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.