Molecular basis for N-type voltage-gated Ca 2 + channel modulation by G q protein-coupled receptors

N-type voltage-gated Ca (CaV2.2) channels, which enable synaptic transmission by triggering neurotransmitter release, are tightly modulated by G protein-coupled receptors (GPCRs) via several downstream signaling messengers, such as G, calmodulin, arachidonic acid and PIP2. However, the molecular mechanism by which Gq/11-coupled receptors (GqPCRs) suppress CaV2.2 currents remains unclear. In this research highlight, we review our recent finding that M1 muscarinic receptors inhibit CaV2.2 channels through both G-mediated voltage-dependent (VD) and Gαq/11/PLC-mediated voltage-independent (VI) pathways. Our photometry results also demonstrate that G-mediated VD inhibition of CaV2.2 channels initiates approximately 3 s earlier than VI inhibition, and is strongly potentiated in cells expressing plasma membrane-localized CaV subunits. Our observations demonstrate a novel mechanism for CaV2.2 channel modulation by GqPCRs where the subcellular location of CaV subunits plays a critical role in determining the voltage-dependence of current suppression by M1 receptors.

N-type voltage-gated Ca 2+ (CaV2.2) channels are central to synaptic transmission [1] in response to the propagation of electrical stimulations and to the processes it underlies, such as learning and memory [2] and gene transcription [3] .CaV2.2 channels are widely expressed throughout the brain [4] and spinal cord [5] , and knockout mice who lack CaV2.2 channels show cardiovascular impairment [6] , hyperactivity [7] , reduced alcohol consumption [8] , and hyperaggressive behavior [9] .The biophysical and pharmacological properties of CaV2.2 channels are determined by diverse combinations of channel subunits.CaVα1B and CaVα2δ are transmembrane proteins.CaVα1B subunits are responsible for forming the voltage-sensitive pore of the channel and CaVα2δ subunits are responsible for promoting CaVα1 subunit stabilization at the plasma membrane [10] .CaV subunits are intracellular components that play an essential role in regulating the gating properties and receptor modulation of CaV channels.They bind to the I-II linker of the CaVα1 subunit and finely tune the trafficking of α1 channel proteins to the plasma membrane, current density, channel inactivation and channel regulation by phospholipids [11][12][13][14] .G-protein coupled receptors (GPCRs) precisely regulate Ca 2+ ion influx through CaV2.2 channels [15,16] .The activation of GPCRs coupled to Gαi/o (Gi/oPCRs) or Gαq/11 (GqPCRs) is known to suppress CaV2.2 current through two distinct pathways.The first operates via G heterodimer dissociation from Gi/oPCR.The G heterodimer then directly binds to the I-II linker of the CaVα1B subunit, which partially overlaps with the binding site of the CaV subunit,

