Gating properties of a sodium channel with three arginines substituted by histidines in the central part of voltage sensor S4D4.

In voltage-dependent sodium channels there is some functional specialization of the four different S4 voltage sensors with regard to the gating process. Whereas the voltage sensors of domains 1 to 3 control activation gating, the movement of the voltage sensor of domain 4 (S4D4) is known to be tightly coupled to sodium channel inactivation, and there is some experimental evidence that S4D4 also participates in activation gating. To further explore its putative multifunctional role in the gating process, we changed the central part of S4D4 in rat brain IIA (rBIIA) sodium channels by the simultaneous replacement of the third (R1632), fourth (R1635) and fifth (R1638) arginine by histidine (mutation R3/4/5H). As a result, the time course of current decay observed in R3/4/5H was about three times slower, if compared to wild type (WT). On the other hand, the recovery, as well as the voltage dependence of fast inactivation, remained largely unaffected by the mutation. This suggests that at physiological pH (7.5) the effective charge of the voltage sensor was not significantly changed by the amino-acid substitutions. The well-known impact of site-3 toxin (ATX-II) on the inactivation was drastically reduced in R3/4/5H, without changing the toxin affinity of the channel. The activation kinetics of WT and R3/4/5H studied at low temperature (8 degrees C) were indistinguishable, while the inactivation time course of R3/4/5H was then clearly more slowed than in WT. These data suggest that the replacement of arginines by histidines in the central part of S4D4 clearly affects the movement of S4D4 without changing the activation kinetics.

Mutation D384N alters recovery of the immobilized gating charge in rat brain IIA sodium channels.

Rat brain (rBIIA) sodium channel fast inactivation kinetics and the time course of recovery of the immobilized gating charge were compared for wild type (WT) and the pore mutant D384N heterologously expressed in Xenopus oocytes with or without the accessory beta1-subunit. In the absence of the beta1-subunit, WT and D384N showed characteristic bimodal inactivation kinetics, but with the fast gating mode significantly more pronounced in D384N. Both, for WT and D384N, coexpression of the beta1-subunit further shifted the time course of inactivation to the fast gating mode. However, the recovery of the immobilized gating charge (Qg) of D384N was clearly faster than in WT, irrespective of the presence of the beta1-subunit. This was also reflected by the kinetics of the slow Ig OFF tail. On the other hand, the voltage dependence of the Qg-recovery was not changed by the mutation. These data suggest a direct interaction between the selectivity filter and the immobilized voltage sensor S4D4 of rBIIA sodium channels.

Variable ratio of permeability to gating charge of rBIIA sodium channels and sodium influx in Xenopus oocytes.

Whole-cell gating current recording from rat brain IIA sodium channels in Xenopus oocytes was achieved using a high-expression system and a newly developed high-speed two-electrode voltage-clamp. The resulting ionic currents were increased by an order of magnitude. Surprisingly, the measured corresponding gating currents were approximately 5-10 times larger than expected from ionic permeability. This prompted us to minimize uncertainties about clamp asymmetries and to quantify the ratio of sodium permeability to gating charge, which initially would be expected to be constant for a homogeneous channel population. The systematic study, however, showed a 10- to 20-fold variation of this ratio in different experiments, and even in the same cell during an experiment. The ratio of P(Na)/Q was found to correlate with substantial changes observed for the sodium reversal potential. The data suggest that a cytoplasmic sodium load in Xenopus oocytes or the energy consumption required to regulate the increase in cytoplasmic sodium represents a condition where most of the expressed sodium channels keep their pore closed due to yet unknown mechanisms. In contrast, the movements of the voltage sensors remain undisturbed, producing gating current with normal properties.

Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization.

