Chih-Yung Tang

Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by δ subunit-containing GABAA receptors

Neuroactive steroids are potent modulators of γ-aminobutyric acid type A receptors (GABAARs), and their behavioral effects are generally viewed in terms of altered inhibitory synaptic transmission. Here we report that, at concentrations known to occur in vivo, neuroactive steroids specifically enhance a tonic inhibitory conductance in central neurons that is mediated by extrasynaptic δ subunit-containing GABAARs. The neurosteroid-induced augmentation of this tonic conductance decreases neuronal excitability. Fluctuations in the circulating concentrations of endogenous neuroactive steroids have been implicated in the genesis of premenstrual syndrome, postpartum depression, and other anxiety disorders. Recognition that δ subunit-containing GABAARs responsible for a tonic conductance are a preferential target for neuroactive steroids may lead to novel pharmacological approaches for the treatment of these common conditions.

Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels.

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder associated with mutations in the gene encoding pore‐forming α1A subunits of human P/Q‐type calcium (CaV2.1) channels. The exact mechanism of how mutant channels cause such clinical EA2 features as cerebellar dysfunctions, however, remains unclear. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert prominent dominant‐negative effects on wild‐type CaV2.1 channels. To further pursue the mechanism underlying this dominant‐negative effect, we examined the effects of EA2 mutants on the subcellular localization pattern of GFP‐tagged wild‐type CaV2.1 channels in HEK293T cells. In the presence of EA2 mutants, wild‐type channels displayed a significant deficiency in membrane targeting and a concurrent increase in cytoplasm retention. Moreover, the cytoplasmic fraction of wild‐type channels co‐localized with an endoplasmic reticulum (ER) marker, suggesting that a significant amount of wild‐type CaV2.1 channels was trapped in the ER. This EA2 mutant‐induced ER retention pattern was reversed by lowering the cell incubation temperature from 37 to 27°C. We also inspected the effects of untagged EA2 mutants on the functional expression of GFP‐tagged wild‐type CaV2.1 channels in HEK293T cells. Whole‐cell current density of wild‐type channels was diminished in the presence of EA2 mutants, which was also reversed by 27°C incubation. Finally, biochemical analyses indicated that EA2 mutants did not significantly affect the protein expression level of wild‐type channels. Taken together, our data suggest that EA2 mutants induce significant ER retention of their wild‐type counterparts, thereby suppressing the functional expression of CaV2.1 channels. J. Cell. Physiol. 214: 422–433, 2008.

CD4+ T Cell-Derived IL-2 Signals during Early Priming Advances Primary CD8+ T Cell Responses

Stimulating naïve CD8+ T cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. In this study, we show that the activation and differentiation of CD8+ T cells require IL-2 provided by activated CD4+ T cells at the initial priming stage within 0–2.5 hours after stimulation. This critical IL-2 signal from CD4+ cells is mediated through the IL-2Rβγ of CD8+ cells, which is independent of IL-2Rα. The activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated CD8+ T-cell into the S phase. Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8+ T cells. Furthermore, IL-2 at priming enhances the ability of P14 effector cells generated by antigen activation to eradicate B16.gp33 tumors in vivo. Therefore, our studies demonstrate that a full CD8+ T-cell response is elicited by a critical temporal function of IL-2 released from CD4+ T cells, providing mechanistic insights into the regulation of CD8+ T cell activation and differentiation.

Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons.

Two isoforms of rat ether-à-go-go (Eag) K+ channels, rEag1 and rEag2, are widely expressed in many regions of the brain. The neurophysiological roles of these channels, however, are unclear. We addressed this issue by studying their subcellular localizations in hippocampal neurons. Immunofluorescence studies using markers for different compartments of neurons demonstrated a differential expression pattern of rEag1 and rEag2 K+ channels in the somatodendritic region. Furthermore, rEag1 K+ channels were in close proximity to synaptophysin and densin-180, but not GAD65. Our data suggest that both rEag1 and rEag2 K+ channels may play a pivotal role in the regulation of the excitability of dendrites and somas, and that rEag1 K+ channels may modulate the postsynaptic signaling of glutamatergic synapses.

Shaker and Ether-à-Go-Go K+ Channel Subunits Fail to Coassemble in Xenopus Oocytes

Members of different voltage-gated K+ channel subfamilies usually do not form heteromultimers. However, coassembly between Shaker and ether-à-go-go (eag) subunits, members of two distinct K+ channel subfamilies, was suggested by genetic and functional studies (Zhong and Wu. 1991. Science. 252: 1562-1564; Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We investigated whether Shaker and eag form heteromultimers in Xenopus laevis oocytes using electrophysiological and biochemical approaches. Coexpression of Shaker and eag subunits produced K+ currents that were virtually identical to the sum of separate Shaker and eag currents, with no change in the kinetics of Shaker inactivation. According to the results of dominant negative and reciprocal coimmunoprecipitation experiments, the Shaker and eag proteins do not interact. We conclude that Shaker and eag do not coassemble to form heteromultimers in Xenopus oocytes.

