Muscarinic Receptor Neuromodulation of KCNQ M-Type K+, and Other, Channels in Hippocampal Principal Neurons Involves Striking Cell-Specific Regulation Controlling Excitability

Abstract

Voltage-gated “M-type” channels are important for neuronal discharge properties and require interactions with phosphatidylinositol 4,5-bisphosphate(PIP2). In sympathetic ganglia, M-current(IM) is strongly depressed by Gq-coupled-M1AChR stimulation via PIP2 depletion by phospholipase C(PLC), and other GqPCRs by Ca2+ signals. Ca2+ rises are suggested to stimulate synthesis of PIP2. In hippocampus, M/KCNQ-channels and M1AChRs are widely expressed, and cholinergic innervation strongly affects circuit excitability. We investigated mAChR neuromodulation of hippocampal neurons using brain-slice electrophysiology and Ca2+ imaging. Using whole-cell voltage-clamp, we observed fraction of deactivating relaxation at −60 mV due to IM/ERG current by kinetics and highly-specific blockers (XE991,E-4031). In dentate gyrus granule cells(DGGCs), >95% of current was from M-channels, whereas in CA1 neurons, M/ERG current ratio was 60/40. We stimulated mAChRs with oxotremorine-M(1-10μM) or M1R-selective agonist 77LH281(3-10μM). Unexpectedly, both agonists enhanced IM in DGGCs (maximum 2.4-fold), whereas IM was suppressed in CA1 neurons (78% block). M1R-induced enhancement of IM was abolished by edelfosine blockade of PLC. Testing M1R-induced increase of PIP2 abundance in DGGCs as underlying increases in IM, we quantified commensurate increase of GIRK current during muscarinic stimulation, thus acting as a PIP2 biosensor. Blockade of PI(4)P5-kinase by UNC3230(3μM) caused muscarinic stimulation to instead suppress IM in DGGCs by ∼60%. Ca2+ imaging revealed robust [Ca2+]i increase in DGGCs by muscarinic stimulation. Despite pronounced increase in IM by mAChR stimulation in DGGCs, it was accompanied by robust increase in excitability (action potential frequency), leading us to hypothesize PLC-induced activation of other channels. When the TRPC-blocker, ML204 (10μM), was applied concurrently with muscarinic agonist, neither excitability nor [Ca2+]i were significantly changed. Thus, DGGC excitability is regulated by muscarinic signals in concert with M-type/TRPC channels, whereas CA1 neurons are regulated in a distinct fashion.