ATP-Inhibition of M Current in Frog Sympathetic Neurons Involves Phospholipase C But Not Ins P3, Ca2+, PKC, orRas

Abstract

Suppression of the voltage-activated, noninactivating K+ conductance (M conductance; g M) by muscarinic agonists, P2Y agonists or bradykinin increases neuronal excitability. All agonist effects are mediated, at least in part, via the Gq/11 class of G protein. We found, using whole cell or perforated patch recording from bullfrog sympathetic B neurons that ATP-induced suppression of gM was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC50 ∼0.14 μM) but not by the inactive isomer, U73343 . The ability of extracellularly applied U73122 to inhibit PLC was confirmed by its antagonism of ATP-induced elevation of intracellular Ca2+ as measured by fura-2 photometry. ATP-induced g M suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 μM extracellular +10 μM intracellular), by the Ca2+-ATPase inhibitor, thapsigargin (5 μM), or by inositol trisphosphate (InsP3) receptor antagonists, heparin (∼300 μM) or xestospongin C (1.8 μM). The effect of ATP on g M was thus dependent on PLC yet independent of PKC and of InsP3-induced release of intracellular Ca2+. We therefore tested the involvement of a PKC-independent action of diacylglycerol (DAG) that could occur via activation of Ras. This low-molecular-weight G protein is activated following DAG binding to Ras-GRP, a neuronal Ras-GTP exchange factor. However, impairment of Ras function by culturing neurons with isoprenylation inhibitors (perillic acid, 0.1 mM, or α-hydroxyfarnesyl-phosphonic acid, 10 μM) failed to affect ATP-induced g M suppression. Inhibition of MEK (mitogen-activated protein kinase), a downstream target of Ras, by using PD 98059 (10 μM) was also ineffective. The transduction mechanism used by ATP to suppress gMin frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca2+-dependent mechanism used by bradykinin and UTP in mammalian ganglia. The possibility remains that “lipid-signaling” mechanisms, perhaps involving PLC-induced depletion of phosphatidylinositol bisphosphate, are involved in PLC-mediated inhibition of g M by ATP in amphibian sympathetic neurons.