Helical structure of the COOH terminus of S3 and its contribution to the gating modifier toxin receptor in voltage-gated ion channels.

The voltage-sensing domains in voltage-gated K(+) channels each contain four transmembrane (TM) segments, termed S1 to S4. Previous scanning mutagenesis studies suggest that S1 and S2 are amphipathic membrane spanning alpha-helices that interface directly with the lipid membrane. In contrast, the secondary structure of and/or the environments surrounding S3 and S4 are more complex. For S3, although the NH(2)-terminal part displays significant helical character in both tryptophan- and alanine-scanning mutagenesis studies, the structure of the COOH-terminal portion of this TM is less clear. The COOH terminus of S3 is particularly interesting because this is where gating modifier toxins like Hanatoxin interact with different voltage-gated ion channels. To further examine the secondary structure of the COOH terminus of S3, we lysine-scanned this region in the drk1 K(+) channel and examined the mutation-induced changes in channel gating and Hanatoxin binding affinity, looking for periodicity characteristic of an alpha-helix. Both the mutation-induced perturbation in the toxin-channel interaction and in gating support the presence of an alpha-helix of at least 10 residues in length in the COOH terminus of S3. Together with previous scanning mutagenesis studies, these results suggest that, in voltage-gated K(+) channels, the entire S3 segment is helical, but that it can be divided into two parts. The NH(2)-terminal part of S3 interfaces with both lipid and protein, whereas the COOH-terminal part interfaces with water (where Hanatoxin binds) and possibly protein. A conserved proline residue is located near the boundary between the two parts of S3, arguing for the presence of a kink in this region. Several lines of evidence suggest that these structural features of S3 probably exist in all voltage-gated ion channels.

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels.

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.

Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel.

Hanatoxin inhibits voltage-gated K(+) channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K(+) channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.

Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins.

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K(+) channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K(+) channel inhibitors. Unlike most other K(+) channel-blocking toxins, HaTx1 adopts an "inhibitor cystine knot" motif and is composed of two beta-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca(2+) and Na(+) channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels.

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel.

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.

alpha-helical structural elements within the voltage-sensing domains of a K(+) channel.

Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

Gating modifier toxins reveal a conserved structural motif in voltage-gated Ca2+ and K+ channels.

Protein toxins from venomous animals exhibit remarkably specific and selective interactions with a wide variety of ion channels. Hanatoxin and grammotoxin are two related protein toxins found in the venom of the Chilean Rose Tarantula, Phrixotrichus spatulata. Hanatoxin inhibits voltage-gated K+ channels and grammotoxin inhibits voltage-gated Ca2+ channels. Both toxins inhibit their respective channels by interfering with normal operation of the voltage-dependent gating mechanism. The sequence homology of hanatoxin and grammotoxin, as well as their similar mechanism of action, raises the possibility that they interact with the same region of voltage-gated Ca2+ and K+ channels. Here, we show that each toxin can interact with both voltage-gated Ca2+ and K+ channels and modify channel gating. Moreover, mutagenesis of voltage-gated K+ channels suggests that hanatoxin and grammotoxin recognize the same structural motif. We propose that these toxins recognize a voltage-sensing domain or module present in voltage-gated ion channels and that this domain has a highly conserved three-dimensional structure.

Inhibition of T-type voltage-gated calcium channels by a new scorpion toxin.

The biophysical properties of T-type voltage-gated calcium channels are well suited to pacemaking and to supporting calcium flux near the resting membrane potential in both excitable and non-excitable cells. We have identified a new scorpion toxin (kurtoxin) that binds to the alpha 1G T-type calcium channel with high affinity and inhibits the channel by modifying voltage-dependent gating. This toxin distinguishes between alpha 1G T-type calcium channels and other types of voltage-gated calcium channels, including alpha 1A, alpha 1B, alpha 1C and alpha 1E. Like the other alpha-scorpion toxins to which it is related, kurtoxin also interacts with voltage-gated sodium channels and slows their inactivation. Kurtoxin will facilitate characterization of the subunit composition of T-type calcium channels and help determine their involvement in electrical and biochemical signaling.

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.

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.

Inhibition of calcium channels in rat central and peripheral neurons by omega-conotoxin MVIIC.

Inhibition of voltage-dependent calcium channels by omega-conotoxin MVIIC (omega-CTx-MVIIC) was studied in various types of rat neurons. When studied with 5 mM Ba2+ as charge carrier, omega-CTx-MVIIC block of N-type calcium channels in sympathetic neurons was potent, with half-block at 18 nM. Block of N-type channels had a rapid onset (tau approximately 1 sec at 1 microM omega-CTx-MVIIC) and quick reversibility (tau approximately 30 sec). The rate of block was proportional to toxin concentration, consistent with 1:1 binding of toxin to channels, with a rate constant (k on) of approximately 1 X 10(6) M-1. sec-1. Both potency and rate of block were reduced dramatically with increasing concentrations of extracellular Ba2+ omega-CTx-MVIIC also blocked P-type calcium channels in cerebellar Purkinje neurons, but both development and reversal of block were far slower than for N-type channels. The rate of block was proportional to toxin concentration, with k on -1.5 x 10(3) M-1. sec-1 at 5 mM Ba2+. From this value and an unblocking time constant of approximately 200 min, a dissociation constant of approximately 50 nM was estimated. Thus, block of P-type channels is potent but very slow. In hippocampal CA3 pyramidal neurons, omega-CTx-MVIIC blocked approximately 50% of the high-threshold calcium channel current; one component (approximately 20%) was blocked with the rapid kinetics expected for N-type channels, whereas the other component was blocked slowly. The component blocked slowly was reduced but not eliminated by preexposure to 200 nM or 1 microM omega-Aga-IVA.

