Action Potential Modulates Ca-Dependent and Ca-Independent Secretion in a Sensory Neuron

Neurotransmitter release normally requires calcium triggering. However, the somata of dorsal root ganglion (DRG) neurons possess a calcium-independent but voltage-dependent secretion (CIVDS) in addition to the classic calcium-dependent secretion (CDS). Here, we investigated the physiological role of CIVDS and the contributions of CIVDS and CDS induced by action potentials (APs) in DRG soma. Using membrane capacitance measurements, caged calcium photolysis, and membrane capacitance kinetics analysis, we demonstrated that AP-induced secretion had both CIVDS and CDS components. Following physiological stimuli, the dominant component of AP-induced secretion was either CIVDS for spontaneous firing or CDS for high-intensity stimuli. AP frequency modulates CDS-coupled exocytosis and CIVDS-coupled endocytosis but not CIVDS-coupled exocytosis and CDS-coupled endocytosis. Finally, CIVDS did not contribute to excitatory postsynaptic currents induced by APs in DRG presynaptic terminals in the spinal cord. Thus, CIVDS is probably an essential physiological component of AP-induced secretion in the soma. These findings bring novel insights into primary sensory processes in DRG neurons.     

Phosphorylation of presynaptic and postsynaptic calcium channels by cAMP-dependent protein kinase in hippocampal neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) and other second messenger-activated protein kinases modulates the activity of a variety of effector proteins including ion channels. Anti-peptide antibodies specific for the alpha 1 subunits of the class B, C or E calcium channels from rat brain specifically recognize a pair of polypeptides of 220 and 240 kDa, 200 and 220 kDa, and 240 and 250 kDa, respectively, in hippocampal slices in vitro. These calcium channels are localized predominantly on presynaptic and dendritic, somatic and dendritic, and somatic sites, respectively, in hippocampal neurons. Both size forms of alpha 1B and alpha 1E and the full-length form of alpha 1C are phosphorylated by PKA after solubilization and immunoprecipitation. Stimulation of PKA in intact hippocampal slices also induced phosphorylation of 25-50% of the PKA sites on class B N-type calcium channels, class C L-type calcium channels and class E calcium channels, as assessed by a back-phosphorylation method. Tetraethylammonium ion (TEA), which causes neuronal depolarization and promotes repetitive action potentials and neurotransmitter release by blocking potassium channels, also stimulated phosphorylation of class B, C and E alpha 1 subunits, suggesting that these three classes of channels are phosphorylated by PKA in response to endogenous electrical activity in the hippocampus. Regulation of calcium influx through these calcium channels by PKA may influence calcium-dependent processes within hippocampal neurons, including neurotransmitter release, calcium-activated enzymes and gene expression.     

Activity of TRPV1 Channels in Primary Nociceptive Neurons of Rats: Modulation by Changes in Intracellular Calcium Level

In rat neurons of the dorsal root ganglia (DRG) with mid- (35 to 25 μm) and small-sized (less than 25 μm) somata, we studied calcium transients induced by application of capsaicin (selective agonist of TRPV1 channels) under conditions of the development of other calcium transients caused by preliminary depolarization of the plasma membrane of these neurons. The above transients in rat DRG neurons were measured using the calcium-sensitive fluorescent dye Fura 2/AM. At delays of 3, 7, and 10 sec with respect to the beginning of preliminary potassium depolarization, the amplitudes of capsaicin-induced responses were smaller, as compared with the control, on average, by 26.8, 22.1, and 4.5%, respectively, in the population of mid-sized neurons and by 35.3, 21.1, and 22.4% in small neurons. Under such conditions, we observed noticeable delays of reactions to applications of capsaicin and a certain decrease in the level of intracellular calcium at the moment of beginning of development of these reactions with respect to the corresponding values in isolated depolarization-induced transients. We conclude that excitation of primary nociceptive neurons and activation of voltage-operated calcium channels result in noticeable modulation of the activity of TRPV1 channels and change their role during pain reception.     

