation for both Ca2+-dependent conductances was phosphorylation dependent, neither conductance appeared in the presence of the broad spectrum kinase inhibitor H-7 (75 mu M). inclusion of the autophosphorylated, Ca2+/calmodulin-dependent protein kinase in the pipette in the presence of ATP induced a rapidly activating current similar to that observed following an elevation in Ca-i. Activation of both conductances would contribute to the changes in membrane potential which accompany stimulation-induced activation of macrophages as well as counteract the decrease in pH(i) during sustained superoxide production.