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.

Intrastriatal injections of quinolinic acid or kainic acid: differential patterns of cell survival and the effects of data analysis on outcome.

There is controversy about the extent to which lesions of the rat striatum with excitatory amino acids mimic the cellular pathology seen in Huntington's Disease (HD). We sought to resolve this debate by determining with cell counts in adjacent sections the patterns of survival of medium spiny and aspiny striatal neurons using enkephalin immunohistochemistry and NADPH-diaphorase histochemistry as markers of these cell populations, respectively. Results showed that 2 weeks after quinolinic acid lesions, cell loss was qualitatively similar for the two cell groups. However, by varying the size of the sampling area for quantitative analyses and its distance from the lesion zone, the outcome of the statistical analyses varied enormously. Thus, a relative sparing of NADPH-diaphorase-labeled cells compared to enkephalin-labeled cells could be detected quantitatively in transition areas bordering the lesion under some but not all analytical conditions. Kainic acid lesions depleted both cell populations similarly, except in regions of transition farthest from the lesion, where enkephalin-containing neurons were more resistant than NADPH-diaphorase-containing cells. The size of the transition area around the lesion also differed depending upon excitotoxin and cell population. These results help to reconcile the controversy and suggest that with highly specified quantitative conditions quinolinic acid-induced injury of the striatum can resemble the histopathology of HD.

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.

Protein kinase C modulates glutamate receptor inhibition of Ca2+ channels and synaptic transmission.

Fast synaptic transmission in the central nervous system can be modulated by neurotransmitters and second-messenger pathways. For example, transmission at glutamatergic synapses can be depressed by the metabotropic glutamate receptor, providing autoreceptor-mediated negative feedback. Metabotropic glutamate receptor inhibition of Ca2+ channels may contribute to this pathway. In contrast, stimulation of protein kinase C can enhance excitatory synaptic transmission, whereas both depression and enhancement of Ca2+ current have been reported. Here we show that in hippocampal CA3 and cortical pyramidal neurons, activation of protein kinase C enhances current through N-type Ca2+ channels and, in addition, dramatically reduces G protein-dependent inhibition of these same channels by the metabotropic glutamate receptor. In parallel experiments on fast excitatory transmission at corticostriatal synapses, kinase C activators were similarly found to reduce the inhibitory effect produced by stimulation of the metabotropic glutamate receptor. The results show that second-to-second control of Ca2+ channels by the metabotropic glutamate receptor can itself be modulated on a slower timescale by protein kinase C. These mechanisms may be used in the control of fast excitatory synaptic transmission.

Inhibition of calcium channels in rat CA3 pyramidal neurons by a metabotropic glutamate receptor.

L-Glutamate rapidly and reversibly suppressed Ca channel current in freshly dissociated pyramidal neurons from the CA3 region of the rat hippocampus. L-Glutamate inhibition of Ca channel current could be distinguished from activation of background conductance by appropriate ionic conditions and by distinct pharmacological profiles. Ca channel inhibition by glutamate was mimicked by quisqualate, ibotenate, racemïct-ACPD and 1S,3R-ACPD but not by kainate, AMPA, L-aspartate, NMDA, L-2-amino-4-phosphonobutyric acid, or 1R,3S-ACPD; 6-cyano-7-nitroquinoxaline-2,3-dione did not inhibit the response. All agonists inhibited a similar fraction of high-voltage-activated Ca channel current, typically approximately 30%. Concentration-response relations for the agonists were consistent with mediation by a metabotropic glutamate receptor. The stereospecific agonist 1S,3R-ACPD was especially useful since it did not activate background conductances. The fraction of Ca channel current sensitive to 1S,3R-ACPD was partially blocked by omega-conotoxin GVIA but was not sensitive to dihydropyridine antagonists or agonists. The suppression of Ca channels by 1S,3R-ACPD became irreversible when cells were dialyzed with GTP-gamma-S. 1S,3R-ACPD suppressed Ca channel currents in outside-out membrane patches but not in cell-attached patches when applied outside the patch. These results suggest that metabotropic glutamate receptors suppress the activity of N-type Ca channels in CA3 neurons by a mechanism involving G-proteins but not readily diffusible second messengers.

Developmental changes in brain kynurenic acid concentrations.

