Elevation in intracellular calcium activates both chloride and proton currents in human macrophages

Abstract

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 (pHi) 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 microM) 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 Ca(2+)-dependent ion conductances based on differences in their characteristic time-dependent kinetics: a rapidly activating Cl- conductance that showed variable inactivation at depolarized potentials and a H+ conductance with delayed activation kinetics. Both conductances were inhibited by the disulfonic acid stilbene DIDS (100 microM). Current activation for both Ca(2+)-dependent conductances was phosphorylation dependent, neither conductance appeared in the presence of the broad spectrum kinase inhibitor H-7 (75 microM). 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 pHi during sustained superoxide production.

Regulation of Human CLC-3 Channels by Multifunctional Ca2+/Calmodulin-dependent Protein Kinase

“…The CaMKII-dependent anion current was absent in vector-transfected tsA cells, and was blocked by the CaMKII specific inhibitor, autocamtide-2-related inhibitory peptide. The relative lyotropic anion permeability (P X /P Cl ) of wild type hClC-3 was I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ and is consistent with previous reports of I Cl,CaMKII from different laboratories (3,16,(25)(26)(27). To confirm that I Cl,CaMKII is directly mediated by hCLC-3 and not due to activation of an ion channel endogenous to tsA cells, a mutation in hCLC-3 (G280E) was made and transiently expressed in tsA cells.…”

“…Previous studies on the characterization of the CaMKIIactivated anion conductance carried out in our laboratory as well as others have shown that I Cl,CaMKII is outwardly rectifying (8, 11, 12, 14 -16, 25), is inhibited in the presence of 500 M DIDS (16,25), and has a P I /P Cl ratio greater than 1 in airway cells (25,26) and human macrophages (16). In this paper, we have cloned and expressed the human isoform of the CLC-3 gene and shown that its properties are identical to the endogenous I Cl,CaMKII in cells of both airway and gastrointestinal origin.…”

Electrical Membrane Properties in the Model Leishmania-Macrophage

“…Thioglycolate-recruited mouse peritoneal cells dialyzed in K + -glutamate containing InsP 3 showed a rapidly activating outward current that depolarized the cells, suggesting that part of the current was carried out by Cl - (Judge et al, 1994). In human monocyte-derived macrophages, rises of [Ca 2+ ] i induced an I Cl current of properties similar to those described previously, and sensitive to www.intechopen.com DIDS (Holevinsky et al, 1994). A third of the total current recorded in our model was inhibited by DIDS involving an anion, most probably Cl - (Camacho et al, 2008).…”

Expression and Functional Significance of the Ca2+-Activated Cl-Channel ANO6 in Dendritic Cells

“…In cells of haematopoetic origin, CaCC currents have been characterized in neutrophils [66], macrophages [67], Jurkat lymphocytes [68] and human mast cell line HMC-1 [69]. In these studies CaCCs were activated by Ca 2+ ionophore or by dialyzing the cell [55].…”