Comparison of the antinociceptive profiles of gabapentin and 3-methylgabapentin in rat models of acute and persistent pain: implications for mechanism of action.

The anticonvulsant gabapentin (GBP) has been shown effective for the treatment of neuropathic pain, although its mechanism of action remains unclear. A recent report has suggested that binding to the alpha(2)delta subunit of voltage-gated calcium channels contributes to its antinociceptive effect, based on the stereoselective efficacy of two analogs: (1S,3R)3-methylgabapentin (3-MeGBP) (IC(50) = 42 nM), which is effective in neuropathic pain models; and (1R,3R)3-MeGBP (IC(50) > 10,000 nM), which is ineffective (Field et al., 2000). The present study was designed to further examine the profiles of GBP and 3-MeGBP in rat models of acute and persistent pain. Systemic administration of GBP or (1S,3R)3-MeGBP inhibited tactile allodynia in the spinal nerve ligation model of neuropathic pain, whereas (1R,3R)3-MeGBP was ineffective. The antiallodynic effect of GBP, but not (1S,3R)3-MeGBP, was blocked by i.t. injection of the GABA(B) receptor antagonist [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl)phosphinic acid (CGP52432). Systemic GBP or (1S,3R)3-MeGBP also inhibited the second phase of formalin-evoked nociceptive behaviors, whereas (1R,3R)3-MeGBP was ineffective. However, both (1S,3R)3-MeGBP and (1R,3R)3-MeGBP, but not GBP, inhibited first phase behaviors. In the carrageenan model of inflammatory pain, systemic GBP or (1R,3R)3-MeGBP failed to inhibit thermal hyperalgesia, whereas (1S,3R)3-MeGBP had a significant, albeit transient, effect. Systemic (1S,3R)3-MeGBP, but not GBP or (1R,3R)3-MeGBP, also produced an antinociceptive effect in the warm water tail withdrawal test of acute pain. These data demonstrate that GBP and 3-MeGBP display different antinociceptive profiles, suggesting dissimilar mechanisms of antinociceptive action. Thus, the stereoselective efficacy of 3-MeGBP, presumably related to alpha(2)delta binding, likely does not completely account for the mechanism of action of GBP.

Differential expression of kcnq2 splice variants: implications to m current function during neuronal development.

The KCNQ family of K(+) channels has been implicated in several cardiac and neurological disease pathologies. KCNQ2 (Q2) is a brain-derived gene, which in association with KCNQ3 (Q3) has been shown to provide a molecular basis for the neuronal M current. We have cloned a long (Q2L) and a short (Q2S) splice variant of the human KCNQ2 gene; these variants differ in their C-terminal tail. Northern blot analysis reveals that Q2L is preferentially expressed in differentiated neurons, whereas the Q2S transcript is prominent in fetal brain, undifferentiated neuroblastoma cells, and brain tumors. Q2L, transfected into mammalian cells, produces a slowly activating, noninactivating voltage-gated K(+) current that is blocked potently by tetraethylammonium (TEA; IC(50), 0.14 mm). Q2S on the other hand produces no measurable potassium currents. Cotransfection of Q2S with either Q2L, Q3, or Q2L/Q3 heteromultimers results in attenuation of K(+) current, the suppression being most profound for Q3. Inclusion of Q2S in the heteromultimer also positively shifts the voltage dependence of current activation and alters affinity for the TEA block, suggesting that under these conditions, some Q2S subunits incorporate into functional channels on the plasma membrane. In view of the crucial role of M currents in modulating neuronal excitability, our findings provide important insight into the functional consequences of differential expression of KCNQ2 splice variants: dampened potassium conductances in the developing brain could shape firing repertoires to provide cues for proliferation rather than differentiation.

Structure-guided transformation of charybdotoxin yields an analog that selectively targets Ca(2+)-activated over voltage-gated K(+) channels.

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca(2+)-activated K(+) channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca(2+)-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys(32), which lies in close proximity to this cluster. These analogs block IKCa1 with approximately 20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1. 6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K(Ca) channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K(+) channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K(+) channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.

Interaction of calicheamicin gamma1(I) and its related carbohydrates with DNA-protein complexes.

