Owen T. Jones

Modulation of High‐Voltage–Activated Calcium Channels in Dentate Granule Cells by Topiramate

Purpose: In this study, we assessed the effects of topiramate (TPM) on high‐voltage‐activated calcium channel (HVACC) currents in vitro.

Absence of Cytochrome b-245 in Chronic Granulomatous Disease

The heme-containing protein cytochrome b-245 has been proposed as a primary component of the microbicidal oxidase system of phagocytes that normally generates superoxide-free radicals but when defective is associated with chronic granulomatous disease. We measured this cytochrome in granulocytes from 27 patients with chronic granulomatous disease and from 64 members of their families. It was undetectable in all 19 of the men in whom the defect appeared to be located on the X chromosome. Female relatives who were heterozygous carriers had reduced concentrations of the cytochrome and variable proportions of cells that were unable to generate superoxide; these two characteristics were closely related (r = 0.93 in the 16 mothers and 0.85 in all 24 carriers, P less than 0.001). In contrast, in all eight patients (seven women) with a probable autosomal recessive inheritance the cytochrome was present but nonfunctional. The properties tested, including midpoint potential, carbon monoxide binding, and organelle distribution, were normal, but the cytochrome did not undergo reduction on cellular stimulation. Thus, absence or malfunction of the cytochrome b-245 may be the causal molecular defect in chronic granulomatous disease, implicating it in the microbicidal oxidase system.

Internalization of the Kv1.4 Potassium Channel Is Suppressed by Clustering Interactions with PSD-95

The contribution of voltage-dependent ion channels to nerve function depends upon their cell-surface distributions. Nevertheless, the mechanisms underlying channel localization are poorly understood. Two phenomena appear particularly important: the clustering of channels by membrane-associated guanylate kinases (MAGUKs), such as PSD-95, and the regional stabilization of cell-surface proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined the effect of PSD-95 on the internalization rate of Kv1.4 K+ channels in transfected HEK293 cells using cell-surface biotinylation assays. When expressed alone Kv1.4 was internalized with a half-life of 87 min, but, in the presence of PSD-95, Kv1.4 internalization was completely suppressed. Immunochemistry and electrophysiology showed PSD-95 had little effect on total or cell-surface levels of Kv1.4 or on current amplitude, activation, or inactivation kinetics. Clustering was necessary and sufficient to suppress Kv1.4 internalization since C35S-PSD-95, a mutant reported to bind but not cluster Kv1.4, (confirmed by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4) restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t 1 2 = 16 min). These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role for PSD-95, and perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.

Abnormal axonal physiology is associated with altered expression and distribution of Kv1.1 and Kv1.2 K+ channels after chronic spinal cord injury

Dysfunction of surviving axons which traverse the site of spinal cord injury (SCI) has been linked to altered sensitivity to the K+ channel blocker 4‐aminopyridine (4‐AP) and appears to contribute to post‐traumatic neurological deficits although the underlying mechanisms remain unclear. In this study, sucrose gap electrophysiology in isolated dorsal column strips, Western blotting and confocal immunofluorescence microscopy were used to identify the K+ channels associated with axonal dysfunction after chronic (6–8 weeks postinjury) clip compresssion SCI of the thoracic cord at T7 in rats. The K+ channel blockers 4‐AP (200 μm, 1 mm and 10 mm) and α‐dendrotoxin (α‐DTX, 500 nm) resulted in a significant relative increase in the amplitude and area of compound action potentials (CAP) recorded from chronically injured dorsal column axons in comparison with control noninjured preparations. In contrast, TEA (10 mm) and CsCl (2 mm) had similar effects on injured and control spinal cord axons. Western blotting and quantitative immunofluorescence microscopy showed increased expression of Kv1.1 and Kv1.2 K+ channel proteins on spinal cord axons following injury. In addition, Kv1.1 and Kv1.2 showed a dispersed staining pattern along injured axons in contrast to a paired juxtaparanodal localization in uninjured spinal cord axons. Furthermore, labelled α‐DTX colocalized with Kv1.1 and Kv1.2 along axons. These findings suggest a novel mechanism of axonal dysfunction after SCI whereby an increased 4‐AP‐ and α‐DTX‐sensitive K+ conductance, mediated in part by increased Kv1.1 and Kv1.2 K+ channel expression, contributes to abnormal axonal physiology in surviving axons.

