L‐ and N‐current but not M‐current inhibition by M₁ muscarinic receptors requires DAG lipase activity

Abstract

Stimulation of postsynaptic M(1) muscarinic receptors (M(1)Rs) increases firing rates of both sympathetic and central neurons that underlie increases in vasomotor tone, heart rate, and cognitive memory functioning. At the cellular level, M(1)R stimulation modulates currents through various voltage-gated ion channels, including KCNQ K+ channels (M-current) and both L- and N-type Ca2+ channels (L- and N-current) by a pertussis toxin-insensitive, slow signaling pathway. Depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) during M(1)R stimulation suffices to inhibit M-current. We found previously that following PIP2 hydrolysis by phospholipase C, activation of phospholipase A2 and liberation of a lipid metabolite, most likely arachidonic acid (AA) are necessary for L- and N-current modulation. Here we examined the involvement of a third lipase, diacylglycerol lipase (DAGL), in the slow pathway. We documented the presence of DAGL in superior cervical ganglion neurons, and then tested the highly selective DAGL inhibitor, RHC-80267, for its capacity to antagonize M(1)R-mediated modulation of whole-cell Ca2+ currents. RHC-80267 significantly reduced L- and N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M) but did not affect their inhibition by exogenous AA. Moreover, voltage-dependent inhibition of N-current by Oxo-M remained in the presence of RHC-80267, indicating selective action on the slow pathway. RHC also blocked inhibition of recombinant N-current. In contrast, RHC-80267 had no effect on native M-current inhibition. These data are consistent with a role for DAGL in mediating L- and N-current inhibition. These results extend our previous findings that the signaling pathway mediating L- and N-current inhibition diverges from the pathway initiating M-current inhibition.

Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors

“…The DAGL ␣ cDNA 2-arachidonoylglycerol, has been implicated in the control of pain mechanisms ( 13 ) and cancer cell proliferation ( 14 ). DAGL enzymes have been found to be important in the production of 2-arachidonoylglycerol and in the control of appetite and pain ( 15 ) and also in the regulation of Ca 2+ infl ux into cells ( 16 ). We recently showed that 1-MAG is a coupling factor linking glucose metabolism to insulin secretion in pancreatic ␤ -cells and that ABHD6 specifi cally controls ␤ -cell 1-MAG levels ( 4 ).…”

G protein inhibition of CaV2 calcium channels

“…Voltage-independent inhibition is generally less well characterized and likely comprised of several distinct mechanisms including channel trafficking, phosphorylation and lipid signaling pathways (reviewed in ref. 34,35,40,[43][44][45][46][47][48]. The primary focus of this review is the direct, voltage-dependent inhibition of Ca V 2 channels.…”

“…97 Likewise, truncating the N-terminal 55 amino acids of Ca V 2.2 prevented voltagedependent inhibition and introducing the Ca V 2.2 N-terminus into the Ca V 1.2 backbone conferred modest inhibition onto these normally resistant channels. 97,121 The N-terminus has a glycine rich stretch of amino acids (9-40) that is followed by eleven amino acids (45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55) that are predicted to form an α-helix.…”

The diacylglycerol lipases: structure, regulation and roles in and beyond endocannabinoid signalling

“…Likewise, 2-AG can be oxygenated by a number of lipooxygenases to generate a ligand that can activate peroxisome-activated receptors [90]. There is also emerging evidence that DAGL-dependent pathways can inhibit L-and Ntype calcium currents [91], and regulate a form of synaptic plasticity mediated by a TRPV-like receptor [92]; however, it remains unclear whether these effects are mediated by 2-AG or by a 2-AG metabolite.…”