Modulation of Ca2+-dependent currents in metabolically stressed cultured sensory neurones by intracellular photorelease of ATP

1 The whole cell recording technique was used to study high voltage-activated Ca2+ currents and Ca2+-activated Cl− tail currents from cultured neonatal dorsal root ganglion neurones of the rat which were metabolically stressed. The neurones were metabolically stressed with 2-deoxy-d-glucose (5 mm) for 30 min to 3 h. The aim of the project was to examine the actions of intracellular photorelease of ATP on the properties of Ca2+-dependent currents and determine if the effects of metabolic stress could be reversed. 2 The mean duration of Ca2+-activated Cl− tail currents was significantly increased by metabolic stress and this effect was reversed by intracellular photorelease of approximately 300 μm ATP. Intracellular photolysis of ‘caged’ photolabile compounds was achieved with a xenon flash lamp. 3 Intracellular photorelease of ATP and adenosine 3′:5′-cyclic monophosphate (cyclic AMP) (about 40 μm) also accelerated the inactivation of high voltage-activated Ca2+ currents evoked by 500 ms depolarizing step commands from −90 mV to 0 mV. This effect was prevented by intracellular application of the calcineurin (protein phosphatase-2B) inhibitor cyclosporin A (14 nm) and cyclophihn A (50 nm) either applied together or individually. In contrast the protein phosphatase 1 and 2A inhibitor, calyculin A, increased voltage-activated Ca2+ currents, but failed to prevent enhanced inactivation induced by intracellular photorelease of ATP. Intracellular photorelease of ATP had no effect on Ca2+ currents recorded from control neurones which were not metabolically stressed and supplied with glucose and ATP in the extracellular and patch pipette solutions respectively. 4 In conclusion, intracellular photorelease of ATP increases the decay of Ca2+-activated Cl− tail currents in metabolically stressed neurones suggesting that the efficiency of intracellular Ca2+ buffering was improved. Additionally, an ATP/cyclic AMP-dependent component of high voltage-activated Ca2+ current inactivation which is mediated by calcineurin is revealed following photolysis of ‘caged’ ATP or cyclic AMP in metabolically stressed neurones.