Seong-Woo Jeong

G Protein α Subunit Gαz Couples Neurotransmitter Receptors to Ion Channels in Sympathetic Neurons

Abstract The functional roles subserved by Gα z , a G protein α subunit found predominantly in neuronal tissues, have remained largely undefined. Here, we report that Gα z coupled neurotransmitter receptors to N-type Ca 2+ channels when transiently overexpressed in rat sympathetic neurons. The Gα z -mediated inhibition was voltage dependent and PTX insensitive. Recovery from Gα z -mediated inhibition was extremely slow but accelerated by coexpression with RGS proteins. Gα z selectively interacted with a subset of receptors that ordinarily couple to N-type Ca 2+ channels via PTX-sensitive G o/i proteins. In addition, Gα z rescued the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that Gα z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions.

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels.

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Cav3 channels: Cav3.1, Cav3.2, and Cav3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 μm) of nickel were found to selectively block the Cav3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Cav3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Cav3.2 and the nickel-insensitive Cav3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Cav3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3–S4 loop of domain I significantly attenuated the nickel block of Cav3.2, mimicking the nickel-insensitive blocking potency of Cav3.1. These findings indicate that His-191 in the S3–S4 loop is a critical residue conferring nickel block to Cav3.2 and reveal a novel role for the S3–S4 loop to control ion permeation through T-type Ca2+ channels.

Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density.

T‐type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol‐12‐myristate‐13‐acetate (PMA) potently enhanced the current amplitude of Cav3.2 T‐type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T‐type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4α‐PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T‐type channel currents via activation of oocyte PKC. The stimulation effect on Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T‐type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II–III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1.

Differential regulation of G protein‐gated inwardly rectifying K+ channel kinetics by distinct domains of RGS8

1 The contribution of endogenous regulators of G protein signalling (RGS) proteins to G protein modulated inwardly rectifying K+ channel (GIRK) activation/deactivation was examined by expressing mutants of GαoA insensitive to both pertussis toxin (PTX) and RGS proteins in rat sympathetic neurons. 2 GIRK channel modulation was reconstituted in PTX‐treated rat sympathetic neurons following heterologous expression of G protein subunits. Under these conditions, noradrenaline‐evoked GIRK channel currents displayed: (1) a prominent lag phase preceding activation, (2) retarded activation and deactivation kinetics, and (3) a lack of acute desensitization. 3 Unexpectedly, heterologous expression of RGS8 in neurons expressing PTX‐i‐RGS‐insensitive GαoA shortened the lag phase and restored rapid activation, but retarded the deactivation phase further. These effects were found to arise from the N‐terminus, but not the core domain, of RGS8 thus suggesting actions on channel modulation independently of GTPase acceleration. 4 These findings indicate that different domains of RGS8 make distinct contributions to the temporal regulation of GIRK channels. The RGS8 core domain accelerates termination of the G‐protein cycle presumably by increasing Gα GTPase activity. In contrast, the N‐terminal domain of RGS8 appears to promote entry into the G protein cycle, possibly by enhancing coupling of receptors to the G protein heterotrimer. Together, these opposing effects should allow for an increase in temporal fidelity without a dramatic decrease in signal strength.

Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A.

Ca2+ influx through T-type Ca2+ channels is crucial for important physiological activities such as hormone secretion and neuronal excitability. However, it is not clear whether these channels are regulated by cAMP-dependent protein kinase A (PKA). In the present study, we examined whether PKA modulates Cav3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 μM forskolin, an adenylyl cyclase stimulant, increased Cav3.2 channel activity by 40 ± 4% over 30 min and negatively shifted the steady-state inactivation curve (V50 = -61.4 ± 0.2 versus -65.5 ± 0.1 mV). Forskolin did not affect other biophysical properties of Cav3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 μM 8-bromo-cAMP, a membrane-permeable cAMP analog. The augmentation of Cav3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 μM N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Cav3.2 channel activity by PKA was mimicked by serotonin when 5HT7 receptor was coexpressed with Cav3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Cav3.2 with those of the Nav1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Cav3.2.

Modulation of Cav3.2 T-type Ca2+ channels by protein kinase C

Although T‐type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Cav3.2 T‐type channels reconstituted in Xenopus oocytes. Phorbol‐12‐myristate‐13‐acetate (PMA) strongly enhanced the amplitude of Cav3.2 channel currents (∼3‐fold). The augmentation effects were not mimicked by 4α‐PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Cav3.2 channel activity via activation of oocyte PKC.

