Expression Of Voltage-gated Ion Channels Research Articles

Properties and Fate of Oligodendrocyte Progenitor Cells in the Corpus Callosum, Motor Cortex, and Piriform Cortex of the Mouse

Oligodendrocyte progenitor cells (OPCs) in the postnatal mouse corpus callosum (CC) and motor cortex (Ctx) reportedly generate only oligodendrocytes (OLs), whereas those in the piriform cortex may also generate neurons. OPCs have also been subdivided based on their expression of voltage-gated ion channels, ability to respond to neuronal activity, and proliferative state. To determine whether OPCs in the piriform cortex have inherently different physiological properties from those in the CC and Ctx, we studied acute brain slices from postnatal transgenic mice in which GFP expression identifies OL lineage cells. We whole-cell patch clamped GFP-expressing (GFP(+)) cells within the CC, Ctx, and anterior piriform cortex (aPC) and used prelabeling with 5-ethynyl-2'-deoxyuridine (EdU) to assess cell proliferation. After recording, slices were immunolabeled and OPCs were defined by strong expression of NG2. NG2(+) OPCs in the white and gray matter proliferated and coexpressed PDGFRα and voltage-gated Na(+) channels (I(Na)). Approximately 70% of OPCs were capable of generating regenerative depolarizations. In addition to OLIG2(+) NG2(+) I(Na)(+) OPCs and OLIG2(+) NG2(neg) I(Na)(neg) OLs, we identified cells with low levels of NG2 limited to the soma or the base of some processes. These cells had a significantly reduced I(Na) and a reduced ability to incorporate EdU when compared with OPCs and probably correspond to early differentiating OLs. By combining EdU labeling and lineage tracing using Pdgfrα-CreER(T2) : R26R-YFP transgenic mice, we double labeled OPCs and traced their fate in the postnatal brain. These OPCs generated OLs but did not generate neurons in the aPC or elsewhere at any time that we examined.

Bidirectional Regulation of Dendritic Voltage-Gated Potassium Channels by the Fragile X Mental Retardation Protein

How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.

Muscle unloading potentiates the effects of acetyl‐L‐carnitine on the slow oxidative muscle phenotype

The effect of acetyl-L-carnitine (ALCAR) supplementation to 3-month-old rats in normal-loading and unloading conditions has been here investigated by a combined morphological, biochemical and transcriptional approach to test whether ALCAR might cause a remodeling of the metabolic/contractile phenotype of soleus muscle. Morphological assessment demonstrated an increase of type I oxidative fiber content and cross-sectional area in ALCAR-treated animals both in normal-loading and in unloading conditions. ALCAR prevented loss of mitochondrial mass in unloaded animals whereas no ALCAR-dependent increase of mitochondrial mass occurred in normal-loaded muscle. Validated microarray analysis delineated an ALCAR-induced maintenance of a slow-oxidative expression program only in unloaded soleus muscle. Indeed, the muscle adjustment of the expression profile of factors underlying mitochondrial oxidative metabolism, protein turnover, fiber type differentiation and an adaptation of voltage-gated ion channel expression was distinguishable with respect to the loading status. This selectivity may suggest a key role of muscle loading status in the manifestation of ALCAR effects. The results extend to a broader level of biological informations the previous notion on ALCAR positive effect in rat soleus muscle during unloading and point to a role of ALCAR for the maintenance of its slow-oxidative fiber character.

Regulation and modulation of electric waveforms in gymnotiform electric fish.

Weakly electric gymnotiform fish specialize in the regulation and modulation of the action potentials that make up their multi-purpose electric signals. To produce communication signals, gymnotiform fish modulate the waveforms of their electric organ discharges (EODs) over timescales spanning ten orders of magnitude within the animal's life cycle: developmental, reproductive, circadian, and behavioral. Rapid changes lasting milliseconds to seconds are the result of direct neural control of action potential firing in the electric organ. Intermediate-term changes taking minutes to hours result from the action of melanocortin peptides, the pituitary hormones that induce skin darkening and cortisol release in many vertebrates. Long-term changes in the EOD waveform taking days to weeks result from the action of sex steroids on the electrocytes in the electric organ as well as changes in the neural control structures in the brain. These long-term changes in the electric organ seem to be associated with changes in the expression of voltage-gated ion channels in two gene families. Electric organs express multiple voltage-gated sodium channel genes, at least one of which seems to be regulated by androgens. Electric organs also express multiple subunits of the shaker (Kv1) family of voltage-gated potassium channels. Expression of the Kv1 subtype has been found to vary with the duration of the waveform in the electric signal. Our increasing understanding of the mechanisms underlying precise control of electric communication signals may yield significant insights into the diversity of natural mechanisms available for modifying the performance of ion channels in excitable membranes. These mechanisms may lead to better understanding of normal function in a wide range of physiological systems and future application in treatment of disease states involving pathology of excitable membranes.

Expressional reprogramming of survival pathways in rat cardiocytes by neuregulin-1β

Neuregulin/ErbB2-induced kinase signaling provides essential survival and protection clues for functional integrity of the adult heart and skeletal muscle. To define the regulatory pathways involved in neuregulin-dependent muscle cell survival, we set out to map the largely unknown transcript targets of this growth/differentiation factor in cardiocytes. Freshly isolated adult primary rat cardiocytes were treated for 24 h with recombinant human neuregulin-1beta (NRG-1beta, 30 ng/ml). Transcript level alterations in NRG-1beta-treated and control cardiocytes (n = 6) were identified with Atlas Rat Toxicology 1.2 cDNA arrays (BD Clontech) and established permutation L1 regression analysis. Selected transcriptional adjustments were confirmed by RT-PCR and Western blotting. Involvement of MAPK pathways was verified with the inhibitor PD-98059. Application of the single dose of NRG-1beta to quiescent cardiocytes induced expressional reprogramming of distinct cellular processes. This response included a prominent 50-100% increase in transcripts of multiple redox systems. It also involved a comparable mRNA augmentation of protein synthetic and folding factors together with augmented message for the trigger of cardiac hypertrophy, cyclin D1 (CCND1). First evidence for a role of neuregulin in promotion of mitochondrial turnover, voltage-gated ion channel expression, and the suppression of fatty acid transporter mRNAs was revealed. Subsequent analysis confirmed a corresponding upregulation of redox factor proteins thioredoxin and the thioredoxin reductase 1, GSTP-1, and CCND1 and demonstrated downregulation of the related transcripts by PD-98059 in neuregulin-stimulated cultures. These MAPK-dependent expressional adjustments point to novel oxidative defense and hypertrophy pathways being involved in the longer lasting protective function of neuregulin in the heart.

Functional expression of a GFP-tagged Kv1.5 alpha-subunit in mouse ventricle.

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+ (Kv) channel alpha-subunit, Kv1.5-GFP, driven by the cardiac-specific alpha-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current (I(K,slow)) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I(K,slow) in wild-type cells. The inactivation rate of the "overexpressed" current, however, is significantly slower than the Kv1.5-encoded component of I(K,slow) in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.

Orchestration of neuronal migration by activity of ion channels, neurotransmitter receptors, and intracellular Ca2+ fluctuations

The real-time observation of cell movement in acute cerebellar slices reveals that granule cells alter their shape concomitantly with changes in the mode and rate of migration as they traverse different cortical layers. Although the origin of local environmental cues responsible for these position-specific changes in migratory behavior remains unclear, several signaling mechanisms involved in controlling granule cell movement have emerged. The onset of one such mechanism is marked by the expression of voltage-gated ion channels and neurotransmitter receptors in postmitotic cells prior to the initiation of their migration. Granule cells start their radial migration after the expression of N-type Ca2+ channels and the N-methyl-D-aspartate subtype of glutamate receptors on the plasmalemmal surface. Blockade of the channel or receptor activity significantly decreases the rate of cell movement, indicating that the activation of these membrane constituents provides an essential signal for the translocation of granule cells. Another signal that controls the rate of cell migration is embedded in the combined amplitude and frequency components of Ca2+ fluctuations in the somata of migrating granule cells. Interestingly, each phase of Ca2+ fluctuation controls a separate phase of saltatory movement in the granule cells: The cells move forward during the phase of transient Ca2+ elevation and remain stationary during the troughs. Consequently, the changes in the amplitude and frequency components of Ca2+ fluctuations directly affect granule cell movement: Reducing the amplitude or frequency of Ca2+ fluctuations slows down the speed of cell movement, while the enhancement of these components accelerates migration. These findings suggest that signaling molecules present in the local cellular milieu encountered on the migratory route control the shape and motility of granule cells by modifying Ca2+ fluctuations in the soma through the activation of specific ion channels and neurotransmitter receptors.

Regulation of voltage-gated ion channels by NGF and ciliary neurotrophic factor in SK-N-SH neuroblastoma cells

Neurotrophic factors have powerful effects on neuronal differentiation and the maintenance of neuronal phenotype, but understanding of their regulation of one important aspect of neuronal function, excitability, remains limited. We have examined the regulation of voltage-gated ion channels by two unrelated neurotrophic factors, NGF and ciliary neurotrophic factor (CNTF), in the SK-N-SH neuroblastoma cell line that is responsive to both factors. NGF and CNTF have strikingly different neuronal specificities and distributions in the nervous system, and might be expected to have significantly different effects on neuronal function. Using whole-cell, perforated-patch, and single-channel recording, we found that treatment with NGF increased levels of voltage-gated sodium, calcium, and potassium currents. In contrast, CNTF treatment increased levels of potassium currents only. NGF and CNTF appeared to regulate the same delayed-rectifier potassium current; in addition, NGF treatment resulted in increased levels of a second potassium current component. Such differential effects of neurotrophic factors on the expression of voltage-gated ion channels would have profound effects on the excitability of target neurons in vivo.

Na+ channel mis‐expression accelerates K+ channel development in embryonic Xenopus laevis skeletal muscle.

1. The normal developmental pattern of voltage-gated ion channel expression in embryonic skeletal muscle cells of the frog Xenopus laevis was disrupted by introduction of cloned rat brain Na+ channels. 2. Following injection of channel mRNA into fertilized eggs, large Na+ currents were observed in muscle cells at the earliest developmental stage at which they could be uniquely identified. Muscle cells normally have no voltage-gated currents at this stage. 3. Muscle cells expressing exogenous Na+ channels showed increased expression of at least two classes of endogenous K+ currents. 4. This increase in K+ current expression was inhibited by the Na+ channel blocker tetrodotoxin, suggesting that increased electrical activity caused by Na+ channel mis-expression triggers a compensatory increase in K+ channel expression. 5. Block of endogenous Na+ channels in later control myocytes retards K+ current development, indicating that a similar compensatory mechanism to that triggered by Na+ channel mis-expression operates to balance Na+ and K+ current densities during normal muscle development.

27] Vaccinia virus as vector to express ion channel genes

This chapter describes the use of vaccinia virus (VV)—a lytic DNA virus—to achieve the transient expression of voltage-gated ion channels in various primary cell cultures and cell lines. Recombinant VV vectors present several advantages for eukaryotic expression technology, such as VV particles have a complex morphology with an outer envelope surrounding the biconcave core and two lateral bodies of unknown function and the genome consists of a linear, double- stranded DNA molecule of 187 kilobases (kb), packaged within the virus core. Another advantage is that VV is able to infect 100% of a eukaryotic cell population. The principal advantage of recombinant VV over other transient expression systems is the broad range of mammalian cell types that can be infected at high success rates. Thus, VV may prove extremely useful in the expression of ion channel and receptor/ionophore proteins that require cell-specific posttranslational processing, or when cell-specific subunits of these excitability proteins are required for complete functional reconstitution.