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Inhibition of synaptic transmission and G protein modulation by synthetic CaV2.2 Ca2+ channel peptides
Abstract: Modulation of presynaptic voltage-dependent Ca+ channels is a major means of controlling neurotransmitter release. The CaV 2.2 Ca2+ channel subunit contains several inhibitory interaction sites for GĪ²Ī³ subunits, including the amino terminal (NT) and IāII loop. The NT and IāII loop have also been proposed to undergo a G protein-gated inhibitory interaction, whilst the NT itself has also been proposed to suppress CaV 2 channel activity. Here, we investigate the effects of an amino terminal (CaV 2.2[45ā55]) āNT peptideā and a IāII loop alpha interaction domain (CaV 2.2[377ā393]) āAID peptideā on synaptic transmission, Ca2+ channel activity and G protein modulation in superior cervical ganglion neurones (SCGNs). Presynaptic injection of NT or AID peptide into SCGN synapses inhibited synaptic transmission and also attenuated noradrenaline-induced G protein modulation. In isolated SCGNs, NT and AID peptides reduced whole-cell Ca2+ current amplitude, modified voltage dependence of Ca2+ channel activation and attenuated noradrenaline-induced G protein modulation. Co-application of NT and AID peptide negated inhibitory actions. Together, these data favour direct peptide interaction with presynaptic Ca2+ channels, with effects on current amplitude and gating representing likely mechanisms responsible for inhibition of synaptic transmission. Mutations to residues reported as determinants of Ca2+ channel function within the NT peptide negated inhibitory effects on synaptic transmission, Ca2+ current amplitude and gating and G protein modulation. A mutation within the proposed QXXER motif for G protein modulation did not abolish inhibitory effects of the AID peptide. This study suggests that the CaV 2.2 amino terminal and IāII loop contribute molecular determinants for Ca2+ channel function; the data favour a direct interaction of peptides with Ca2+ channels to inhibit synaptic transmission and attenuate G protein modulation. Non-technical summary: Nerve cells (neurones) in the body communicate with each other by releasing chemicals (neurotransmitters) which act on proteins called receptors. An important group of receptors (called G protein coupled receptors, GPCRs) regulate the release of neurotransmitters by an action on the ion channels that let calcium into the cell. Here, we show for the first time that small peptides based on specific regions of calcium ion channels involved in GPCR signalling can themselves inhibit nerve cell communication. We show that these peptides act directly on calcium channels to make them more difficult to open and thus reduce calcium influx into native neurones. These peptides also reduce GPCR-mediated signalling. This work is important in increasing our knowledge about modulation of the calcium ion channel protein; such knowledge may help in the development of drugs to prevent signalling in pathways such as those involved in pain perception
RGS2 Determines Short-Term Synaptic Plasticity in Hippocampal Neurons by Regulating Gi/o- Mediated Inhibition of Presynaptic Ca2+ Channels
SummaryRGS2, one of the small members of the regulator of G protein signaling (RGS) family, is highly expressed in brain and regulates Gi/o as well as Gq-coupled receptor pathways. RGS2 modulates anxiety, aggression, and blood pressure in mice, suggesting that RGS2 regulates synaptic circuits underlying animal physiology and behavior. How RGS2 in brain influences synaptic activity is unknown. We therefore analyzed the synaptic function of RGS2 in hippocampal neurons by comparing electrophysiological recordings from RGS2 knockout and wild-type mice. Our study provides a general mechanism of the action of the RGS family containing RGS2 by demonstrating that RGS2 increases synaptic vesicle release by downregulating the Gi/o-mediated presynaptic Ca2+ channel inhibition and therefore provides an explanation of how regulation of RGS2 expression can modulate the function of neuronal circuits underlying behavior
Facilitation versus depression in cultured hippocampal neurons determined by targeting of Ca2+ channel CavĪ²4 versus CavĪ²2 subunits to synaptic terminals
Ca2+ channel Ī² subunits determine the transport and physiological properties of high voltageāactivated Ca2+ channel complexes. Our analysis of the distribution of the CavĪ² subunit family members in hippocampal neurons correlates their synaptic distribution with their involvement in transmitter release. We find that exogenously expressed CavĪ²4b and CavĪ²2a subunits distribute in clusters and localize to synapses, whereas CavĪ²1b and CavĪ²3 are homogenously distributed. According to their localization, CavĪ²2a and CavĪ²4b subunits modulate the synaptic plasticity of autaptic hippocampal neurons (i.e., CavĪ²2a induces depression, whereas CavĪ²4b induces paired-pulse facilitation [PPF] followed by synaptic depression during longer stimuli trains). The induction of PPF by CavĪ²4b correlates with a reduction in the release probability and cooperativity of the transmitter release. These results suggest that CavĪ² subunits determine the gating properties of the presynaptic Ca2+ channels within the presynaptic terminal in a subunit-specific manner and may be involved in organization of the Ca2+ channel relative to the release machinery
Molecular Basis of Inward Rectification: Polyamine Interaction Sites Located by Combined Channel and Ligand Mutagenesis
Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter
Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand
We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a Gi-coupled designer receptor in hippocampal neurons (hM4D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo
Contribution of the kinetics of G protein dissociation to the characteristic modifications of N-type calcium channel activity
Direct G protein inhibition of N-type calcium channels is recognized by
characteristic biophysical modifications. In this study, we quantify and
simulate the importance of G protein dissociation on the phenotype of G
protein-regulated whole-cell currents. Based on the observation that the
voltage-dependence of the time constant of recovery from G protein inhibition
is correlated with the voltage-dependence of channel opening, we depict all G
protein effects by a simple kinetic scheme. All landmark modifications in
calcium currents, except inhibition, can be successfully described using three
simple biophysical parameters (extent of block, extent of recovery, and time
constant of recovery). Modifications of these parameters by auxiliary beta
subunits are at the origin of differences in N-type channel regulation by G
proteins. The simulation data illustrate that channel reluctance can occur as
the result of an experimental bias linked to the variable extent of G protein
dissociation when peak currents are measured at various membrane potentials. To
produce alterations in channel kinetics, the two most important parameters are
the extents of initial block and recovery. These data emphasize the
contribution of the degree and kinetics of G protein dissociation in the
modification of N-type currents
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