The highly charged transmembrane segments in each of the four homologous domains (S4D1-S4D4) represent the principal voltage sensors for sodium channel gating. Hitherto, the existence of a functional specialization of the four voltage sensors with regard to the control of the different gating modes, i.e., activation, deactivation, and inactivation, is problematic, most likely due to a functional coupling between the different domains. However, recent experimental data indicate that the voltage sensor in domain 4 (S4D4) plays a unique role in sodium channel fast inactivation. The correlation of fast inactivation and the movement of the S4D4 voltage sensor in rat brain IIA sodium channels was examined by site-directed mutagenesis of the central arginine residues to histidine and by analysis of both ionic and gating currents using a high expression system in Xenopus oocytes and an optimized two-electrode voltage clamp. Mutation R1635H shifts the steady state inactivation to more hyperpolarizing potentials and drastically increases the recovery time constant, thereby indicating a stabilized inactivated state. In contrast, R1638H shifts the steady state inactivation to more depolarizing potentials and strongly increases the inactivation time constant, thereby suggesting a preferred open state occupancy. The double mutant R1635/1638H shows intermediate effects on inactivation. In contrast, the activation kinetics are not significantly influenced by any of the mutations. Gating current immobilization is markedly decreased in R1635H and R1635/1638H but only moderately in R1638H. The time courses of recovery from inactivation and immobilization correlate well in wild-type and mutant channels, suggesting an intimate coupling of these two processes that is maintained in the mutations. These results demonstrate that S4D4 is one of the immobilized voltage sensors during the manifestation of the inactivated state. Moreover, the presented data strongly suggest that S4D4 is involved in the control of fast inactivation.

A novel protein tag from herpes simplex virus type 1 DNA polymerase.

An epitope (HPOL) derived from the so-called thumb region of the herpes simplex virus type 1 DNA polymerase in combination with a monoclonal antibody (MAb 1051c) was tested for protein tagging. Using a conventional expression vector, a DNA cassette encoding the HPOL epitope was fused to the C-terminus of the dihydrofolate reductase (DHFR) gene such that the recombinant DHFR contained both a N-terminal HIS-tag and a C-terminal HPOL tag. Expression of recombinant DHFR in Escherichia coli cells was compared by Western blot analysis using either mouse RGS.HIS antibody or MAb 1051c. Immunostaining revealed that both antibodies reacted specifically with DHFR, but the detection sensitivity achieved with MAb 1051c was about 15-fold greater using a standard staining protocol. An HPOL antibody column was successfully applied for affinity purification of DHFR, demonstrating the usefulness of the HPOL epitope/MAb 1051c system for protein tagging, expression monitoring and purification of HPOL-tagged recombinant proteins.

DNA and protein interactions of the small subunit of herpes simplex virus type 1 DNA polymerase.

Herpes simplex virus DNA polymerase (HSV pol) holoenzyme consists of a large catalytic (UL30 gene product) and a small auxiliary subunit (UL42 gene product). The DNA binding of HSV pol, its cofactor, and the assembled holoenzyme complex was studied by bandshift analysis using purified proteins expressed via recombinant baculovirus. The functional activity of the recombinant UL42, purified by phenyl-Sepharose chromatography, was confirmed by its ability (1) to convert the salt sensitivity of both, 3'-5' exonuclease and polymerase, activities of HSV pol and (2) to enhance the processivity of polymerization. Bandshift analyses revealed that the HSV pol holoenzyme-DNA complex was stably formed up to a salt concentration of 50 mM ammonium sulfate, indicating that the restricted DNA and protein interactions of both HSV pol and UL42 are responsible for the observed salt preference of the HSV pol holoenzyme under standard assay conditions in vitro. Studies of the assembly of the holoenzyme complex demonstrated that initial DNA binding of HSV pol was advantageous for the formation of the HSV pol holoenzyme-DNA complex.

Herpes simplex virus type 1 DNA polymerase. Mutational analysis of the 3'-5'-exonuclease domain.