We report studies of the contribution of DNA structure, holding the sequence constant, to the affinity of calicheamicin gamma(1)(I) and its aryltetrasaccharide moiety for DNA. We used polynucleotide chains as models of known protein-binding sequences [the catabolite activator protein (CAP) consensus sequence, AP-1 and cAMP response element (CRE) sites] in their free and protein-bound forms. The proteins were selected to provide examples in which the minor-groove binding site for the carbohydrate is (CAP) or is not (GCN4) covered by the protein. Additionally, peptides related to the GCN4 and CREB families, which have different bending effects on their DNA-binding sites, were used. We observe that proteins of the CREB class, which induce a tendency to bend toward the minor groove at the center of the site, inhibit drug-cleavage sites located at the center of the free AP-1 or CRE DNA sites. In the case of GCN4, which does not induce DNA bending, there is no effect on calicheamicin cleavage of the CRE site, but we observe a GCN4-induced rearrangement of the cutting pattern in the AP-1 site. This effect may arise from either a subtle local conformational rearrangement not accompanied by bending or a localized reduction in DNA flexibility. Whereas GCN4 binding is not inhibited by the calicheamicin aryltetrasaccharide, binding of CAP to its DNA target is significantly inhibited, and calicheamicin cutting of DNA at the center of the CAP-DNA complex site is strongly reduced by protein binding. This result probably reflects steric inhibition of drug binding by the protein.

Calmodulin mediates calcium-dependent activation of the intermediate conductance KCa channel, IKCa1.

Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail of hSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+ channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1. 3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed with hIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

Regulation of mammalian Shaker-related K+ channels: evidence for non-conducting closed and non-conducting inactivated states.

1. Using the whole-cell recording mode we have characterized two non-conducting states in mammalian Shaker-related voltage-gated K+ channels induced by the removal of extracellular potassium, K+o. 2. In the absence of K+o, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of K+o had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour. 3. To characterize the nature of the interaction between Kv1.3 and K+o concentration ([K+]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of K+o, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mM K+o, possibly by making the channel unstable in the absence of K+o. Mutation at a neighbouring position (400) had a similar effect. 4. The effect of removing K+o on current amplitude does not seem to be correlated with the rate of C-type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K+]o dependence as the wild-type Kv1.3 channel. 5. CP-339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of K+o unless the channels were inactivated through depolarizing pulses. 6. We conclude that removal of K+o induces the Kv1.3 channel to transition to a non-conducting 'closed' state which can switch into a non-conducting 'inactivated' state upon depolarization.

Purification, visualization, and biophysical characterization of Kv1.3 tetramers.

The voltage-gated K+ channel of T-lymphocytes, Kv1.3, was heterologously expressed in African Green Monkey kidney cells (CV-1) using a vaccinia virus/T7 hybrid expression system; each infected cell exhibited 10(4) to 5 x 10(5) functional channels on the cell surface. The protein, solubilized with detergent (3-[cholamidopropyl)dimethylammonio]-1-propanesulfonic acid or cholate), was purified to near-homogeneity by a single nickel-chelate chromatography step. The Kv1.3 protein expressed in vaccinia virus-infected cells and its purified counterpart are both modified by a approximately 2-kDa core-sugar moiety, most likely at a conserved N-glycosylation site in the external S1-S2 loop; absence of the sugar does not alter the biophysical properties of the channel nor does it affect expression levels. Purified Kv1.3 has an estimated size of approximately 64 kDa in denaturing SDS-polyacrylamide electrophoresis gels, consistent with its predicted size based on the amino acid sequence. By sucrose gradient sedimentation, purified Kv1.3 is seen primarily as a single peak with an approximate mass of 270 kDa, compatible with its being a homotetrameric complex of the approximately 64-kDa subunits. When reconstituted in the presence of lipid and visualized by negative-staining electron microscopy, the purified Kv1.3 protein forms small crystalline domains consisting of tetramers with dimensions of approximately 65 x 65 A. The center of each tetramer contains a stained depression which may represent the ion conduction pathway. Functional reconstitution of the Kv1.3 protein into lipid bilayers produces voltage-dependent K+-selective currents that can be blocked by two high affinity peptide antagonists of Kv1.3, margatoxin and stichodactylatoxin.

A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes.

