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

Abstract

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 pM 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 gM) 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 cyclophilin A (50 nM) either applied together or individually. In contrast the protein phosphatase I 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 Ca2c urrent inactivation which is mediated by calcineurin is revealed following photolysis of 'caged' ATP or cyclic AMP in metabolically stressed neurones.

Cellular Distribution and Developmental Expression of AMP‐Activated Protein Kinase Isoforms in Mouse Central Nervous System

“…During metabolic stress, ATP‐modulated K + channels play an important role in modulating membrane excitability, with different ATP concentrations modulating different K + channels (Jiang et al, 1994; Jiang and Haddad, 1997). In addition, ATP modulates Ca 2+ ‐dependent currents in metabolically stressed neurons (Stapleton et al, 1995). As AMPK is activated by decreased levels of ATP (Corton et al, 1994), it seems reasonable that it functions to monitor local metabolic stress and ATP depletion in neurites.…”

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

“…However, prolonged activation of calcium-activated conductances holds the cell membrane potential at about -30 mV and promotes inactivation of voltage-dependent conductances. The prolonged activation of after-depolarizations and I Cl(Ca) probably reflects inefficient intracellular calcium handling, observed in older cultured DRG neurones, or when neurones have been metabolically compromised, by for example 2-deoxyglucose Stapleton et al 1995). The modulation of CICR and Ca 2+ -activated conductances in the cell bodies of DRG neurones may be important in somatosensory functions.…”