Extra- and intracellular proton-binding sites of volume-regulated anion channels

We have investigated the effects of extracellular and intracellular pH on s ingle channel and macroscopic (macropatches) currents through volume-regula ted anion channels (VRAC) in endothelial cells. Protonation of extracellular binding sites with an apparent pK of 4.6 incre ased voltage independent of the single-channel amplitude. Cytosolic acidifi cation had a dual effect on VRAC cut-rents: on the one hand, it increased s ingle channel conductance by similar to 20% due to protonation of a group w ith an apparent pK of 6.5 and a Hill coefficient of 2. On the other hand, i t reduced channel activity due to protonation of a group with an apparent p K of 6.3 and a Hill coefficient of 2.1. This dual effect enhances the macro scopic current at a slightly acidic pH but inhibits it at more acidic DH. C ytosolic 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 condu ctance. These data show that VRAC channels are maintained in an active state in a n arrow pH range around the normal physiological pH and shut down outside thi s range. They also show that HEPES-buffered pipette solutions do not effect ively buffer pH in the vicinity of the VRAC channels.