Extra- and intracellular proton-binding sites of volume-regulated anion channels.
We have investigated the effects of extracellular and intracellular pH on single channel and macroscopic (macropatches) currents through volume-regulated anion channels (VRAC) in endothelial cells. Protonation of extracellular binding sites with an apparent pK of 4.6 increased voltage independent of the single-channel amplitude. Cytosolic acidification had a dual effect on VRAC currents: on the one hand, it increased single channel conductance by approximately 20% due to protonation of a group with an apparent pK of 6.5 and a Hill coefficient of 2. On the other hand, it reduced channel activity due to protonation of a group with an apparent pK of 6.3 and a Hill coefficient of 2.1. This dual effect enhances the macroscopic current at a slightly acidic pH but inhibits it at more acidic pH. Cytosolic alkalization also reduced channel activity with a pK of 8.4 and a Hill coefficient of 1.9, but apparently did not affect single-channel conductance. These data show that VRAC channels are maintained in an active state in a narrow pH range around the normal physiological pH and shut down outside this range. They also show that HEPES-buffered pipette solutions do not effectively buffer pH in the vicinity of the VRAC channels.
Sabirov
R Z
RZ
Laboratorium voor Fysiologie, KU Leuven, B-3000 Leuven, Belgium.
Prenen
J
J
Droogmans
G
G
Nilius
B
B
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002478:Cells, Cultured; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology; D005110:Extracellular Space / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000502:physiology; D007473:Ion Channels / Q000502:physiology*; D011522:Protons
D007473:Ion Channels; D011522:Protons