We have isolated a novel gene, hKCa4, encoding an intermediate conductance, calcium-activated potassium channel from a human lymph node library. The translated protein comprises 427 amino acids, has six transmembrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shares 41-42% similarity at the amino acid level with three small conductance calcium-activated potassium channels cloned from brain. Northern blot analysis of primary human T lymphocytes reveals a 2.2-kilobase transcript that is highly up-regulated in activated compared with resting cells, concomitant with an increase in KCa current. hKCa4 transcript is also detected by Northern blots or by polymerase chain reaction in placenta, prostate, thymus, spleen, colon, and many cell lines of hematopoietic origin. Patch-clamp recordings of hKCa4-transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassium current that is blocked by externally applied tetraethylammonium (Kd = 30 +/- 7 mM), charybdotoxin (Kd = 10 +/- 1 nM), and clotrimazole (Kd = 387 +/- 34 nM), but is resistant to apamin, iberiotoxin, kaliotoxin, scyllatoxin (Kd > 1 microM), and margatoxin (Kd > 100 nM). Single hKCa4 channels have a conductance of 33 +/- 2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (Kd = 270 +/- 8 nM) with a highly cooperative interaction of approximately three calcium ions per channel. These properties of the cloned channel are very similar to those reported for the native KCa channel in activated human T lymphocytes, indicating that hKCa4 encodes this channel type.

Novel nonpeptide agents potently block the C-type inactivated conformation of Kv1.3 and suppress T cell activation.

The nonpeptide agent CP-339,818 (1-benzyl-4-pentylimino-1,4-dihydroquinoline) and two analogs (CP-393,223 and CP-394,322) that differ only with respect to the type of substituent at the N1 position, potently blocked the Kv1.3 channel in T lymphocytes. A fourth compound (CP-393,224), which has a smaller and less-lipophilic group at N1, was 100-200-fold less potent, suggesting that a large lipophilic group at this position is necessary for drug activity. CP-339,818 blocked Kv1.3 from the outside with a IC50 value of approximately 200 nM and 1:1 stoichiometry and competitively inhibited 125I-charybdotoxin from binding to the external vestibule of Kv1.3. This drug inhibited Kv1.3 in a use-dependent manner by preferentially blocking the C-type inactivated state of the channel. CP-339,818 was a significantly less potent blocker of Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv3.1-4, and Kv4.2; the only exception was Kv1.4, a cardiac and neuronal A-type K+ channel. CP-339,818 had no effect on two other T cell channels (I(CRAC) and intermediate-conductance K(Ca)) implicated in T cell mitogenesis. This drug suppresses human T cell activation, suggesting that blockade of Kv1.3 alone is sufficient to inhibit this process.

The signature sequence of voltage-gated potassium channels projects into the external vestibule.

A highly conserved motif, GYGD, contributes to the formation of the ion selectivity filter in voltage-gated K+ channels and is thought to interact with the scorpion toxin residue, Lys27. By probing the pore of the Kv1.3 channel with synthetic kaliotoxin-Lys27 mutants, each containing a non-natural lysine analog of a different length, and using mutant cycle analysis, we determined the spatial locations of Tyr400 and Asp402 in the GYGD motif, relative to His404 located at the base of the outer vestibule. Our data indicate that the terminal amines of the shorter Lys27 analogs lie close to His404 and to Asp402, while Lys27 itself interacts with Tyr400. Based on these data, we developed a molecular model of this region of the channel. The junction between the outer vestibule and the pore is defined by a ring ( approximately 8-9-A diameter) formed from alternating Asp402 and His404 residues. Tyr400 lies 4-6 A deeper into the pore, and its interaction with kaliotoxin-Lys27 is in competition with K+ ions. Studies with dimeric Kv1.3 constructs suggest that two Tyr400 residues in the tetramer are sufficient to bind K+ ions. Thus, at least part of the K+ channel signature sequence extends into a shallow trough at the center of a wide external vestibule.

Characterization of the transcription unit of mouse Kv1.4, a voltage-gated potassium channel gene.