Cell Surface Targeting and Clustering Interactions between Heterologously Expressed PSD-95 and the Shal Voltage-gated Potassium Channel, Kv4.2

Kv4.2 is a voltage-gated potassium channel that is critical in controlling the excitability of myocytes and neurons. Processes that influence trafficking and surface distribution patterns of Kv4.2 will affect its ability to contribute to cellular functions. The scaffolding/clustering protein PSD-95 regulates trafficking and distribution of several receptors and Shaker family Kv channels. We therefore investigated whether the C-terminal valine-serine-alanine-leucine (VSAL) of Kv4.2 is a novel binding motif for PSD-95. By using co-immunoprecipitation assays, we determined that full-length Kv4.2 and PSD-95 interact when co-expressed in mammalian cell lines. Mutation analysis in this heterologous expression system showed that the VSAL motif of Kv4.2 is necessary for PSD-95 binding. PSD-95 increased the surface expression of Kv4.2 protein and caused it to cluster, as shown by deconvolution microscopy and biotinylation assays. Deleting the C-terminal VSAL motif of Kv4.2 eliminated these effects, as did substituting a palmitoylation-deficient PSD-95 mutant. In addition to these effects of PSD-95 on Kv4.2 distribution, the channel itself promoted redistribution of PSD-95 to the cell surface in the heterologous expression system. This work represents the first evidence that a member of the Shal subfamily of Kv channels can bind to PSD-95, with functional consequences.

Suppression of the rat microglia Kv1.3 current by src-family tyrosine kinases and oxygen/glucose deprivation.

Microglia activate following numerous acute insults to the brain, including oxygen/glucose deprivation (OGD), and both protein tyrosine kinases (PTKs) and K+ channels have been implicated in their activation. We identified Kv1.3 (voltage‐gated potassium channel) protein in cultured rat microglia and confirmed that the native current is biophysically and pharmacologically similar to Kv1.3. To explore whether src‐family PTKs regulate the microglial Kv current, we first heterologously expressed Kv1.3 in a microglia‐like cell line derived from neonatal rat brain (MLS‐9). The resulting large Kv1.3 current was eliminated by co‐transfecting the constitutively active PTK, v‐src, then rapidly restored by the PTK inhibitor, lavendustin A. Acute activation of endogenous src kinases by a peptide activator significantly reduced the current, an effect that was mimicked by OGD. Similarly, in primary cultures of rat microglia, the endogenous Kv1.3‐like current was inhibited by activating endogenous src‐family PTKs and by OGD. Biochemical analysis showed that OGD increased the tyrosine phosphorylation of native Kv1.3 protein, which was alleviated by PTK inhibitors or reactive oxygen species (ROS) scavengers. Conversely, the basal level of Kv1.3 phosphorylation was decreased by PTK inhibitors or scavengers of ROS. Together, our results point to a post‐insertional downregulation of the microglial Kv1.3‐like current by oxidative stress and tyrosine phosphorylation. This interaction may be facilitated by a multiprotein complex because, in cultured microglia, the endogenous Kv1.3 and src proteins both bind to the scaffolding protein, post‐synaptic density protein 95 (PSD‐95). By associating with, and phosphorylating Kv1.3, src is well positioned to regulate microglial responses to oxidative stress.

Assays of plasma membrane NADPH oxidase.

