Regulation and molecular identification of a lipid-gated cation channel in Aplysia bag cell neurons

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Stevens, Elise Mary MacLeod
Aplysia , ion channel , cation channel , transient receptor potential channel , TRP channel , electrophysiology , phospholipid , calcium , phospholipase C , afterdischarge
Non-selective cation currents are key to the control of neuronal activity, and can be regulated by numerous intracellular messengers, including lipid metabolites and Ca2+. In neuroendocrine bag cell neurons from the sea snail, Aplysia californica, a diacylglycerol (DAG)-gated cation current supports an ~30 min afterdischarge and hormone secretion to induce egg-laying. To understand how this current is regulated, cultured bag cell neurons were whole-cell voltage-clamped or sharp-electrode current-clamped and exposed to N-(3-trifluoromethyl phenyl)-2,4,6-trimethylbenzenesulfonamide (m-FBS), which direcly stimulates phospholipase C (PLC) to hydrolyse phosphatidylinositol 4,5-bisphosphate (PIP2) into DAG and inositol trisphosphate. Consistent with cation channel opening, m-FBS induced an ~6-fold increase in membrane conductance with a voltage-independent current between -80 and 0 mV that reversed at -25.1 mV. Low extracellular Ca2+, replacing Ca2+ with Ba2+, or low intracellular Ca2+ potentiated the m-FBS-induced current by 2-3 times, suggesting Ca2+-dependent inactivation via channel-mediated Ca2+ entry. PIP2 appeared to be a basal negative regulator, as depletion with wortmannin, which inhibits PIP2 synthesis, augmented the current (+120%) or depolarization (+30%) evoked by the DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol. The m-FBS-induced current may in part be coded for by an Aplysia transient receptor potential channel, ApTRPC5, which our laboratory previously cloned and found responsive to PLC activation with an inward current in a heterologous expression system. Here, real-time quantitative polymerase chain reactions showed ApTRPC5 to be expressed in bag cell neurons and adjacent abdominal ganglion. Human embryonic kidney cells expressing ApTRPC5 responded to m-FBS with a current that reversed at -10.8 mV and, like the native current, was doubled by low extracellular Ca2+. Mutation of Arg627, a residue suspected to be involved in PLC gating of human TRPC5, nearly eliminated the response to m-FBS but did not interfere with ApTRPC5 expression or trafficking as assayed by immunoblotting or epi-fluorescent microscopy, respectively. Moreover, disruption of ApTRPC5 expression in bag cell neurons with double-stranded RNA halved the m-FBS-induced current. In summary, the reproductive afterdischarge is promoted through PLC activation, which generates DAG to open a TRP-like non-selective cation channel, which in turn is both facilitated by PIP2 breakdown and, to prevent aberrant signalling, limited by Ca2+ influx.
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