The mouse voltage-gated K+ channel gene, Kv1.4, is expressed in brain and heart as approximately 4.5- and approximately 3.5-kilobase (kb) transcripts. Both mRNAs begin at a common site 1338 bp upstream of the initiation codon, contain 3477 and 4411 nucleotides, respectively, and are encoded by two exons; exon 1 contains 0.5 kb of the 5'-noncoding region (NCR), while exon 2 encodes the remaining 0.8 kb of the 5'-NCR, the entire coding region (2 kb), and all of the 3'-NCR. The 3.5-kb transcript terminates at a polyadenylation signal 177 bp 3' of the stop codon, while the 4.5-kb mRNA utilizes a signal 94 bp farther downstream. Although the proteins generated from either transcript are identical, the two mRNAs are functionally different, the 3.5-kb transcript producing approximately 4-5-fold larger currents when expressed in Xenopus oocytes compared to the 4. 5-kb mRNA. The decreased expression of the longer transcript is due to the presence of five ATTTA repeats in the 3'-NCR which inhibit translation; such motifs have also been reported to destabilize the messages of many other genes and might therefore shorten the life of the 4.5-kb transcript during its natural expression. The Kv1.4 basal promoter is GC-rich, contains three SP1 repeats (CCGCCC, -65 to -35), lacks canonical TATAAA and GGCAATCT motifs, and has no apparent tissue specificity. One region enhances activity of this promoter. Thus, transcriptional and post-transcriptional regulation of mKv1.4, coupled with selective usage of the two alternate Kv1.4 mRNAs, may modulate the levels of functional Kv1.4 channels.

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins.

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

The P-region and S6 of Kv3.1 contribute to the formation of the ion conduction pathway.

The loop between transmembrane regions S5 and S6 (P-region) of voltage-gated K+ channels has been proposed to form the ion-conducting pore, and the internal part of this segment is reported to be responsible for ion permeation and internal tetraethylammonium (TEA) binding. The two T-cell K+ channels, Kv3.1 and Kv1.3, with widely divergent pore properties, differ by a single residue in this internal P-region, leucine 401 in Kv3.1 corresponding to valine 398 in Kv1.3. The L401V mutation in Kv3.1 was created with the anticipation that the mutant channel would exhibit Kv1.3-like deep-pore properties. Surprisingly, this mutation did not alter single channel conductance and only moderately enhanced internal TEA sensitivity, indicating that residues outside the P-region influence these properties. Our search for additional residues was guided by the model of Durell and Guy, which predicted that the C-terminal end of S6 formed part of the K+ conduction pathway. In this segment, the two channels diverge at only one position, Kv3.1 containing M430 in place of leucine in Kv1.3. The M430L mutant of Kv3.1 exhibited permeant ion- and voltage-dependent flickery outward single channel currents, with no obvious changes in other pore properties. Modification of one or more ion-binding sites located in the electric field and possibly within the channel pore could give rise to this type of channel flicker.

Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines.

We have analyzed the biophysical and pharmacological properties of five cloned K+ (Kv) channels (Kv1.1, Kv1.2, Kv1.3, Kv1.5, and Kv3.1) stably expressed in mammalian cell lines. Kv1.1 is biophysically similar to a K+ channel in C6 glioma cells and astrocytes, Kv1.3 and Kv3.1 have electrophysiological properties identical to those of the types n and l K+ channels in T cells, respectively, and Kv1.5 closely resembles a rapidly activating delayed rectifier in the heart. Each of these native channels may be formed from the homomultimeric association of the corresponding Kv subunits, and pharmacological compounds that selectively modulate them may be useful for the treatment of neurological, immune, and cardiac disorders. The cell lines described in this report could be used to identify such drugs and we have therefore embarked on a pharmacological characterization of the five cloned channels. The compounds tested in this study include 4-aminopyridine, capsaicin, charybdotoxin, cromakalim, dendrotoxin, diltiazem, D-sotalol, flecainide, kaliotoxin, mast cell degranulating peptide, nifedipine, noxiustoxin, resiniferatoxin, and tetraethylammonium.

Genomic organization, nucleotide sequence, biophysical properties, and localization of the voltage-gated K+ channel gene KCNA4/Kv1.4 to mouse chromosome 2/human 11p14 and mapping of KCNC1/Kv3.1 to mouse 7/human 11p14.3-p15.2 and KCNA1/Kv1.1 to human 12p13.

