Inactivation properties of ORIC, VRAC-like current of filamentous fungus P. blakesleeanus: the role of ATP and the first glimpse of the single channel behavior

Abstract

Outwardly rectifying, inactivating current (ORIC), recorded in cytoplasmatic droplets of P.blakesleeanus is characterized by a mild outward rectification, selectivity for anions, and a characteristic inactivation on depolarizing potentials. In absence of ATP, ORIC has a fast rundown with increasing speed of inactivation, while both processes are delayed by ATPpip (for at least 8min) and the non-hydrolysable AM-PCP (minimum of 5min). Extracellularly applied, ATP has a blocking effect. Flavonoid querzetin leads to a gradual loss of current despite the presence of intracellular ATP. Our research group has shown azide to be the potent respiration inhibitor of P. blakesleeanus, leaving the cells viable but significantly depleted from ATP. We are presenting data recorded in whole cell configuration, showing that: 1.ORIC can be activated despite the depletion of ATP in the cytoplasmatic droplet. However, its inactivation profile is significantly different comparing to normal conditions with or without ATP in the pipette in the first minute of recording, with reduced τ on lower depolarizing potentials. The fraction of current blocked by querzetin with AM-PCP in pipette solution is greater comparing to experiments with ATPpip, even with the 5-fold increase of AM-PCPpip concentration. The time required for current to recover from depolarizing steps is shorter at more hyperpolarized potentials. We have also started to look for the first insights into ORIC single channel activity in out-out configuration, and recorded, upon stimulation of ORIC in whole cell, and patch excision, the step-like pattern of inactivation that strongly resembles one of ORIC. Based on current data, we can conclude that ATP has a significant role in the inactivation process, with a more potent effect comparing to AM-PCP, whether by the means of greater affinity for the binding site or an additional mechanism involving kinase activity.