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, P(2Y) 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 g(M) was attenuated by the phospholipase C (PLC) inhibitor, U73122 (IC(50) approximately 0.14 microM) 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 Ca(2+) as measured by fura-2 photometry. ATP-induced g(M) suppression was not antagonized by the protein kinase C (PKC) inhibitor, chelerythrine (5 microM extracellular +10 microM intracellular), by the Ca(2+)-ATPase inhibitor, thapsigargin (5 microM), or by inositol trisphosphate (InsP(3)) receptor antagonists, heparin (approximaterly 300 microM) or xestospongin C (1.8 microM). The effect of ATP on g(M) was thus dependent on PLC yet independent of PKC and of InsP(3)-induced release of intracellular Ca(2+). 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 alpha-hydroxyfarnesyl-phosphonic acid, 10 microM) 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 microM) was also ineffective. The transduction mechanism used by ATP to suppress g(M) in frog sympathetic neurons therefore differs from the PLC-independent mechanism used by muscarine and from the PLC and Ca(2+)-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.

Inositol Triphosphate-Mediated Ca2+Signals Direct Purinergic P2Y Receptor Regulation of Neuronal Ion Channels

“…The mechanism of action of neuronal P2Y receptors on I M has been controversial and may depend on precise neuronal type or receptor isoform. Previous work in the rat SCG cells studied here, in which the dominant subtype is likely P2Y 6 , suggested the requirement for [Ca 2ϩ ] i signals (BofillCardona et al, 2000), consistent with our results, but, in frog sympathetic neurons in which the subtype is not clear, the PIP 2 -depletion mode is suggested (Stemkowski et al, 2002). Recently, P2Y 1 receptor-mediated suppression of M current in hippocampus was studied, and the lack of [Ca 2ϩ ] i signals seen during purinergic stimulation in those cells points toward a PIP 2 -depletion mechanism there as well.…”

“…In many neuronal types, P2Y receptor stimulation depresses M current via PLC activation, but the intracellular mechanism used from that point on appears intriguingly divergent. In the rat SCG cells also studied here, the action involves intracellular Ca 2ϩ signals (Bofill-Cardona et al, 2000) but does not in frog ganglia (Stemkowski et al, 2002). Moreover, in rat hippocampus, the P2Y 1 receptor-mediated suppression of I M is not associated with [Ca 2ϩ ] i signals and is suggested to be via PIP 2 depletion (Filippov et al, 2006).…”

“…Although all three receptor types couple to G q/11 and activate phospholipase C, it is hypothesized that colocalization of B 2 , but not M 1 or AT 1 , receptors with IP 3 receptors in "microdomains" accounts for this selectivity for eliciting [Ca 2ϩ ] i signals . Previous work has demonstrated P2Y receptor-mediated suppression of I M to be via G q/11 and to require PLC activity (Bofill-Cardona et al, 2000;Stemkowski et al, 2002). We thus began our investigation of the intracellular mechanism linking P2Y receptor stimulation with suppression of I M by asking whether UTP elicits [Ca 2ϩ ] i signals in SCG neurons.…”

Does diacylglycerol regulate KCNQ channels?

“…Our early work on frog neurons showed that exogenous DAGs did reduce M current somewhat, an effect blocked by blockers of PKC [11]. We also showed that the PKC blockers did not block the acute muscarinic suppression of M current (see also [26]). …”

Differential Neurotrophic Regulation of Sodium and Calcium Channels in an Adult Sympathetic Neuron

“…Moreover, U-73122 has already been shown to inhibit agonist-induced PLC activation in BFSG neurons (Stemkowski et al 2002). Inclusion of 20 M U-73122 in the cultures prevented the increase in I Ba produced by 0.45 M LHRH (P Ͼ 0.05, Table 1).…”