A genomic clone encoding the Shaker-related potassium channel gene, Kcna4/mKv1.4, was isolated from mice. Its coding region is contained in a single exon, encodes a protein of 654 amino acids, and shares approximately 91% nucleotide sequence identity with human KCNA4/hKv1.4. We show that 0.8 kb of the 5' noncoding region (NCR), the entire protein coding region (approximately 2.0 kb), and all of the known 3' NCR (approximately 1.1 kb) are contained within a single exon; the remaining 0.5 kb of the 5' NCR is separated from this exon by a 3.4-kb intron. The sequenced genomic region thus accounts for essentially all of the longest known transcript (4.5 kb), although the precise ends of this transcript have not been defined. The 3' NCR contains several ATTTA and ATTTG motifs that are thought to destabilize mRNAs, and these are also present in rat, bovine, and human Kcna4/Kv1.4 cDNAs. It also contains three conserved polyadenylation signals, alternate utilization of which could generate mRNAs of differing stabilities. The 5' NCR of Kcna4/mKv1.4 may also serve to regulate channel expression. This region is approximately 85% identical to KCNA4/hKv1.4 and contains eight consensus translation start sites [(G, A)NNATG] that, based on the 5'-3' scanning model, would lead to a lowering of translational efficiency. The shortest Kcna4/Kv1.4 transcript (2.4 kb) can contain at most 400 bp of NCR and should lack the 3' ATTTAs and most of the 5' ATGs; this transcript might therefore exhibit increased stability and translational efficiency. The Kcna4/mKv1.4 channel exhibited biophysical and pharmacological properties indistinguishable from its rat and human homologues. Kcna4/mKv1.4 lies on mouse chromosome 2, near the Fshb locus, and in humans on the proximal half of chromosome 11p14 near human FSHB. Another K+ channel gene, Kcnc1/mKv3.1, lies approximately 1.8 cM from the Myod-1 gene on mouse chromosome 7, and in situ hybridization localizes KCNC1/hKv3.1 to the homologous region on human chromosome 11p14.3-p15.2. A third gene, KCNA1/hKv1.1, was mapped to human 12p13.

Full-length and truncated Kv1.3 K+ channels are modulated by 5-HT1c receptor activation and independently by PKC.

In T-cells, the Shaker-related gene, Kv1.3 encodes the type n K+ channel, whereas the type l channel is a product of the Shaw. subfamily gene, Kv3.1. Both these genes are also expressed in the brain. We have used the Xenopus oocyte heterologous expression system to study the modulatory effects of serotonin (5-hydroxytryptamine, 5-HT) on both these cloned channels. In oocytes coexpressing the mouse 5-HT1c receptor and mouse Kv1.3 channel, addition of 100 nM 5-HT causes a complete and sustained suppression of Kv1.3 currents in approximately 20 min. In contrast, 5-HT has no effect on mouse Kv3.1 currents when coexpressed with 5-HT1c receptor. The 5-HT-mediated suppression of Kv1.3 currents proceeds via activation of a pertussis toxin-sensitive G protein and a subsequent rise in intracellular Ca2+, but Ca2+ does not directly block the channel. Protein kinase (PK) C activation is not part of the pathway linking 5-HT1c receptor to Kv1.3 channels. However, phorbol esters independently suppress Kv1.3 currents. Deletion of the first 146 amino acids from the NH2-terminal, containing putative tyrosine kinase and PKA phosphorylation sites, does not alter the time course of 5-HT-mediated suppression of Kv1.3 currents, indicating that these residues are not necessary for modulation. Treatment of oocytes with calmodulin or phosphatase inhibitors does not alter 5-HT-mediated modulation. Collectively, these experiments indicate that the mouse Kv1.3 channel is capable of being modulated by 5-HT via 5-HT1c receptor in a G protein and Ca(2+)-dependent manner, but the subsequent steps in the pathway remain elusive.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of chromium(VI) to chromium(V) by rat liver cytosolic and microsomal fractions: is DT-diaphorase involved?

