On the activation of outwardly rectifying anion channels in excised patches

Previous studies have shown that outwardly rectifying anion channels can be activated in excised patches by exposure to protein kinases or large depolarizing voltage pulses and by raising the bath temperature to 37 degrees C. These maneuvers presumably induce some conformational change in the channel or a regulatory molecule. However, the mechanisms underlying stimulation have not been defined. We have tested several procedures known to influence the structure and solubility of proteins for their effects on activation of anion channels in excised patches. Spontaneous and voltage-induced activation of outward rectifiers was enhanced by increasing the ionic strength or the pH of the bath solution. These maneuvers had no effect on the activity of calcium-activated cation nonselective channels. Activation of outward rectifiers depended on the anion used and followed the (inverse) Hofmeister series consistent with a salting-in process. Divalent cations also enhanced activation with relative potencies Ba greater than Ca greater than Mg but at a much lower concentration (4 meq/l). Exposing patches from T84 cells to purified catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent kinase had no effect on the outward rectifier but did activate the low-conductance Cl channel. The results indicate that the outward rectifier is labile and raise the possibility that nonspecific physical mechanisms may contribute to its activation in excised patches by kinases and other stimuli.