M1 muscarinic receptors inhibit L-type Ca2+ current and M-current by divergent signal transduction cascades

Ion channels reside in a sea of phospholipids. During normal fluctuations in membrane potential and periods of modulation, lipids that directly associate with channel proteins influence gating by incompletely understood mechanisms. In one model, M-1-muscarinic receptors ( M(1)Rs) may inhibit both Ca2+ (L- and N-) and K-= (M-) currents by losing a putative interaction between channels and phosphatidylinositol-4,5-bisphosphate(PIP2). However, we found previously that M1R inhibition of N- current in superior cervical ganglion (SCG) neurons requires loss of PIP2 and generation of a free fatty acid, probably arachidonic acid ( AA) by phospholipase (2) (PLA(2)). It is not known whether PLA(2) activity and AA also participate in L- and M- current modulation in SCG neurons. To test whether PLA(2) plays a similar role in M1R inhibition of L- and M- currents, we used several experimental approaches and found unanticipated divergent signaling. First, blocking resynthesis of PIP2 minimized M- current recovery from inhibition, whereas L- current recovered normally. Second, L- current inhibition required group IVa PLA(2) [ cytoplasmic PLA(2) ( cPLA(2))], whereas M- current did not. Western blot and imaging studies confirmed acute activation of cPLA(2) by muscarinic stimulation. Third, in type IIa PLA(2) [secreted (sPLA(2))](-/-)/cPLA(2) double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M- current over-recovery after washout of muscarinic agonist. Our findings support a model of M1R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.