Like true DNA replicases, herpes simplex virus type 1 DNA polymerase is equipped with a proofreading 3'-5'-exonuclease. In order to assess the functional significance of conserved residues in the putative exonuclease domain, we introduced point mutations as well as deletions within and near the conserved motifs' exonuclease (Exo) I, II, and III of the DNA polymerase gene from a phosphonoacetic acid-resistant derivative of herpes simplex virus-1 strain ANG. We examined the catalytic activities of the partially purified enzymes after overexpression by recombinant baculovirus. Mutations of the motifs' Exo I (D368A, E370A) and Exo III (Y577F, D581A) yielded enzymes without detectable and severely impaired 3'-5'-exonuclease activities, respectively. Except for the Exo I mutations, all other Exo mutations examined affected both exonuclease and polymerization activities. Mutant enzymes D368A, E370A, Y557S, and D581A showed a significant ability to extend mispaired primer termini. Mutation Y557S resulted in a strong reduction of the 3'-5'-exonuclease activity and in a polymerase activity that was hyperresistant to phosphonoacetic acid. The results of the mutational analysis provide evidence for a tight linkage of polymerase and 3'-5'-exonuclease activity in the herpesviral enzyme.

WAL 2014--a muscarinic agonist with preferential neuron-stimulating properties.

The ability of WAL 2014 to elicit muscarinic responses was investigated in various in vitro and in vivo models. In CHO cells transfected with human m1- or m3- receptor genes, WAL 2014 was clearly more effective in stimulating the M1-mediated PI response. In isolated tissue preparations, WAL 2014 exhibited full agonist properties in the rabbit vas deferens (putative M1 receptor) and behaved like a partial agonist at M2 receptors in the atrium and M3 receptors in the ileum of guinea-pigs. In the pithed rat, in which the increase in blood pressure is mediated through a stimulation of M1 receptors in sympathetic ganglia, WAL 2014 produced a full dose response curve, whereas the reference compounds RS 86 and arecoline exhibited a bell-shaped behaviour. This is in accord with the view that WAL 2014 selectively activates M1 receptors in sympathetic ganglia, whereas conventional agonists in the same dose range stimulate both ganglionic M1 and vascular M3 receptors. The preferential neuron-stimulating properties were confirmed by EEG recording in the rabbit, in which muscarinic activation occurred at doses similar to those for ganglionic stimulation in the pithed rat. On the other hand, higher doses of WAL 2014 were needed to elicit muscarinic effects in peripheral effector organs, i.e. bradycardia, urinary bladder contraction and increase in airway resistance. It is concluded that WAL 2014 due to its preferential neuronal activity is a promising candidate for a cholinergic substitution therapy in Alzheimer's disease.

The ERG as a complementary diagnostic tool in ophthalmoscopy in albino rats.

Using light induced retinal damage in albino rats as a model, the time of occurrence of lesions was investigated by ophthalmoscopy, electroretinography and light microscopy. Changes in the electroretinogram correlated well with histopathological lesions in the first retinal neuron. In contrast, ophthalmoscopy revealed no evidence of retinal damage even at a time, when the animals were apparently blind. It is concluded that electroretinography is an appropriate experimental tool for detection of retinal damage at a very early stage in toxicological investigation.

In vitro and in vivo studies of the non-sedating antihistamine epinastine.

Epinastine (3-amino-9,13b-dihydro-1H-dibenz [c,f]imidazo[1,5-a]azepine hydrochloride, WAL 801 CL) was tested in vitro and in vivo in comparison with other H1-receptor antagonists. In the guinea pig ileum and in receptor binding studies the test substance showed a high affinity to H1-receptors. The following rank order was determined: WAL 801 CL greater than astemizole greater than terfenadine. These results were confirmed in vivo. The studies were carried out with oral and intravenous administration of WAL 801 CL to assess the inhibition of histamine-induced reactions in the skin or the lung of rats, dogs and guinea pigs. 10- to 100fold antihistaminic doses of WAL 801 CL showed no effect on the sleeping-waking behaviour of cats. From this and other results it is suggested that the compound does not penetrate in the central nervous system. The action pattern of WAL 801 CL as a non-sedating antihistamine corresponds more to that of terfenadine than that of ketotifen.

Pharmacology of WEB 1881-FU, a central cholinergic agent, which enhances cognition and cerebral metabolism.