Akt/PKB controls the activity-dependent bulk endocytosis of synaptic vesicles

Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode during intense neuronal activity. The dephosphorylation of Ser774 on dynamin I is essential for triggering of ADBE, as is its subsequent rephosphorylation by glycogen synthase kinase 3 (GSK3). We show that in primary cultures of cerebellar granule neurons the protein kinase Akt phosphorylates GSK3 during intense neuronal activity, ensuring that GSK3 is inactive during intense stimulation to aid dynamin I dephosphorylation. Furthermore, when a constitutively active form of Akt was overexpressed in primary neuronal cultures, ADBE was inhibited with no effect on clathrin-mediated endocytosis. Thus Akt has two major regulatory roles (i) to ensure efficient dynamin I dephosphorylation via acute activity-dependent inhibition of GSK3 and (ii) to negatively regulate ADBE when activated in the longer term. This is the first demonstration of a role for Akt in SV recycling and suggests a key role for this protein kinase in modulating synaptic strength during elevated neuronal activity.     

Developmental change in the calcium sensor for synaptic vesicle endocytosis in central nerve terminals

Synaptic vesicle endocytosis is stimulated by calcium influx in mature central nerve terminals via activation of the calcium-dependent protein phosphatase, calcineurin. However, in different neuronal preparations calcineurin activity is either inhibitory, stimulatory or irrelevant to the process. We addressed this inconsistency by investigating the requirement for calcineurin activity in synaptic vesicle endocytosis during development, using vesicle recycling assays in isolated nerve terminals. We show that endocytosis occurs independently of calcineurin activity in immature nerve terminals, and that a calcineurin requirement develops 2-4 weeks after birth. Calcineurin-independent endocytosis is not due to the absence of calcineurin activity, since calcineurin is present in immature nerve terminals and its substrate, dynamin I, is dephosphorylated on depolarization. Calcineurin-independent endocytosis is calcium-dependent, since substitution of the divalent cation, barium, inhibits the process. Finally, we demonstrated that in primary neuronal cultures derived from neonatal rats, endocytosis that was initially calcineurin-independent developed a calcineurin requirement on maturation in culture. Our data account for the apparent inconsistencies regarding the role of calcineurin in synaptic vesicle endocytosis, and we propose that an unidentified calcium sensor exists to couple calcium influx to endocytosis in immature nerve terminals.     

The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction

High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.     

Capsaicin Up-Regulates Protease-Activated Receptor-4 mRNA and Protein in Primary Cultured Dorsal Root Ganglion Neurons

Previous study has shown that there is a functional link between the transient receptor potential vanilloid type 1 (TRPV1) receptor and protease-activated receptor-4 (PAR4) in modulation of inflammation and pain. Capsaicin activation of TRPV1 is involved in enhancement of the expression of TRPV1 in mRNA and protein in dorsal root ganglion (DRG) in vivo. Whether capsaicin could influence expression of PAR4 in primary sensory neurons remains unknown. In the present study, expression of PAR4 in cultured rat DRG neurons was observed using immunofluorescence, real-time PCR and Western blots to examine whether increases in PAR4 mRNA and protein levels are induced by capsaicin treatment with or without pre-treatment of forskolin, a cyclic AMP/protein kinase A (cAMP/PKA) activator or PKA inhibitor fragment 14-22 (PKI14-22), a PKA inhibitor. Capsaicin treatment of cultured DRG neurons significantly increased the expression of PAR4 in mRNA and protein levels. The percentage of PAR4-, TRPV1-immunoreactive neurons and their co-localization in cultured DRG neurons increased significantly in the presence of capsaicin as compared with that in the absence of capsaicin. Compared with capsaicin-only group, pre-incubation with forskolin strongly enhanced the capsaicin-induced increase of PAR4 in mRNA and protein levels. Consistent with the involvement of PKA in the modulation of PAR4 expression, this evoked expression both at mRNA and protein levels was significantly inhibited after PKA was inhibited by pre-incubation with PKI14-22. Taken together, these results provide evidence that TRPV1 activation significantly increases the expression of PAR4 mRNA and protein levels in primary cultures of DRG neurons after capsaicin incubation. Effects of capsaicin on PAR4 expression appear to be mediated by cAMP/PKA signal pathways in DRG neurons.     

Homodimerization of the Src homology 3 domain of the calcium channel β-subunit drives dynamin-dependent endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.     

