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

Abstract

In the hemaphroditic sea snail, <i>Aplysia californica</i>, reproduction is initiated when the bag cell neurons secrete egg-laying hormone during a protracted afterdischarge. A source of depolarization for the afterdischarge is a voltage-gated, nonselective cation channel, similar to transient receptor potential (TRP) channels. Once the afterdischarge is triggered, phospholipase C (PLC) is activated to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol trisphosphate (IP₃). We previously reported that a DAG analog, 1-oleoyl-2-acetyl-<i>sn</i>-glycerol (OAG), activates a prominent, inward whole-cell cationic current that is enhanced by IP₃. To examine the underlying mechanism, we investigated the effect of exogenous OAG and IP₃, as well as PLC activation, on cation channel activity and voltage dependence in excised, inside-out patches from cultured bag cell neurons. OAG transiently elevated channel open probability (P_O) when applied to excised patches; however, coapplication of IP₃ prolonged the OAG-induced response. In patches exposed to OAG and IP₃, channel voltage dependence was left-shifted; this was also observed with OAG, but not to the same extent. Introducing the PLC activator, m-3M3FBS, to patches increased channel P_O, suggesting PLC may be physically linked to the channels. Accordingly, blocking PLC with U-73122 ablated the m-3M3FBS-induced elevation in P_O. Treatment with m-3M3FBS left-shifted cation channel voltage dependence to a greater extent than exogenous OAG and IP₃. Finally, OAG and IP₃ potentiated the stimulatory effect of PKC, which is also associated with the channel. Thus, the PLC-PKC signaling system is physically localized such that PIP₂ breakdown products liberated during the afterdischarge modulate the cation channel and temporally influence neuronal activity. <b>SIGNIFICANCE STATEMENT</b> Using excised patches from <i>Aplysia</i> bag cell neurons, we present the first evidence of a nonselective cation channel physically associating with phospholipase C (PLC) at the single-channel level. PLC-mediated breakdown of phospholipids generates diacylglycerol and inositol trisphosphate, which activate the cation channel. This is mimicked by exogenous lipids; furthermore, these second messengers left-shift channel voltage dependence and enhance the response of the channel to protein kinase C. PLC-mediated lipid signaling controls single-channel currents to ensure depolarization is maintained for an extended period of firing, termed the afterdischarge, when the bag cell neurons secrete egg-laying hormone to trigger reproduction.