CPLA 2 α -/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca 2+ current inhibition by M 1 muscarinic receptor signaling

Abstract

Group IVa cytosolic phospholipase A 2 (cPLA 2 α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP 2 ) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA 2 activity is required for voltage-independent N-type Ca 2+ (N-) current inhibition by M 1 muscarinic receptors (M 1 Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP 2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA 2 α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA 2 α antagonists or dialyzing cPLA 2 α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca 2+ -independent PLA 2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA 2 α -/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA 2 α. In contrast, cPLA 2 α antibodies or the absence of cPLA 2 α had no effect on voltage-dependent N-current inhibition by M 2 / M 4 Rs or on M-current inhibition by M 1 Rs. These findings document divergent M 1 R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA 2 α acts locally to metabolize PIP 2 intimately associated with N- but not M-channels. To determine cPLA 2 α’s functional importance more globally, we examined action potential firing of cPLA 2 α +/+ and cPLA 2 α -/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA 2 α -/- neurons. These unanticipated findings identify cPLA 2 α as a tonic regulator of neuronal membrane excitability.