Kenton J. Swartz

Ion channel expression by white matter glia: The O-2A glial progenitor cell

We describe electrophysiological properties of the O-2A glial progenitor cell in a new serum-free culture system. O-2A progenitors have many properties characteristic of neurons: they have glutamate-activated ion channels, express the neuronal form of the sodium channel, fire single regenerative potentials, and synthesize the neurotransmitter GABA by an alternative synthetic pathway. Nearly identical properties were observed in acutely isolated O-2A progenitors, indicating that this phenotype is not an artifact of culture. The O-2A did not express a simple subset of channel types found in its descendant cells, the type-2 astrocyte and oligodendrocyte, studied in the same culture system. During development, these electrophysiological properties may contribute to O-2A function in vivo.

Kynurenine Pathway Measurements in Huntington's Disease Striatum: Evidence for Reduced Formation of Kynurenic Acid

Abstract: Recent evidence suggests that there may be over‐activation of the N‐methyl‐D‐aspartate (NMDA) subtype of excitatory amino acid receptors in Huntingtons disease (HD). Tryptophan metabolism by the kynurenine pathway produces both quinolinic acid, an NMDA receptor agonist, and kynurenic acid, an NMDA receptor antagonist. In the present study, multiple components of the tyrosine and tryptophan metabolic pathways were quantified in postmortem putamen of 35 control and 30 HD patients, using HPLC with 16‐sensor electrochemical detection. Consistent with previous reports in HD putamen, there were significant increases in 5‐hydroxyindoleacetic acid, 5‐hydroxytryptophan, and serotonin concentrations. Within the kynurenine pathway, the ratio of kynurenine to kynurenic acid was significantly (p < 0.01) increased twofold in HD patients as compared with controls, consistent with reduced formation of kynurenic acid in HD. CSF concentrations of kynurenic acid were significantly reduced in HD patients as compared with controls and patients with other neurologic diseases. Because kynurenic acid is an endogenous inhibitor of excitatory neurotransmission and can block excitotoxic degeneration in vivo, a relative deficiency of this compound could directly contribute to neuronal degeneration in HD.

AN INHIBITOR OF THE KV2.1 POTASSIUM CHANNEL ISOLATED FROM THE VENOM OF A CHILEAN TARANTULA

The Kv2.1 voltage-activated K+ channel, a Shab-related K+ channel isolated from rat brain, is insensitive to previously identified peptide inhibitors. We have isolated two peptides from the venom of a Chilean tarantula, G. spatulata, that inhibit the Kv2.1 K+ channel. The two peptides, hanatoxin1 (HaTx1) and hanatoxin2 (HaTx2) are unrelated in primary sequence to other K+ channel inhibitors. The activity of HaTx was verified by synthesizing it in a bacterial expression system. The concentration dependence for both the degree of inhibition at equilibrium (Kd = 42 nM) and the kinetics of inhibition (kon = 3.7 x 10(4) M-1s-1; koff = 1.3 x 10(-3) s-1), are consistent with a bimolecular reaction between HaTx and the Kv2.1 K+ channel. Shaker-related, Shaw-related, and eag K+ channels were relatively insensitive to HaTx, whereas a Shal-related K+ channel was sensitive. Regions outside the scorpion toxin binding site (S5-S6 linker) determine sensitivity to HaTx. HaTx introduces a new class of K+ channel inhibitors that will be useful probes for studying K+ channel structure and function.

Hanatoxin Modifies the Gating of a Voltage-Dependent K+ Channel through Multiple Binding Sites

We studied the mechanism by which Hanatoxin (HaTx) inhibits the drk1 voltage-gated K+ channel. HaTx inhibits the K+ channel by shifting channel opening to more depolarized voltages. Channels opened by strong depolarization in the presence of HaTx deactivate much faster upon repolarization, indicating that toxin bound channels can open. Thus, HaTx inhibits the drk1 K+ channel, not by physically occluding the ion conduction pore, but by modifying channel gating. Occupancy of the channel by HaTx was studied using various strength depolarizations. The concentration dependence for equilibrium occupancy as well as the kinetics of onset and recovery from inhibition indicate that multiple HaTx molecules can simultaneously bind to a single K+ channel. These results are consistent with a simple model in which HaTx binds to the surface of the drk1 K+ channel at four equivalent sites and alters the energetics of channel gating.

Modulation of Ca2+ channels by protein kinase C in rat central and peripheral neurons: Disruption of G protein-mediated inhibition

Activation of protein kinase C (PKC) reduced G protein-dependent inhibition of Ca2+ channels by glutamate, GA-BAB, adenosine, muscarinic, alpha-adrenergic, and LHRH receptors in a variety of central and peripheral neurons. PKC stimulation also relieved the inhibitory effect of internal GTP gamma S and reduced tonic G protein-mediated inhibition observed with internal GTP in the absence of transmitter receptor agonist. Basal Ca2+ channel currents were enhanced by PKC stimulation in most neurons studied. The PKC-induced enhancement of basal current was voltage dependent, and enhanced currents displayed altered kinetics. Inhibition of G proteins with GDP beta S attenuated the PKC-induced enhancement of basal Ca2+ channel current. These results show that PKC regulates the inhibitory effects of G proteins, possibly by disrupting the coupling of G proteins to Ca2+ channels. The PKC-induced enhancement of Ca2+ channel current results, at least in part, from the removal of tonic G protein-mediated inhibition.