Increased GTP-binding to dynamin II does not stimulate receptor-mediated endocytosis

Regarding the molecular mechanism of dynamin in receptor-mediated endocytosis, GTPase activity of dynamin has been thought to have a critical role in endocytic vesicle internalization. However, a recent report suggested that GTP-binding to dynamin itself activates the dynamin to recruit molecular machinery necessary for endocytosis. In this study, to investigate the role of GTP binding to dynamin II, we generated two mutant dynamin II constructs: G38V and K44E. G38V, its GTP binding site might be mainly occupied by GTP caused by reduced GTPase activity, and K44E mutant, its GTP binding site might be vacant, caused by its decreased affinity for GTP and GDP. From the analysis of the ratio of GTP vs GDP bound to dynamin, we confirmed these properties. To test the effect of these mutant dynamins on endocytosis, we performed flow cytometry and confocal immunofluorescence analysis and found that these two mutants have inhibitory effect on transferrin-induced endocytosis. Whereas fluorescent transferrin was completely internalized in wild-type (WT) dynamin II expressing cells, no intracellular accumulation of fluorescent transferrin was found in the cells overexpressing K44E and G38V mutant. Interestingly, the amount of GTP bound to K44E was increased when endocytosis was induced than that bound to WT. The present results suggested that the GTPase activity of dynamin II is required for formation of endocytic vesicle and GTP-binding to dynamin II per se is not sufficient for stimulating endocytosis.     

Modulation on tetrodotoxin-resistant sodium current of loureirin B in rat dorsal root ganglion neurons via cyclic AMP–dependent protein kinase A

To search the modulation mechanism of loureirin B, a flavonoid is extracted from Dracaena cochinchinensis, on tetrodotoxin-resistant (TTX-R) sodium channel in dorsal root ganglion (DRG) neurons of rats. Experiments were carried out based on patch-clamp technique and molecular biological methods. We observed the time-dependent inhibition of loureirin B on TTX-R sodium currents in DRG neurons and found that neither occupancy theory nor rate theory could well explain the time-dependent inhibitory effect of loureirin B on TTX-R sodium currents. It suggested that a second messenger-mediated signaling pathway may be involved in the modulation mechanism. So the cyclin AMP (cAMP) level of the DRG neurons before and after incubation with loureirin B was tested by ELISA Kit. Results showed that loureirin B could increase the cAMP level and the increased cAMP was caused by the enhancement of adenylate cyclase (AC) induced by loureirin B. Immunolabelling experiments further confirmed that loureirin B can promote the production of PKA in DRG neurons. In the presence of the PKA inhibitor H-89, the inhibitory effect of loureirin B on TTX-R sodium currents was reversed. Forskolin, a tool in biochemistry to raise the levels of cAMP, also could reduce TTX-R sodium currents similar to that of loureirin B. These studies demonstrated that loureirin B can modulate the TTX-R sodium channel in DRG neurons via an AC/cAMP/PKA pathway involving the activation of AC and PKA, which also can be used to explain the other pharmacological effects of loureirin B.     

Overlapping role of dynamin isoforms in synaptic vesicle endocytosis

The existence of neuron-specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. Although lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2.     

Building a Better Dynasore: The Dyngo Compounds Potently Inhibit Dynamin and Endocytosis

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC₅₀ ? 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC₅₀ = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo? compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37‐fold improvement in potency over dynasore for liposome‐stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36‐fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin‐dependent endocytosis of transferrin in multiple cell types (IC₅₀ of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin‐independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity‐dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non‐specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin‐mediated endocytosis .     

Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1–Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin’s GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.     

Neuromedin U type 1 receptor stimulation of A-type K+ current requires the βγ subunits of Go protein, protein kinase A, and extracellular signal-regulated kinase 1/2 (ERK1/2) in sensory neurons

Although neuromedin U (NMU) has been implicated in analgesia, the detailed mechanisms still remain unclear. In this study, we identify a novel functional role of NMU type 1 receptor (NMUR1) in regulating the transient outward K(+) currents (I(A)) in small dorsal root ganglion (DRG) neurons. We found that NMU reversibly increased I(A) in a dose-dependent manner, instead the sustained delayed rectifier K(+) current (I(DR)) was not affected. This NMU-induced I(A) increase was pertussis toxin-sensitive and was totally reversed by NMUR1 knockdown. Intracellular application of GDPβS (guanosine 5'-O-(2-thiodiphosphate)), QEHA peptide, or a selective antibody raised against the Gα(o) or Gβ blocked the stimulatory effects of NMU. Pretreatment of the cells with the protein kinase A (PKA) inhibitor or ERK inhibitor abolished the NMU-induced I(A) response, whereas inhibition of phosphatidylinositol 3-kinase or PKC had no such effects. Exposure of DRG neurons to NMU markedly induced the phosphorylation of ERK (p-ERK), whereas p-JNK or p-p38 was not affected. Moreover, the NMU-induced p-ERK increase was attenuated by PKA inhibition and activation of PKA by foskolin would mimic the NMU-induced I(A) increase. Functionally, we observed a significant decrease of the firing rate of neuronal action potential induced by NMU and pretreatment of DRG neurons with 4-AP could abolish this effect. In summary, these results suggested that NMU increases I(A) via activation of NMUR1 that couples sequentially to the downstream activities of Gβγ of the G(o) protein, PKA, and ERK, which could contribute to its physiological functions including neuronal hypoexcitability in DRG neurons.     