The pharmacological effects of the novel compound WEB 1881 FU (4-amino-methyl-1-benzyl-pyrrolidine-2-one-fumarate) were investigated. The tests performed indicate that the compound has cytoprotective as well as metabolism and cognition enhancing and central cholinomimetic properties. The nootropic effects in all tests were more pronounced than those of piracetam, while the central cholinomimetic effects were generally weaker than those of directly acting cholinomimetic agents. However, the typical peripheral cholinergic side effects were not observed. From the results we believe that the stimulating effect of WEB 1881 FU upon the central cholinergic system is modulatory rather than direct. The combination of nootropic and cholinomimetic properties appears favorable for treatment of brain dysfunction in the elderly. Side effects are less serious than with other known cholinomimetics.

Effects of brotizolam on the sleep-waking cycle of the cat.

The effects of brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) on the sleep-waking behavior of freely moving cats was investigated by electroencephalographic and behavioral methods. In the cat brotizolam and triazolam increased the time asleep. With diazepam, sleep enhancement could be observed only within a very limited period. After nitrazepam and flurazepam sleep was decreased. REM-sleep was not changed with 0.1 mg/kg brotizolam, slightly reduced after 0.3 and moderately shortened with 1 mg/kg. After triazolam REM-sleep was reduced to a higher degree than after brotizolam. The experiments show, that brotizolam has sleep-inducing properties in the cat and is in this respect different from other diazepine derivatives.

General pharmacology of brotizolam in animals.

Brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) is a thienotriazolo-diazepine with profound sedative and hypnogenic properties. The side effects of the drug on general behavior, motocoordination, feeding pattern, body temperature, uropoietic and gastrointestinal functions, cardiovascular system, and respiration, as well as interactions with some biogenic amines are reported and discussed. The findings correlate with those known for other diazepines. Accordingly, effects on motocoordination were prominent, but were limited to an ataxia, whereas even extremely high doses scarcely eliminated the postural reflexes. Sleeping animals could invariably be woken and were capable of locomotion; thus, no comatose condition developed. The cardiovascular functions were not appreciably altered by brotizolam in anesthetized cats, while in conscious dogs minor fluctuations of blood pressure and heart rate occurred. Respiration was clearly inhibited when brotizolam was given intravenously. The cardiovascular effects of acetylcholine, norepinephrine, epinephrine, isoprenaline, and histamine were only slightly modulated. The orexigenic and hypothermic effects equalled those of other diazepines. The functions of kidney, stomach, and intestines were not affected. The entirety of the observations procured in ten different species suggest that brotizolam is well tolerated when given orally.

Antiemotional and anticonvulsant activity of brotizolam and its effects on motor performance in animals.

Brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) is a thienotriazolo diazepine with predominantly sleep-inducing properties. Additionally, brotizolam, attenuated conflict behavior in rats and inhibited aggressive behavior in mice and cats. Brotizolam prevented audiogenic seizures in mice, seizures provoked by electroshock in rats and inhibited convulsions elicited by electrical stimulation in the limbic system of cats. Furthermore, brotizolam antagonized seizures induced by the convulsant drugs pentetrazol, bicuculline and strychnine in mice. Motocoordination was not impaired within the effective dose range. Muscle relaxant effects appeared at higher doses only. The onset of effect of brotizolam occurred in the different experiments within 15-30 min, thus indicating a fast enteral absorption and penetration of the blood-brain barrier. The duration of action within the therapeutic dose range was between 2-6 h. The effect of brotizolam was compared with other diazepine derivatives. Brotizolam was more active than diazepam, nitrazepam, estazolam, flurazepam and clonazepam and nearly as active as triazolam.

Pharmacological and neurochemical properties of 1,4-diazepines with two annelated heterocycles ('hetrazepines').