MAPPING THE RECEPTOR SITE FOR HANATOXIN, A GATING MODIFIER OF VOLTAGE-DEPENDENT K+ CHANNELS

Hanatoxin (HaTx) binds to multiple sites on the surface of the drk1 voltage-gated K+ channel and modifies channel gating. We set out to identify channel residues that contribute to form these HaTx binding sites. Chimeras constructed using the drk1 and shaker K+ channels suggest that the S3-S4 linker may contain influential residues. Alanine scanning mutagenesis of the region extending from the C terminal end of S3 through S4 identified a number of residues that likely contribute to form the HaTx binding sites. The pore blocker Agitoxin2 and the gating modifier HaTx can simultaneously bind to individual K+ channels. These results suggest that residues near the outer edges of S3 and S4 form the HaTx binding sites and are eccentrically located at least 15 A from the central pore axis on the surface of voltage-gated K+ channels.

Aminooxyacetic Acid Results in Excitotoxin Lesions by a Novel Indirect Mechanism

Aminooxyacetic acid (AOAA) is an inhibitor of several pyridoxal phosphate‐dependent enzymes in the brain. In the present experiments intrastriatal injections of AOAA produced dose‐dependent excitotoxic lesions. The lesions were dependent on a pyridoxal phosphate mechanism because pyridoxine blocked them. The lesions were blocked by the noncompetitive N‐methyl‐D‐aspartate (NMDA) antagonist MK‐801 and by coinjection of kynurenate, a result indicating an NMDA receptor‐mediated excitotoxic process. Electrophysiologic studies showed that AOAA does not directly activate ugand‐gated ion channels in cultured cortical or striatal neurons. Pentobarbital anesthesia attenuated the lesions. AOAA injections resulted in significant increases in lactate content and depletions of ATP levels. AOAA striatal lesions closely resemble Huntingtons disease both neurochemically and histologically because they show striking sparing of NADPH‐diaphorase and large neurons within the lesioned area. AOAA produces excitotoxic lesions by a novel indirect mechanism, which appears to be due to impairment of intracellular energy metabolism, secondary to its ability to block the mitochondrial malate‐aspartate shunt. These results raise the possibility that a regional impairment of intracellular energy metabolism may secondarily result in excitotoxic neuronal death in chronic neurodegenerative illnesses, such as Huntingtons disease.

Kynurenine metabolites of tryptophan: Implications for neurologic diseases

Over the past 2 decades, a number of studies have demonstrated that amino acids act as precursors for the biosynthesis of a variety of neuroactive compounds, including catecholamines and indoleamines. For example, the aromatic amino acid l-tryptophan is a precursor for serotonin biosynthesis. Based on this observed precursor relationship, dietary tryptophan supplementation is used to treat a number of neurologic disorders attributed to alterations in serotoninergic neurotransmission. Recent studies have revealed that, in addition to serotonin, a number of neuroactive compounds, the kynurenines, are metabolities of tryptophan. Of these, perhaps the most important is quinolinic acid, a neurotoxin that acts at the N-methyl-d-aspartate (NMDA) receptor and whose precursor responsiveness to tryptophan far exceeds that of serotonin. In the central nervous system, kynurenines, and in particular quinolinic acid, may modulate excitatory amino acid transmission, and may act as neurotoxic agents implicated in the pathogenesis of several neurologic diseases.

Regional brain and cerebrospinal fluid quinolinic acid concentrations in Huntington's disease.

Many of the characteristics neuroanatomical and neurochemical features of Huntingtons disease (HD) are produced in experimental animals by an intrastriatal injection of the endogenous N-methyl-D-aspartate receptor agonist quinolinic acid (QUIN). Conceivably, a chronic over-production of QUIN in brain could be involved in the pathogenesis of HD. To investigate this hypothesis, concentrations of QUIN were measured both in cerebrospinal fluid (CSF) and postmortem tissue from patients with HD and neurologically normal age-matched controls. CSF QUIN concentrations were slightly lower in patients with HD, however the changes were not significant. Mean concentrations of QUIN tended to be lower in HD putamen, dentate nucleus and several cortical regions, although significant reductions were found only in Brodmann areas 17, 20 and 28. The mechanisms responsible for these small reductions in brain QUIN concentrations remain to be determined. These results do not support the hypothesis that a chronic increase of QUIN production is responsible for neurodengeneration in HD.

A detailed examination of substance P in pathologically graded cases of Huntington's disease

Substance P concentrations have been found to be reduced in the basal ganglia in Huntingtons disease (HD). In order to further examine this finding in the present study we measured substance P-like immunoreactivity (SPLI) in cases of HD which had been graded as to the severity of pathological changes in the striatum. Marked significant reductions of SPLI were found in all striatal nuclei which were significantly correlated with the percentage of neuronal loss in the varying pathologic grades. Similar changes were found in the projection sites of striatal substance P neurons, the globus pallidus interna and the substantia nigra. These changes are consistent with a loss of striatal substance P containing projection neurons in HD. Significant reductions in SPLI were also found in the external pallidum, bed nucleus of the stria terminalis and the subthalamic nucleus. Small significant increases in SPLI (20-30%) were found in 3 frontal cortical regions (Brodmann areas 6, 8 and 9). The finding of neurochemical changes in the subthalamic nucleus is of particular interest since lesions in this nucleus are known to result in chorea and therefore might contribute to the chorea which is a cardinal symptom of HD.