A simulation study on the Ca2+-independent but voltage-dependent exocytosis and endocytosis in dorsal root ganglion neurons

In patch-clamped somata of dorsal root ganglion (DRG) neurons, two types of secretion have been proposed: Ca²⁺-dependent secretion and Ca²⁺-independent but voltage-dependent secretion (CIVDS). The Ca²⁺-induced and the depolarization-induced membrane capacitance (C_m) increases contribute 80 and 20% to the total C_m increase, respectively (Zhang and Zhou in Nat Neurosci 5:425, 2002). In order to explore the mechanism of the voltage-dependent C_m change (C_m), we constructed a model with sequential states. The simulation with this model closely approximates all the experimental data. The model predicts that the majority of fusion events (approximately 80%) are so-called kiss-and-run events, which account for the fast recovery or the rapid retrieval feature of the signals. The remaining 20% are attributed to full fusion events, which account for a slow retrieval feature. On the basis of the model, one mechanism of the activity-dependent endocytosis has revealed a differential distribution of vesicles between the kiss-and-run and full fusion states at different stimulation frequencies. The quantitative model presented in this study may help us to understand the mechanism of the CIVDS and the tightly coupled endocytosis found in mammalian DRG neurons.     

Clathrin-dependent endocytosis is required for TrkB-dependent Akt-mediated neuronal protection and dendritic growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.     

Specific role for the PH domain of dynamin-1 in the regulation of rapid endocytosis in adrenal chromaffin cells.

Dynamin plays a key role in the scission event common to various types of endocytosis. We demonstrate that the pleckstrin homology (PH) domain of dynamin-1 is critical in the process of rapid endocytosis (RE) in chromaffin cells. Introduction of this isolated PH domain into cells at concentrations as low as 1 microM completely suppressed RE. PH domains from other proteins, including that from the closely related dynamin-2, were ineffective as inhibitors, even at high concentrations. Mutational studies indicated that a pair of isoform-specific amino acids, located in a variable loop between the first two beta-strands, accounted for the differential effect of the two dynamin PH domains. Switching these amino acids in the dynamin-2 PH domain to the equivalent residues in dynamin-1 (SL-->GI) generated a molecule that blocked RE. Thus, the PH domain of dynamin-1 is essential for RE and exhibits a precise molecular selectivity. As chromaffin cells express both dynamin-1 and -2, we speculate that different isoforms of dynamin may regulate distinct endocytotic processes and that the PH domain contributes to this specificity.     

Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism

We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.     

PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels.     

Bisphenol A Modulates Calcium Currents and Intracellular Calcium Concentration in Rat Dorsal Root Ganglion Neurons

The endocrine-disrupting chemical bisphenol A (BPA) is used to manufacture plastics including food containers, and it may leach into these containers. Consumption of BPA that has leached out of plastics may be harmful as recent research highlighted that BPA can induce alterations in the nervous system. In the present work, we studied the effects of BPA on Ca²⁺ channels in dorsal root ganglion (DRG) neurons. Using whole-cell patch-clamp recordings, we found that I _Ca could be reduced by BPA in a concentration-dependent manner. Additionally, BPA shifted the activation curve of calcium currents toward a depolarizing direction and increased the slope factor of the curve. The inactivation curve for the currents was also assessed, and the curve shifted toward the depolarizing direction, although it was not significant. Moreover, inhibitory effects of BPA on the increments of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by 50 mM KCl were observed in DRG neurons using a laser scanning confocal microscopy assay. Further work revealed that the PKA and PKC pathways may be involved in the inhibitory effects of BPA since the PKA antagonist GÖ-6983 and the PKC antagonist H-89 significantly alleviated the inhibitory effects of BPA on I _Ca. As such, the results of the present study provide direct evidence that BPA decreases I _Ca and impairs calcium homeostasis, which may be involved in any toxic effects of BPA on DRG neurons.