ATP dependency of osmotically activated outwardly rectified current in the membrane of cytoplasmic droplets obtained from sporangiophore of model filamentous fungus Phycomyces blakesleeanus

ORIC is outwardly rectified, rapidly inactivating anionic current that is activated in hypo-osmotic conditions in the membrane of cytoplasmic droplets obtained from sporangiophore of filamentous fungus Phycomyces blakesleeanus . It shares biophysical properties (rectification and inactivation dynamics, ion selectivity order) and activating stimuli (hypo-osmotic conditions or presence of intracellular GTP under isoosmotic conditions) with volume regulated anionic current (VRAC) present in animals. Since VRAC is ATP regulated channel, we tested if ATP affects ORIC as well. For these purposes patch-clamp experiments in whole-cell configuration with 2mM MgATP in the patch pipette were performed. Inclusion of ATP increased maximal ORIC while strikingly reducing its run-down. Time to full activation of ORIC was longer in the presence of MgATP. Additionally, decrease of Mg +2 to 32μM, with 2mM ATP present, did not affect the activation speed, but ORIC activated to a larger value and plateau of maximal activity was longer compared to 2mM Mg +2 with ATP. This suggests that the intracellular Mg +2 exert an inhibiting effect on ORIC, also similar to the effect of Mg +2 on VRAC. Furthermore, in order to establish if respiration blockers affect ORIC, we tested the efficiency of two respiration blockers, antimycin A and azide, on ORIC in concentrations that inhibit cytochrome respiratory pathway completely. Both metabolic blockers inhibited ORIC, thus confirming the ATP is necessary for ORIC activity. Financing: Serbian Ministry of Education, Science and Technological Development, Grant no. 173040