RESEARCH HIGHLIGHT
and triggers fast current inhibition [17] .Since the G binding to the α1B subunit slows channel activation and shifts the voltage dependence of the channel opening towards a positive charge, a stronger depolarization of the plasma membrane is needed for the CaV2.2 channels to open.This inhibition can be relieved by supplying a large depolarizing pulse [18,19] and is thus referred to as "voltage-dependent" (VD) [15] .The second pathway, for GqPCRs, depends on phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C (PLC) [20][21][22] and/or arachidonic acid (AA) generation by phospholipase A2 (PLA2) activation [23] following Gαq/11 subunit activation.This pathway is responsible for relatively slow and voltage-independent (VI) Ca 2+ channel inhibition.The role of the G subunit in GqPCR modulation of CaV2.2 channels remains unclear.It is possible that the G subunit released from the Gq/11 protein can produce fast and voltage-dependent inhibition of the CaV2.2 current [21,22,[24][25][26] .
Previous studies have revealed that the slow component of CaV2.2 channel modulation by the Gq/11-coupled M1 muscarinic acetylcholine receptor (M1R) relies on PIP2 depletion through Gαq/PLC activation and that the CaV subunit is an important regulator of the muscarinic modulation of the channel [13,22] .Indeed, since GqPCRs activation accompanies many downstream signals, such as Ca 2+ release from the endoplasmic reticulum and PKC activation via diacylglycerol generation, discriminating between PIP2 effects and those of other signals is almost impossible.However, with the aid of several genetically encoded tools, such as voltage-sensing phosphatase from zebra fish (Dr-VSP) [22,27,28] and the chemically-inducible dimerization system (Lyn-FRB/FKBP-Inp54p) [29] which cleaves the 5-phosphate of PIP2 to generate phosphatidylinositol 4-monophosphate (PIP) on demand, the contribution of PI(4,5)P2 depletion alone to CaV channel inhibition was uncovered in single cells.Interestingly, M1R activation resulted in stronger CaV current inhibition than direct PI(4,5)P2 depletion through Dr-VSP activation or through the rapamycin inducible dimerization system, which implies that pathways besides Gαq/PLC are involved.To verify this hypothesis, N-type CaV2.2 channels, those are regulated by both VI and VD pathways, were studied [30] .We also adopted C-terminus of -adrenergic receptor kinase (ARK-ct) as a G subunit scavenger [25,31,32] .When Ba 2+ currents were measured in tsA201 cells expressing M1R, CaV α1B, α2δ1 and 2a subunits upon muscarinic stimulation, the co-expression of ARK-ct resulted in the disappearance of the VD component of M1R-induced inhibition; channel inhibition was consequently attenuated to the level due to  [31] .
Dr-VSP activation alone (approximately10% of total).Similarly, substituting G-insensitive chimeric CaV2.2 (α1C-1B) for the wild type CaV α1B subunit also decreased M1R-induced CaV current depression.In contrast, pertussis-toxin (PTX) sensitive Gαi/o-coupled M2R-induced CaV2.2 current inhibition was completely abolished by the co-expression of ARK-ct.Therefore, we concluded that unlike M2R that mainly inhibits CaV2.2 channels through G-mediated VD pathway, M1R inhibits the channels through both VI and VD components, such that the VI component is subject to Gαq/PLC activation followed by PIP2 depletion, while the VD component is mainly affected by the G subunit (Fig. 1 A).
To further examine the two M1R-mediated regulatory pathways, we simultaneously measured FRET (Förster resonance energy transfer) between the eCFP-and eYFP-tagged pleckstrin homology (PH) domain from phospholipase C-δ (PH-PLCδ), which sufficiently reflects the extent of plasma membrane PIP2 [22,[33][34][35][36] , and CaV2.2 current inhibition in single control and ARK-ct-expressing cells.In this single cell assay, CaV2.2 current depression due to M1R activation was attenuated in cells co-expressing ARK-ct by approximately 50%.In addition, we found that current inhibition started 3-4 s before PIP2 hydrolysis in control cells, but not in ARK-ct expressing cells, where current inhibition and PIP2 hydrolysis started at almost the same time.Although we did not find any differences in the kinetics of current inhibition and PIP2 hydrolysis between control and ARK-ct expressing cells, we did discriminate between the fast (τ = 1.6 s) and slow (τ = 4.1 s) components of M1R induced CaV2.2 inhibition by subtracting the scaled CaV current of ARK-ct co-expressing cells from that of control cells, considering 'lag time'.The findings clearly indicated that M1R activation mediates both slow and fast N-type CaV2.2 current suppression through the Gαq/11 and G subunits, respectively.Furthermore, our findings suggest that under physiological conditions, the downstream effects of GqPCRs may be affected by the duration, frequency or intensity of stimulation.
As mentioned earlier, the CaV subunit determines the CaV2.2 channel modulation by membrane PIP2 turnover [13] .CaV2.2 channels expressed with membrane-localized  subunits were only very slightly inhibited by PIP2 depletion while channels expressed with cytosolic  subunits were dramatically inhibited by PIP2 depletion.Additionally, we also realized that the extent of M1R-induced VI or VD inhibition of N-type CaV current varied with the  subunit isotype expressed.This was confirmed by measuring the CaV2.2 current suppression by M1R in cells transfected with α2δ1, 2a, palmitoylation resistant mutant 2a(C3,4S), 2b, 3 or membrane targeted Lyn-3.Firstly, VD inhibition by M1R was measured by applying a strong depolarizing pulse between the pre-and post-test pulses with and without the presence of a muscarinic receptor agonist.Current inhibition in cells co-expressing membrane-localized 2a or chimeric Lyn-3 was approximately 30%, while the inhibition in cells with cytosolic 2b, β2a(C3,4S) or 3 was 11-15%.However, the PIP2 dependence of the current inhibition was 10-25% and 41-59% for membrane-localized and cytosolic  subunits, respectively.As depicted in Fig. 1 B, although CaV2.2 channels are inhibited by both VI and VD pathways, the channels with membrane-localized  subunits are more sensitive to the G-mediated VD pathway, whereas cytosolic  subunits are largely affected by the PIP2-dependent VI pathway.

Figure 1 .
Figure 1.Diagram of inhibitory signaling to CaV2.2 channels by acetylcholine muscarinic receptors.(A) M1 and M2 muscarinic receptors suppress CaV2.2 current via different pathways.M1R suppresses CaV2.2 currents through both PIP2-dependent VI and G-mediated VD pathways, while M2R suppresses currents only through the G-mediated VD pathway.The G scavenger ARK-ct inhibits the G-mediated pathway and PTX inhibits M2 receptor signaling by blocking the activation of Gi/o proteins.(B) M1R modulates CaV2.2 channels through two separate pathways independently.The predominance of each type of modulation is determined by the CaV subunit.Membrane-associated  subunits decrease PIP2-dependent VI regulation and enhance G-mediated VD regulation.The cytosolic  subunit increases VI regulation and decreases the effects of VD regulation.Thick line: major inhibitory pathway.Thin line: minor or weak inhibitory pathway.AC, adenylyl cyclase; PLC, phospholipase C; PTX, pertussis toxin; VD, voltage-dependent inhibition; VI, voltage-independent inhibition.Images are modified from the original work[31] .