Publisher Summary Phagocytic cells, including neutrophils, eosinophils, and monocytes/macrophages, form part of the host defense system against infection. On binding to the target organism, there is an increase in oxygen consumption accompanied by the release of superoxide. This production of superoxide is catalyzed by a plasma membrane-bound enzyme: NADPH oxidase. Superoxide can undergo spontaneous or catalyzed dismutation to form hydrogen peroxide, or it can be converted to highly reactive oxidants, such as . OH or HOCl by a series of secondary reactions. The NADPH oxidase is normally dormant but can be activated by the addition of both soluble and particulate stimuli. The preferred electron donor is NADPH. The enzyme complex is composed of a membrane-bound b -type cytochrome together with at least three cytosolic components, which translocate to the membrane on activation. This complex is known to contain flavin adenine dinucleotide (FAD). Other nonphagocytic cells are also found to contain an NADPH oxidase-like enzyme. These include B lymphocytes, fibroblasts, mesangial cells, carotid body cells, and thyroid cells.

Biosynthesis of the dystonia-associated AAA +} ATPase torsinA at the endoplasmic reticulum

TorsinA is a widely expressed AAA(+) (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

Stromal-derived factor 1 signalling regulates radial and tangential migration in the developing cerebral cortex

Stromal-derived factor 1 (SDF-1), a known chemoattractant, and its receptor CXCR4 are widely expressed in the developing and adult cerebral cortex. Recent studies have highlighted potential roles for SDF-1 during early cortical development. In view of the current findings, our histological analysis has revealed a distinct pattern of SDF-1 expression in the developing cerebral cortex at a time when cell proliferation and migration are at peak. To determine the role of chemokine signalling during early cortical development, embryonic rat brain slices were exposed to a medium containing secreted SDF-1 to perturb the endogenous levels of chemokine. Alternatively, brain slices were treated with 40 µM of T140 or AMD3100, known antagonists of CXCR4. Using these experimental approaches, we demonstrate that chemokine signalling is imperative for the maintenance of the early cortical plate. In addition, we provide evidence that both neurogenesis and radial migration are concomitantly regulated by this signalling system. Conversely, interneurons, although not dependent on SDF-1 signalling to transgress the telencephalic boundary, require the chemokine to maintain their tangential migration. Collectively, our results demonstrate that SDF-1 with its distinct pattern of expression is essential and uniquely positioned to regulate key developmental events that underlie the formation of the cerebral cortex.

Upregulation of Kv 1.4 protein and gene expression after chronic spinal cord injury

After spinal cord injury (SCI), white matter tracts are characterized by demyelination and increased sensitivity to the K+ channel blocker 4‐aminopyridine (4‐AP). These effects appear to contribute to neurological impairment after SCI, although the molecular changes in K+ channel subunit expression remain poorly understood. We examined changes in gene expression of the 4‐AP‐sensitive voltage‐gated K+ channel Kv 1.4 after chronic SCI in the rat. Quantitative immunoblotting showed that Kv 1.4 protein was significantly increased at 6 weeks, but not at 1 week, after SCI in spinal cord white matter. Kv 1.4 was localized to astrocytes, oligodendrocytes, and oligodendrocyte progenitor cells but not to axons in both the normal and the injured spinal cord white matter. Because glial cells proliferate after SCI, we used immunogold electron microscopy to quantify Kv 1.4 protein in individual glial cells and found a sixfold increase of Kv 1.4 in cells of the oligodendrocyte lineage after chronic injury. Finally, quantitative in situ hybridization showed that Kv 1.4 mRNA was significantly upregulated in spinal cord white matter, but not gray matter, after SCI. In summary, we show that Kv 1.4 is expressed in glial cells and not in axons in the rat spinal cord white matter and that its expression is markedly increased in cells of the oligodendrocyte lineage after chronic SCI. Given that K+ channels play a role in glial cell proliferation, cells exhibiting changes in Kv 1.4 expression may represent proliferating oligodendroglia in the chronically injured spinal cord. J. Comp. Neurol. 443:154–167, 2002.