The cerebral distribution and regulation of excitatory amino acid levels may play a crucial role in neuronal development. In the present study we examined concentrations of the endogenous excitatory amino acid antagonist kynurenic acid and related substances during development in fetal and neonatal rat brain and fetal non-human primate cerebral cortex. Kynurenic acid concentrations in rat fetal whole brain were significantly increased 4-5 fold prenatally, then declined rapidly at 1 day after birth, and reached adult concentrations at 7 days after birth. L-Kynurenine concentrations were also markedly increased prior to birth and then declined to adult concentrations at 1 day after birth. L-Tryptophan was increased 3 fold before birth, and decreased to adult concentrations 1 day after birth. In contrast concentrations of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid and homovanillic acid increased 1 day prior to birth and continued to increase following birth. Fetal baboon cerebral cortex showed significant increases in kynurenic acid concentrations both pre-term and near-term as compared with adult concentrations. These results show that marked changes in kynurenic acid concentrations occur prior to and following birth. It is possible that high levels of kynurenic acid prior to birth inhibit neurite branching and development of excitatory synapses, which then develop rapidly in parallel with the decrease in kynurenic acid levels.

Competitive antagonism of glutamate receptor channels by substituted benzazepines in cultured cortical neurons.

Whole-cell recordings from rat cortical neurons in dissociated cell culture were used to study the antagonism of glutamate receptors by several lipophilic benzazepine analogues of 2,5-dihydro-2,5-dioxo-3-hydroxy-1H-benzazepine (DDHB). DDHB and three substituted derivatives, 4-bromo-, 7-methyl-, and 8-methyl-DDHB, inhibited the activation of N-methyl-D-aspartate (NMDA) receptors at both the NMDA recognition site and the glycine allosteric site. In addition, all four compounds blocked the activation of non-NMDA receptors by kainate and L-glutamate. Antagonism by the four benzazepines was equivalent at holding potentials from -80 mV to +50 mV. Both the onset of and recovery from block of the agonist-gated currents were complete within seconds. Antagonist affinity was calculated from the displacement of steady state concentration-response curves for kainate, L-glutamate, glycine, and NMDA, based on the Gaddum-Schild relationship (dose ratio = 1 + [antagonist]/KB). The most potent blocker, 8-Me-DDHB, had an apparent dissociation constant (KB) of 470 nM at the glycine allosteric site and 27 microM at the NMDA recognition site. The apparent dissociation constant of 8-Me-DDHB for non-NMDA receptors was 6.4 microM when kainate was the agonist and 9.6 microM when L-glutamate was the agonist. Unsubstituted DDHB showed slightly higher affinity for the NMDA recognition site (KB = 16 microM) but was less potent than 8-Me-DDHB at the glycine allosteric site and at non-NMDA receptors (KB = 3 and 65 microM, respectively). At all three sites, the inhibitory actions of these benzazepine derivatives were consistent with a simple competitive mechanism of antagonism. In addition, the antagonist potency of the parent compound, DDHB, against kainate, NMDA, and glycine was equal to or greater than that of other bicyclic antagonists, including kynurenic acid, indole-2-carboxylic acid, and quinoxaline-2,3-dione. Substituted benzazepines represent a new class of glutamate receptor antagonists that show competitive action, significant potency at multiple sites, and a high degree of lipophilicity.

Aminooxyacetic acid results in excitotoxin lesions by a novel indirect mechanism.

Aminooxyacetic acid (AOAA) is an inhibitor of several pyridoxal phosphate-depedent 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 mechanisms 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 ligand-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 Huntington's 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 Huntington's disease.

Chronic quinolinic acid lesions in rats closely resemble Huntington's disease.

We previously found a relative sparing of somatostatin and neuropeptide Y neurons 1 week after producing striatal lesions with NMDA receptor agonists. These results are similar to postmortem findings in Huntington's disease (HD), though in this illness there are two- to threefold increases in striatal somatostatin and neuropeptide Y concentrations, which may be due to striatal atrophy. In the present study, we examined the effects of striatal excitotoxin lesions at 6 months and 1 yr, because these lesions exhibit striatal shrinkage and atrophy similar to that occurring in HD striatum. At 6 months and 1 yr, lesions with the NMDA receptor agonist quinolinic acid (QA) resulted in significant increases (up to twofold) in concentrations of somatostatin and neuropeptide Y immunoreactivity, while concentrations of GABA, substance P immunoreactivity, and ChAT activity were significantly reduced. In contrast, somatostatin and neuropeptide Y concentrations did not increase 6 months after kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) lesions. At both 6 months and 1 yr, QA lesions showed striking sparing of NADPH-diaphorase neurons as compared with both AMPA and KA lesions, neither of which showed preferential sparing of these neurons. Long-term QA lesions also resulted in significant increases in concentrations of both 5-HT and 5-hydroxyindoleacetic acid (HIAA), similar to findings in HD. Chronic QA lesions therefore closely resemble the neurochemical features of HD, because they result in increases in somatostatin and neuropeptide Y and in 5-HT and HIAA. These findings strengthen the possibility that an NMDA receptor-mediated excitotoxic process could play a role in the pathogenesis of HD.