Modulation of G_q-Protein-Coupled Inositol Trisphosphate and Ca²⁺Signaling by the Membrane Potential

Abstract

G q protein-coupled receptors (G q PCRs) are widely distributed in the CNS and play fundamental roles in a variety of neuronal processes. Their activation results in PIP 2 hydrolysis and Ca 2+ release from intracellular stores via the PLC-IP 3 signaling pathway. Since early G q PCR signaling events occur at the plasma membrane of neurons, they might be influenced by changes in membrane potential. In this study we use combined patch-clamp and imaging methods to investigate whether membrane potential changes can modulate G q PCR signaling in neurons. Our results demonstrate that G q PCR signaling in the human neuronal cell line SH-SY5Y and in rat cerebellar granule neurons is directly sensitive to changes in membrane potential, even in the absence of extracellular Ca 2+ . Depolarization has a bi-directional effect on G q PCR signaling, potentiating thapsigargin-sensitive Ca 2+ responses to muscarinic receptor activation but attenuating those mediated by bradykinin receptors. The depolarization-evoked potentiation of the muscarinic signaling is graded, bipolar, non-inactivating and with no apparent upper limit, ruling out traditional voltage-gated ion channels as the primary voltage sensors. Flash photolysis of caged IP 3 /GPIP 2 places the voltage-sensor before the level of the Ca 2+ store and measurements using the fluorescent bioprobe eGFP-PH PLCδ directly demonstrate that voltage affects muscarinic signaling at the level of the IP 3 production pathway. The sensitivity of G q PCR IP 3 signaling in neurons to voltage itself may represent a fundamental mechanism by which ionotropic signals can shape metabotropic receptor activity in neurons and influence processes such as synaptic plasticity in which the detection of coincident signals is crucial.

Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice

“…Because M 2 and M 4 are coupled to G i/o proteins, stimulation of these two subtypes can inhibit synaptic glutamate release through inhibition of voltage-gated calcium channels (40 -42). On the other hand, M 3 and M 5 are coupled to G q/11 proteins, and activation of these two subtypes could increase presynaptic glutamate release through stimulation of phospholipase Cinositol triphosphate, which increase the intracellular calcium level (43,44). It would be interesting to determine whether M 3 /M 5 stimulation "antagonizes" the muscarinic analgesic effect at the spinal level by comparing the effects of mAChR agonists on nociception in WT, M 3 -KO, and M 5 -KO mice.…”

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca 2+ release in smooth muscle

“…These findings are consistent with the well-known fact that M 3 Rs, but not M 2 Rs and M 1 Rs, normally provide predominant neuronal control of Ca 2ϩ and contractile responses in SMCs. Billups et al have shown that voltage depolarization significantly increases the muscarinic agonist oxotremorine-evoked Ca 2ϩ release by augmenting IP 3 production in neurons (14). It is also known that membrane depolarization can affect other multiple GPCRdependent signaling cascade sites to enhance muscarinic agonistinduced Ca 2ϩ release in a variety of cells (8).…”

Fast Regulation of Axonal Growth Cone Motility by Electrical Activity

“…The remaining Ca 2ϩ release pathway from the ER involves IP 3 -sensitive receptors. Interestingly, it has been shown recently in cerebellar granule neurons that depolarization alone can activate phospholipase C (PLC) via G q -proteins, leading to phosphatidylinositol-4,5-biphosphate hydrolysis and IP 3 production in a mechanism completely independent of Ca 2ϩ influx (Billups et al, 2006). To block IP 3 -mediated Ca 2ϩ release, we used the widely used IP 3 channel blocker 2-APB.…”