Modulation of human cardiovascular outward rectifying chloride channel by intra- and extracellular ATP

Abstract

Liu GX, Vepa S, Artman M, Coetzee WA. Modulation of human cardiovascular outward rectifying chloride channel by intra-and extracellular ATP. Am J Physiol Heart Circ Physiol 293: H3471-H3479, 2007. First published October 12, 2007; doi:10.1152/ajpheart.00357.2007.-The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl Ϫ channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out singlechannel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl Ϫ solutions, exhibited a slope conductance of ϳ90 -100 pS at positive potentials and ϳ22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, "extracellular" ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATP␥S Ͼ ATP ϭ UTP Ͼ ADP Ͼ AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions. human heart; atrial myocytes; ventricular myocytes; endothelial cells; smooth muscle cells CHLORIDE CHANNELS in the cardiovascular system play an important role in maintaining the resting potential and by participating in repolarization of the action potential (15,17,27). There are several types of macroscopic Cl Ϫ currents identified by whole cell patch-clamp recordings, which are distinguished from each other in terms of their regulation. These include Cl

ATP release during cell swelling activates a Ca2+-dependent Cl− current by autocrine mechanism in mouse hippocampal microglia

“…This indicates that ATP is not necessary as P i -donor as in phosphorylation-based regulation mechanisms 27 . Conversly, we hypothesize that ATP is released by microglia cells during swelling, contributing to I Swell activation in autocrine fashion 39, 42, 44 (Fig. 8), as proposed for the mechanisms leading to cell volume regulation in different cellular systems 45 and immune cells activation 46 .This conclusion is based on the absence of I Swell when slices are treated with apyrase, an enzyme that degrades ATP and ADP to AMP and free inorganic phosphate 47 .…”

“…The role of intracellular ATP in the regulation of chloride currents activated by mechanical membrane deformation has been already highlighted in microglia and other cell types and is generally described in terms of an ATP-dependent current run down 40 . However, ATP effect is apparently not dependent on ATP hydrolysis 11, 14, 40–42 . Differently from what reported in rat carotid body cells 43 , in our conditions, intracellular Mg-ATP could be substituted by analogs, like Na-ATP, ATPɣS or ADP, impairing phosphate transfer to substrate.…”

Swelling activated Cl- channels in microglia

“…For VRAC, the ATP requirement is not through a phosphorylation event because ATP allows channel activation under Mg 2+ free conditions. 23 In MLS-9 cells, spontaneous activation of the Cl -current by low internal ionic strength occurred when GTPγS was present in the pipette solution, but we cannot rule out residual ATP inside the cell.…”

Regulation of Coronary Blood Flow

“…In that negative feedback scheme, ATP is postulated to act on coronary vascular endothelial cells and transmit a hyperpolarizing signal to the smooth muscle in upstream resistance vessels. Determining what that signal is and how it is transmitted will require additional study, but human coronary endothelial cells possess a Cl − channel that is inhibited by ATP (617). In human coronary myocytes, ATP activates BK Ca and Cl − channels by releasing stored Ca 2+ (892).…”