Use of RGS-Insensitive Gα Subunits to Study Endogenous RGS Protein Action on G-Protein Modulation of N-Type Calcium Channels in Sympathetic Neurons

Regulators of G-protein signaling (RGS) proteins are a large family of signaling proteins that control both the magnitude and temporal characteristics of heterotrimeric G-protein-mediated signaling. A current challenge is to define how endogenous RGS protein function impacts G-protein modulation of ionic channels in mammalian neurons. The experimental strategy described here utilizes distinct mutations in Galpha subunits that confer Bordetella pertussis toxin (PTX) and RGS protein insensitivity. The native signaling pathway in rat sympathetic neurons that mediates voltage-dependent modulation of N-type Ca2+ channels is ablated by PTX treatment and the signaling is reconstituted by expressing a PTX/RGS-insensitive Galpha mutant along with Gbeta and Ggamma subunits. As neurons are resistant to conventional transfection modalities, heterologous expression is accomplished by the direct microinjection of plasmids into the nucleus of the neuron. An advantage of this approach is that knowledge of the specific RGS subtypes participating in the pathway is not required. From the resulting alterations in the kinetics and pharmacology of G-protein-coupled receptor modulation of N-type Ca2+ channels, we can infer the role endogenous RGS proteins play in the signaling pathway.

Neuregulin 1 up-regulates the expression of nicotinic acetylcholine receptors through the ErbB2/ErbB3-PI3K-MAPK signaling cascade in adult autonomic ganglion neurons

We investigated effects of Neuregulin 1 (NRG1) on the expression of nicotinic acetylcholine receptor (nAChR) in major pelvic ganglion (MPG) from adult rat. MPG neurons were found to express transcripts for type I and III NRG1s as well as α and β‐type epidermal growth factor (EGF)‐like domains. Of the four ErbB receptor isoforms, ErbB1, ErbB2, and ErbB3 were expressed in MPG neurons. Treating MPG with NRG1β significantly increased the transcript and protein level of the nAChR α3 and β4 subunits. Consistent with these molecular data, nicotinic currents (IACh) were significantly up‐regulated in NRG1β‐treated sympathetic and parasympathetic MPG neurons. In contrast, the type III NRG1 and the α form of the NRG1 failed to alter the IACh. Inhibition of the ErbB2 tyrosine kinase completely abolished the effects of NRG1β on the IACh. Stimulation of the ErbB receptors by NRG1β activated the phosphatidylinositol‐3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK). Immunoblot analysis revealed that PI3K‐mediated activation of Akt preceded Erk1/2 activation in NRG1β‐treated MPG neurons. Furthermore, specific PI3K inhibitors abrogated the phosphorylation of Erk1/2, while inhibition of MEK did not prevent the phosphorylation of Akt. Taken together, these findings suggest that NRG1 up‐regulates nAChR expression via the ErbB2/ErbB3‐PI3K‐MAPK signaling cascade and may be involved in maintaining the ACh‐mediated synaptic transmission in adult autonomic ganglia.

Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system.

Among the autonomic ganglia, major pelvic ganglia (MPG) innervating the urogenital system are unique because both sympathetic and parasympathetic neurons are colocalized within one ganglion capsule. Sympathetic MPG neurons are discriminated from parasympathetic ones by expression of low voltage-activated Ca2+ channels that primarily arise from T-type α1H isoform and contribute to the generation of low-threshold spikes. Until now, however, expression profiles of high voltage-activated (HVA) Ca2+ channels in these two populations of MPG neurons remain unknown. Thus, in the present study, we dissected out HVA Ca2+ channels using pharmacological and molecular biological tools. Reverse transcription-polymerase chain reaction analysis showed that MPG neurons contained transcripts encoding all of the known HVA Ca2+ channel isoforms (α1B, α1C, α1D and α1E), with the exception of α1A. Western blot analysis and pharmacology with ω-agatoxin IVA (1 μM) confirmed that MPG neurons lack the α1A Ca2+ channels. Unexpectedly, the expression profile of HVA Ca2+ channel isoforms was identical in the sympathetic and parasympathetic neurons of the MPG. Of the total Ca2+ currents, ω-conotoxin GVIA-sensitive N-type (α1B) currents constituted 57 ± 5% (n = 9) and 60 ± 3% (n = 6), respectively; nimodipine-sensitive L-type (α1C and α1D) currents made up 17 ± 4% and 14 ± 2%, respectively; and nimodipine-resistant and ω-conotoxin GVIA-resistant R-type currents were 25 ± 3% and 22 ± 2%, respectively. The R-type Ca2+ currents were sensitive to NiCl2 (IC50 = 22 ± 0.1 μM) but not to SNX-482, which was able to potently (IC50 = 76 ± 0.4 nM) block the recombinant α1E/β2a/α2δ Ca2+ currents expressed in human embryonic kidney 293 cells. Taken together, our data suggest that sympathetic and parasympathetic MPG neurons share a similar but unique profile of HVA Ca2+ channel isoforms.

Nerve injury alters profile of receptor-mediated Ca2+ channel modulation in vagal afferent neurons of rat nodose ganglia.

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague-Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of alpha2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of micro-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury.