Heterologous expression of a glial Kir channel (KCNJ10) in a neuroblastoma spinal cord (NSC-34) cell line.

Heterologous expression of Kir channels offers a tool to modulate excitability of neurons which provide insight into Kir channel functions in general. Inwardly-rectifying K+ channels (Kir channels) are potential candidate proteins to hyperpolarize neuronal cell membranes. However, heterologous expression of inwardly-rectifying K+ channels has previously proven to be difficult. This was mainly due to a high toxicity of the respective Kir channel expression. We investigated the putative role of a predominantly glial-expressed, weakly rectifying Kir channel (Kir4.1 channel subunit; KCNJ10) in modulating electrophysiological properties of a motoneuron-like cell culture (NSC-34). Transfection procedures using an EGFP-tagged Kir4.1 protein in this study proved to have no toxic effects on NSC-34 cells. Using whole cell-voltage clamp, a substantial increase of inward rectifying K+ currents as well as hyperpolarization of the cell membrane was observed in Kir4.1-transfected cells. Na+ inward currents, observed in NSC-34 controls, were absent in Kir4.1/EGFP motoneuronal cells. The Kir4.1-transfection did not influence the NaV1.6 sodium channel expression. This study demonstrates the general feasibility of a heterologous expression of a weakly inward-rectifying K+ channel (Kir4.1 subunit) and shows that in vitro overexpression of Kir4.1 shifts electrophysiological properties of neuronal cells to a more glial-like phenotype and may therefore be a candidate tool to dampen excitability of neurons in experimental paradigms.

Severe demyelinating hypertrophic polyneuropathy caused by a de novo frameshift mutation within the intracellular domain of myelin protein zero (MPZ/P0).

Hereditary motor and sensory neuropathy (HMSN), also known as Charcot-Marie-Tooth disease (CMT) is a group of clinically and genetically heterogeneous neuropathies classically divided into demyelinating (CMT1) and axonal forms (CMT2). The most common demyelinating form is CMT1A with an underlying duplication in the gene coding for the peripheral myelin protein 22 (PMP22). Less frequently, mutations in the myelin protein zero gene (MPZ/P(0)) account for demyelinating CMT1B, Dejerine-Sottas syndrome (DSS), or congenital hypomyelinating neuropathy (CHN). Here, we report a patient with a severe, early-onset hypertrophic and dysmyelinating neuropathy. The patient exhibits a novel frameshift mutation with an insertion of a single T-nucleotide on position c.618_619 of the MPZ gene resulting in a premature stop M207fsX38.

[Ventilator-induced diaphragmatic dysfunction (VIDD)]. / Ventilator induzierter Zwerchfellschaden.

Although life-saving, mechanical ventilation is associated with numerous complications. Recently, it was shown in animal models that controlled mechanical ventilation (CMV) can cause the so-called ventilator-induced diaphragmatic dysfunction (VIDD). The decrease in diaphragmatic strength proceeds along with prolonged mechanical ventilation. The decrease in diaphragmatic force occurs during CMV rather than during assisted modes of ventilation. Different mechanisms may cause VIDD: muscle atrophy, oxidative stress, structural injury and muscle fibre remodelling have been demonstrated in animal models. Although most critically ill patients receiving mechanical ventilation show profound diaphragmatic weakness, clear evidence supporting the existence of VIDD is still lacking. Since VIDD is particularly caused by comorbidity, a series of other factors may lead to diaphragmatic dysfunction. Whether or not VIDD causes weaning failure is difficult to decide since other reasons may also cause respiratory muscle weakness. However, based on pathophysiology, treatment options may be assisted mechanical ventilation, administration of antioxidative agents or stimulation of the phrenic nerve.

Mitofusin 2 gene mutation (R94Q) causing severe early-onset axonal polyneuropathy (CMT2A).

Charcot-Marie-Tooth disease (CMT) has been classified into two types: demyelinating forms (CMT1) and axonal forms (CMT2). Mutations in the CMT2A locus have been linked to the KIF1B and the mitofusin 2 (MFN2) genes. Here, we report a German patient with CMT2 with an underlying spontaneous mutation (c.281G-->A) in the MFN2 gene. Clinically, the patient presented with early-onset CMT that was not associated with additional central nervous system pathology. The disease course was rapidly progressive in the first years and slowed afterwards. We also suggest that single patients with early-onset axonal polyneuropathies should be screened for MFN2 mutations.

Cyclin-dependent kinase 5 (CDK5) and neuronal cell death.

Many neurological disorders like Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis (ALS) or stroke have in common a definite loss of CNS neurons due to apoptotic or necrotic neuronal cell death. Previous studies suggested that proapoptotic stimuli may trigger an abortive and, therefore, eventually fatal cell cycle reentry in postmitotic neurons. Neuroprotective effects of small molecule inhibitors of cyclin-dependent kinases (CDKs), which are key regulators of cell cycle progression, support the cell cycle theory of neuronal apoptosis. However, growing evidence suggests that deregulated CDK5, which is not involved in cell cycle control, rather than cell cycle relevant members of the CDK family, promotes neuronal cell death. Here we summarize the current knowledge about the involvement of CDK5 in neuronal cell death and discuss possible up- or downstream partners of CDK5. Moreover, we discuss potential therapeutic options that might arise from the identification of CDK5 as an important upstream element of neuronal cell death cascades.

Kir channels in the CNS: emerging new roles and implications for neurological diseases.

Inwardly rectifying potassium (Kir) channels have long been regarded as transmembrane proteins that regulate the membrane potential of neurons and that are responsible for [K(+)] siphoning in glial cells. The subunit diversity within the Kir channel family is growing rapidly and this is reflected in the multitude of roles that Kir channels play in the central nervous system (CNS). Kir channels are known to control cell differentiation, modify CNS hormone secretion, modulate neurotransmitter release in the nigrostriatal system, may act as hypoxia-sensors and regulate cerebral artery dilatation. The increasing availability of genetic mouse models that express inactive Kir channel subunits has opened new insights into their role in developing and adult mammalian tissues and during the course of CNS disorders. New aspects with respect to the role of Kir channels during CNS cell differentiation and neurogenesis are also emerging. Dysfunction of Kir channels in animal models can lead to severe phenotypes ranging from early postnatal death to an increased susceptibility to develop epileptic seizures. In this review, we summarize the in vivo data that demonstrate the role of Kir channels in regulating morphogenetic events, such as the proliferation, differentiation and survival of neurons and glial cells. We describe the way in which the gating of Kir channel subunits plays an important role in polygenic CNS diseases, such as white matter disease, epilepsy and Parkinson's disease.

Kir4.1 potassium channel subunit is crucial for oligodendrocyte development and in vivo myelination.

To understand the cellular and in vivo functions of specific K(+) channels in glia, we have studied mice with a null mutation in the weakly inwardly rectifying K(+) channel subunit Kir4.1. Kir4.1-/- mice display marked motor impairment, and the cellular basis is hypomyelination in the spinal cord, accompanied by severe spongiform vacuolation, axonal swellings, and degeneration. Immunostaining in the spinal cord of wild-type mice up to postnatal day 18 reveals that Kir4.1 is expressed in myelin-synthesizing oligodendrocytes, but probably not in neurons or glial fibrillary acidic protein-positive (GFAP-positive) astrocytes. Cultured oligodendrocytes from developing spinal cord of Kir4.1-/- mice lack most of the wild-type K(+) conductance, have depolarized membrane potentials, and display immature morphology. By contrast, cultured neurons from spinal cord of Kir4.1-/- mice have normal physiological characteristics. We conclude that Kir4.1 forms the major K(+) conductance of oligodendrocytes and is therefore crucial for myelination. The Kir4.1 knock-out mouse is one of the few CNS dysmyelinating or demyelinating phenotypes that does not involve a gene directly involved in the structure, synthesis, degradation, or immune response to myelin. Therefore, this mouse shows how an ion channel mutation could contribute to the polygenic demyelinating diseases.

The dopamine D2 receptor agonist alpha-dihydroergocryptine modulates voltage-gated sodium channels in the rat caudate-putamen.

Alpha-Dihydroergocryptine (alpha-DHEC), a Dopamine (DA) D2 receptor agonist, is widely used as dopaminergic drug in the treatment of Parkinson's disease. To study the mechanisms involved in the signal transduction process induced by alpha-DHEC on the presynaptic site of the dopaminergic neuron, we incubated slices of the rat caudate-putamen with alpha-DHEC and the indicated substances in static chambers. Following incubation the resulting DA outflow was measured by high-performance-liquid chromatography with electrochemical detection. The addition of alpha-DHEC (10 microM-0.1mM) did not modulate basal DA outflow. Activation of voltage-gated sodium channels by veratridine (VER) from low to relatively high concentrations (1-10 microM) led to a concentration-dependent increase of DA outflow. Using concentrations as high as 10 microM a dramatic increase of DA levels (600% of baseline levels) was observed. The ability of VER to provoke DA release was sensitive to the addition of tetrodotoxin (TTX) and was completely blocked by 1 mM TTX. Coincubation of alpha-DHEC (10microM-0.1mM) and VER (10microM) reduced VER-stimulated DA outflow in a concentration-dependent manner. The time-concentration course of VER-induced DA outflow was not modulated by alpha-DHEC. As described in our earlier studies, the specific D2 receptor antagonist (-)sulpiride (SLP) concentration-dependently enhances extracellular DA levels. Addition of alpha-DHEC almost completely blocked SLP-induced DA-outflow. When slices were incubated with the non-selective DA receptor agonist haloperidol (HLP, 0.1 mM) the effect of alpha-DHEC on VER-induced DA outflow was partially but not completely abolished. These data strongly suggest that the effect of alpha-DHEC on the presynaptic site implies an activation of D2 receptors as well as an inhibitory action on voltage-gated sodium channels. Alpha-DHEC seems to modulate voltage-gated sodium channels in part independently from DA receptors since blockade of D2 receptors with saturating concentrations of haloperidol did not completely abolish its effect. Based on our data we have no evidence that voltage-gated potassium channels, N-type calcium channels or D1, D3-receptors are involved in the action of alpha-DHEC at the presynaptic site of the dopaminergic neuron. The results give one rationale for the proposed neuroprotective effect of alpha-DHEC.

Expression of GIRK (Kir3.1/Kir3.4) channels in mouse fibroblast cells with and without beta1 integrins.

G protein-activated K+ channel (GIRK) subunits possess a conserved extracellular integrin-binding motif (RGD) and bind directly to beta1 integrins. We expressed GIRK1/GIRK4 channels labeled with green fluorescent protein in fibroblast cell lines expressing or lacking beta1 integrins. Neither plasma membrane localization nor agonist-evoked GIRK currents were affected by the absence of beta1 integrins or by incubation with externally applied RGD-containing peptide. Mutation of the aspartate (D) of RGD impaired currents, GIRK glycosylation, and membrane localization, but the interaction with beta1 integrins remained intact. Thus, beta1 integrins are not essential for functional GIRK expression; and the GIRK-integrin interactions involve structural elements other than the RGD motif.

G proteins modulate D2 receptor-coupled K(ATP) channels in rat dopaminergic terminals.

The presynaptic dopamine (DA) D2 receptor-mediated regulation of ATP-sensitive potassium (K+ATP) channels was examined in slices of the rat caudate-putamen. When slices were incubated with the specific D2 receptor antagonist (-)-sulpiride (SLP), a concentration-dependent increase of extracellular DA release was observed. SLP-induced enhancement was completely antagonized by coincubation with the K+ATP channel opener diazoxide (DIA). Treatment of slices with the D2 receptor agonist quinpirole (QUI) almost completely inhibited DA outflow induced by the K+ATP channel blocker butanedione-monoxime (BDM). Coincubation of SLP and guanosine triphosphate (GTP) or its non-hydrolizable analogue guanylyl-5'-imidodiphosphate [Gpp(NH)p], significantly reduced the SLP-induced effect on DA levels. Furthermore, we observed that BDM-induced DA outflow was markedly inhibited by G protein activators suggesting an additional receptor-independent regulation of K+ATP channel gating. Our results suggest that PTX-sensitive G proteins are involved in the signal transduction between D2 receptors and K+ATP channels. Furthermore, K+ATP channels can be modulated in a receptor-independent mechanism by G protein activators.

Selegiline induces dopamine release through ATP-sensitive potassium channels in the rat caudate-putamen in vitro.

We used superfusion chambers to investigate the role of ATP-sensitive potassium (KATP) channels in dopamine (DA) release elicited by the monoamine oxidase inhibitor selegiline in the rat caudate-putamen in vitro. Selegiline (R[-]-deprenyl], but not the S[+] enantiomer, concentration-dependently induced increases in extracellular concentrations of DA, with a maximal increase to 185% in comparison to basal outflow at 0.1 mM selegiline. Since in our experimental conditions exclusive MAO inhibition does not lead to an enhancement of extracellular DA levels, the effect of selegiline on DA levels seems not to be related to MAO inhibition. Butanedione (0.1 mM), a specific KATP channel blocker, also significantly enhanced extracellular DA levels in the rat caudate-putamen to approx. 260%. Selegiline only led to an additional increase of DA outflow, when added to submaximal concentrations of butanedione or tolbutamide, implying that selegiline is acting on identical sites. When the KATP channel opener cromakalim was added to the incubation medium, basal as well as butanedione-enhanced DA levels markedly decreased to about 40% when compared to baseline values. Selegiline-activated DA release was also antagonized by cromakalim. The selegiline effect was neither modulated by preincubation with the uptake inhibitor nomifensine nor by the DA agonist quinpirole and antagonist sulpiride. In conclusion these results suggest that selegiline is able to modulate KATP channels in the caudate-putamen of the rat in vitro resulting in an enhancement of striatal DA release.