Physiology and pathophysiology of CLC-1: mechanisms of a chloride channel disease, myotonia.

The CLC-1 chloride channel, a member of the CLC-channel/transporter family, plays important roles for the physiological functions of skeletal muscles. The opening of this chloride channel is voltage dependent and is also regulated by protons and chloride ions. Mutations of the gene encoding CLC-1 result in a genetic disease, myotonia congenita, which can be inherited as an autosmal dominant (Thomsen type) or an autosomal recessive (Becker type) pattern. These mutations are scattered throughout the entire protein sequence, and no clear relationship exists between the inheritance pattern of the mutation and the location of the mutation in the channel protein. The inheritance pattern of some but not all myotonia mutants can be explained by a working hypothesis that these mutations may exert a “dominant negative” effect on the gating function of the channel. However, other mutations may be due to different pathophysiological mechanisms, such as the defect of protein trafficking to membranes. Thus, the underlying mechanisms of myotonia are likely to be quite diverse, and elucidating the pathophysiology of myotonia mutations will require the understanding of multiple molecular/cellular mechanisms of CLC-1 channels in skeletal muscles, including molecular operation, protein synthesis, and membrane trafficking mechanisms.

Membrane targeting and coupling of NHE1-integrin α llbβ-NCX1 by lipid rafts following integrin-ligand interactions trigger Ca2+ oscillations

The cyclic calcium release and uptake during calcium oscillation are thought to result from calcium-induced calcium release (CICR); however, it is unclear, especially in nonexcitable cells, how the initial calcium mobilization that triggers CICR occurs. We report here a novel mechanism, other than conventional calcium channels or the phopholipase C-inositol trisphosphate system, for initiating calcium oscillation downstream of integrin signaling. Upon integrin αIIbβ3 binding to fibrinogen ligand or the disintegrin rhodostomin, sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 are actively transported to the plasma membrane, and they become physically coupled to integrin αIIbβ3. Lipid raft-dependent mechanisms modulate the membrane targeting and formation of the NHE1-integrin αIIbβ3-NCX1 protein complex. NHE1 and NCX1 within such protein complex are functionally coupled, such that a local increase of sodium concentration caused by NHE1 can drive NCX1 to generate sodium efflux in exchange for calcium influx. The resulting calcium increase inside the cell can then trigger CICR as a prelude to calcium oscillation downstream of integrin αIIbβ3 signaling. Fluorescence resonance energy transfer based on fluorescence lifetime measurements is employed here to monitor the intermolecular interactions among NHE1-integrin αIIbβ3-NCX1, which could not be properly detected using conventional biochemical assays.

The cullin 4A/B-DDB1-cereblon E3 ubiquitin ligase complex mediates the degradation of CLC-1 chloride channels

Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

Depressor effect on blood pressure and flow elicited by electroacupuncture in normal subjects

To clarify the effect of electroacupuncture (Ea) on the activity of the cardiovascular system in normal individuals, hemodynamic parameters including arterial blood pressure (BP), finger blood flow (FBF) and heart rate (HR) as well as paravertebral temperature (PVT) were non-invasively recorded under Ea stimulation. Surface stimulation electrode was placed on the Hoku point (Li-4). Square wave pulses (0.05 ms) were applied from a stimulator with a stimulation frequency of 2 Hz (3 min). The stimulation intensity was five times of sensory threshold. BP and FBF were decreased (68.5+/-6.0%, P<0.01 and 96.8+/-1.1%, P<0.01 of control, respectively, n=7) while HR and PVT were increased significantly (115.0+/-5.1 of control, P<0.05 and 0.054+/-0.004 degree C, P<0.01, respectively, n=7) during Ea treatment. The results suggested an inhibition in sympathetic outflow, which induced vasodilatation of systemic arteriole and decrease in BP and FBF were elicited by Ea stimulation.

Myotonia Congenita Mutation Enhances the Degradation of Human CLC-1 Chloride Channels

Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl−) channel CLC-1. Myotonia congenita can be inherited in an autosomal recessive (Becker type) or dominant (Thomsen type) fashion. One hypothesis for myotonia congenita is that the inheritance pattern of the disease is determined by the functional consequence of the mutation on the gating of CLC-1 channels. Several disease-related mutations, however, have been shown to yield functional CLC-1 channels with no detectable gating defects. In this study, we have functionally and biochemically characterized a myotonia mutant: A531V. Despite a gating property similar to that of wild-type (WT) channels, the mutant CLC-1 channel displayed a diminished whole-cell current density and a reduction in the total protein expression level. Our biochemical analyses further demonstrated that the reduced expression of A531V can be largely attributed to an enhanced proteasomal degradation as well as a defect in protein trafficking to surface membranes. Moreover, the A531V mutant protein also appeared to be associated with excessive endosomal-lysosomal degradation. Neither the reduced protein expression nor the diminished current density was rescued by incubating A531V-expressing cells at 27°C. These results demonstrate that the molecular pathophysiology of A531V does not involve anomalous channel gating, but rather a disruption of the balance between the synthesis and degradation of the CLC-1 channel protein.