Incubation of rat liver cytosolic or microsomal fractions with chromium(VI) led to a dramatic decrease in chromium(VI) mutagenicity, as determined by the Ames Salmonella assay using the TA100 tester strain. The cytosol-dependent decrease in chromium(VI) mutagenicity was found to be counteracted in the presence of dicumarol, an inhibitor of the cytosolic enzyme NAD(P)H:quinone oxidoreductase (DT-diaphorase). In order to determine whether DT-diaphorase is a significant factor in enzymatic reduction of chromium(VI) in rat liver tissue, cytosolic and microsomal fractions were analyzed for NAD(P)H-dependent chromium (VI) reductase activity leading to chromium(V) formation by using electron paramagnetic resonance (EPR) spectroscopy. Reaction of chromium(VI) with NADH or NADPH in the presence of either cytosolic or microsomal fractions led to the formation of stable chromium(V)--NAD(P)H complexes. When glucose 6-phosphate (G6P) was present in the reaction as part of a NADPH-generating system, stable chromium(V)--G6P complexes were formed in addition to the chromium(V)--NAD(P)H complexes. The chromium(V) complexes had g values of 1.980-1.982 and superhyperfine splitting constants of 0.8-0.9 characteristic of bis(diol)oxochromium(V) complexes. Inhibition of 90% of the cytosolic DT-diaphorase activity by dicumarol led to only partial (20-22%) inhibition of chromium(V) formation. Visible and EPR spectroscopic studies showed that purified DT-diaphorase had no detectable chromium(VI) reductase activity and did not catalyze formation of chromium(V). Inhibition of 69% of microsomal aryl hydrocarbon hydroxylase activity by ketoconazole led to partial (10%) inhibition of chromium(V) formation. These results indicate that intracellular NAD(P)H-dependent enzymatic reduction of chromium(VI) in rat liver cannot be attributed to the activity of any one enzyme in the cytosolic or microsomal fractions. DT-diaphorase appears to play an indirect role in decreasing chromium(VI)-induced mutagenicity in Salmonella, possibly through interaction with other redox active cellular components. The involvement of diols such as sugars and pyridine nucleotides in stabilizing intracellularly generated chromium(V) is discussed.

Reaction of chromium(VI) with glutathione or with hydrogen peroxide: identification of reactive intermediates and their role in chromium(VI)-induced DNA damage.

The types of reactive intermediates generated upon reduction of chromium(VI) by glutathione or hydrogen peroxide and the resulting DNA damage have been determined. In vitro, reaction of chromium(VI) with glutathione led to formation of two chromium(V) complexes and the glutathione thiyl radical. When chromium(VI) was reacted with DNA in the presence of glutathione, chromium-DNA adducts were obtained, with no DNA strand breakage. The level of chromium-DNA adduct formation correlated with chromium(V) formation. Reaction of chromium(VI) with hydrogen peroxide led to formation of hydroxyl radical. No chromium(V) was detectable at 24 degrees C (297 K); however, low levels of the tetraperoxochromium(V) complex were detected at 77 K. Reaction of chromium(VI) with DNA in the presence of hydrogen peroxide produced significant DNA strand breakage and the 8-hydroxydeoxyguanosine adduct, whose formation correlated with hydroxyl radical production. No significant chromium-DNA adduct formation was detected. Thus, the nature of chromium(VI)-induced DNA damage appears to be dependent on the reactive intermediates, i.e. chromium(V) or hydroxyl radical, produced during the reduction of chromium(VI).

K+ channel expression in primary cell cultures mediated by vaccinia virus.

A recombinant vaccinia virus (VV) was used to express functional Drosophila Shaker H4 K+ channels in primary cell cultures from rat heart (atrial and ventricular myocytes, fibroblasts), autonomic ganglia (SCG neurons) and CNS (hippocampal neurons, cerebral astroglia). In most cells the expressed currents possessed the typical characteristics of the native Drosophila muscle A currents; a few cells showed evidence of hetero-oligomers with new properties. The maximum current density corresponded to a channel density of 2-3/microns 2. Voltage recordings in heart cells showed altered action potential waveforms after successful infection. VV vectors thus are useful for studying altered excitability and cell-specific processing of ion channel proteins.

Rotavirus-specific antibody response in saliva of infants with rotavirus diarrhea.

The reliability of saliva as an indicator of rotavirus infection was assessed among 15 infants (3-12 months) with rotaviral and 15 with nonrotaviral diarrhea. Paired salivary samples collected during acute and convalescent phases were tested for rotavirus-specific IgA and IgM by an ELISA. The sensitivity of IgA or IgM alone to predict infection was 53.3% and 46.6%, respectively; used in conjunction, the sensitivity rose to 80%. It seems that infants with rotaviral diarrhea mount mucosal antibody responses as reflected in their saliva; possibly salivary antibodies could be used to evaluate vaccine "take" in rotavirus vaccine trials.