Diacylglycerol‐mediated regulation of Aplysia bag cell neuron excitability requires protein kinase C

Abstract

Following synaptic input, the bag cell neurons of Aplysia undergo a long-term afterdischarge of action potentials to secrete egg-laying hormone and initiate reproduction. Early in the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3 ) and diacylglycerol (DAG). In Aplysia, little is known about the action of DAG, or any interaction with IP3 ; thus, we examined the effects of a synthetic DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), on whole-cell voltage-clamped cultured bag cell neurons. OAG induced a large, prolonged, Ca(2+) -permeable, concentration-dependent inward current (IOAG ) that reversed at ∼-20 mV and was enhanced by intracellular IP3 . A similar current was evoked by either another DAG analogue, 1,2-dioctanoyl-sn-glycerol (DOG), or activating PLC with N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide (m-3M3FBS). IOAG was reduced by the general cation channel blockers Gd(3+) or flufenamic acid. Work in other systems indicated that OAG activates channels independently of protein kinase C (PKC); however, we found pretreating bag cell neurons with any of the PKC inhibitors bisindolylmaleimide, sphinganine, or H7, attenuated IOAG . However, stimulating PKC with phorbol 12-myristate 13-acetate (PMA) did not evoke current or enhance IOAG ; moreover, unlike PMA, OAG failed to trigger PKC, as confirmed by an independent bioassay. Finally, OAG or m-3M3FBS depolarized cultured neurons, and while OAG did not provoke afterdischarges from bag cell neurons in the nervous system, it did double the duration of synaptically elicited afterdischarges. To our knowledge, this is the first report of obligate PKC activity for IOAG gating. An interaction between phosphoinositol metabolites and PKC could control the cation channel to influence afterdischarge duration.

Hydrogen Peroxide Gates a Voltage-Dependent Cation Current inAplysiaNeuroendocrine Cells

“…Last, ethanol (100% v/v) was used to dissolve N-ethylmaleimide (NEM; 50 mM; E3876, Sigma-Aldrich). The final concentration of DMSO or ethanol in the bath was Յ0.2% (v/v), which in control experiments here or in prior studies was found to have no effect on bag cell neuron holding current, membrane conductance, or membrane potential (Hickey et al, 2010(Hickey et al, , 2013Tam et al, 2011;Sturgeon and Magoski, 2016;White et al, 2018).…”

“…With the slow-phase, phospholipase C cleaves PIP 2 into IP 3 and DAG (Fink et al, 1988). DAG gates a second, voltageindependent cation channel, with a distinct pharmacology and reversal potential of ϳϪ20 mV (Sturgeon and Magoski, 2016). This channel may be similar to TRPC3/6/7 channels, which are well established as being activated by DAG (Hofmann et al, 1999;Okada et al, 1999).…”

“…The hormone engages both central neurons and peripheral organs to induce behaviors that culminate in the deposition of fertilized eggs (Arch and Smock, 1977;Stuart and Strumwasser, 1980;Rothman et al, 1983). The afterdischarge is maintained by opening of three, distinct cation channels: a voltage-independent channel gated by calmodulin-kinase (Hung and Magoski, 2007;Hickey et al, 2010), a separate voltage-independent channel triggered by diacylglycerol (DAG; Sturgeon and Magoski, 2016), and a Ca 2ϩ -permeable, Ca 2ϩ -activated, voltage-dependent channel (Wilson et al, 1996;Lupinsky and Magoski, 2006;Geiger et al, 2009). Munnamalai et al (2014) showed that NADPH oxidase is present in bag cell neurons, and protein kinase C (PKC), which is activated early in the slow-phase of the afterdischarge (Wayne et al, 1999), stimulates H 2 O 2 production.…”

A Closely Associated Phospholipase C Regulates Cation Channel Function through Phosphoinositide Hydrolysis

“…The presence of phosphatidylinositols in Aplysia neuronal membranes was established based on lipid profile studies (Piomelli et al, 1987) and the liberation of arachidonic acid from PIP 2 by PLC (Carlson and Levitan, 1990), as well as previous whole-cell experiments, suggesting that PLC activity can influence a voltageindependent bag cell neuron cation current (Sturgeon and . Given that PLC is activated during the afterdischarge and results in the formation of IP 3 and DAG (Fink et al, 1988), we investigated the effect of these metabolites on the voltagedependent cation channel.…”

“…Ongoing research indicates that DAG and IP 3 also directly gate or modulate various ion channels, including gap junctions and inwardly rectifying K ϩ channels, as well as native and transient receptor potential (TRP) nonselective cation channels (Albert and Large, 2003;Gamper and Shapiro, 2007;Decrock et al, 2013). TRP channels are a diverse cation channel superfamily, some of which are voltage-dependent, permeable to both monovalent and divalent cations, sensitive to intracellular Ca 2ϩ , and play a role in a myriad of cellular processes (Clapham, 2003;Montell, 2005;Hardie, 2007). Both PIP 2 and DAG directly gate TRP channels, either by modulation of the lipid microenvironment surrounding the channels or via classic agonist-like binding to the channel (Hofmann et al, 1999;Hansen, 2015;Hille et al, 2015).…”