1,4-Diazepines with two annelated heterocycles ('hetrazepines') such as brotizolam (WE 941), WE 973 and WE 1008 bind with high affinities to benzodiazepine receptors in the central nervous system. Brotizolam has a pharmacologic spectrum of action similar to clinically useful benzodiazepines, while the closely related derivatives WE 973 and WE 1008 appear to lack hypnotic action. Unlike other benzodiazepine receptor ligands which share common pharmacologic properties with the benzodiazepines, the apparent affinities of WE 973 and WE 1008 are not increased significantly in the presence of GABA, even at an elevated incubation temperature. Furthermore, the apparent affinities of these compounds do not appear to be reduced as a result of increasing the incubation temperature. Brotizolam, like the benzodiazepines, facilitates GABAergic transmission in zona recitulata neurons of the substantia nigra. In contrast, at a dose which inhibits cell firing, WE 973 does not appear to significantly augment the inhibitory action of GABA in these cells. These observations suggest that the so-called 'GABA shift' may not be a valid means of distinguishing benzodiazepine-like compounds in vitro. Furthermore, these data suggest that facilitation of GABAergic transmission may be necessary for the hypnotic action of benzodiazepine receptor ligands, but not for the anticonflict or the anticonvulsant actions of such compounds.

Pharmacology and hypnogenic properties of brotizolam in animals.

Brotizolam differs in pharmacological profile from other diazepines by virtue of its hypnogenic potency. It increases, whereas other diazepines reduce, sleep in the cat. With increasing doses, the latency to rapid eye movement sleep is lengthened and the proportion is reduced. Brotizolam has anxiolytic, anticonvulsant and muscle relaxant properties. The effective anxiolytic and muscle relaxant doses are somewhat lower than those of diazepam, and the effective anti-convulsant dose is ten times lower. Barbiturate synergism in mice is lower with brotizolam than with diazepam, while alcohol-induced coma is prolonged over the same dose range. Side-effects of brotizolam are similar to those of other diazepines, but the therapeutic range is more favourable. Physical dependence as tested in the monkey seems to be low.

[General pharmacology and secretion inhibitory action of (8r)-3alpha-hydroxy-8-isopropyl-1alphaH,5alphaH-tropanium bromide-(+/-)-tropate (ipratropiumbromide) (author's transl)]. / Allgemeine Pharmakologie und sekretionshemmende Wirkung von (8r)-3alpha-Hydroxy-8-isopropyl-1alphaH,5alphaH-tropaniumbromid-(+/-)-tropat (Ipratropiumbromid)

(8r)-3alpha-Hydroxy-8-isopropyl-1alphaH,5alphaH-tropaniumbromide-(+/-)-tropate (ipratropiumbromide, Sch 1000) is a quaternary tropic acid tropane ester with pronounced anticholinergic activities (inhibition of secretion and spasmolysis). The effect of the substance of all parasympathetically innervated organs ist 1.4 to 2 times stronger than that of atropine. As an inhibitor of the secretion of free hydrochloric acid in the stomach it proved to be 5 times more effective than atropine. Central activities were not observed. The duration of action of Sch 1000 exceeds that of atropine by far.

[Study on novel morphine antagonists in the animal experiment (author's transl)]. / Prüfung einiger neuer Morphinantagonisten im Tierexperiment

The morphine-antagonistic properties of 5 novel compounds, (--)N-(3-Furylmethyl)-nordesomorphine-hydrochloride-monohydrate (Wa 494-Cl), (--)N-(3-Furylmethyl)-3-hydroxy-morphinan-methansulfonate (Mr 1257 MS), (--)2-(3-Furylmethyl)-2'-hydroxy-5,9alpha-dimethyl-6,7-benzomorphan-methansulfonate (Mr 1452 MS), (+/-)-2-(3-Furylmethyl)-2'-hydroxy-5,9alpha-diethyl-6,7-benzomorphan-hydrochloride (Mr 1302 MS), (--)-N-(3-Furylmethyl)-noroxymorphon-methanesulfonate (Mr 1767 MS), have been examined with different methods on mice and cats. Their properties have been compared with those of naloxone, cyclazocine and nalorphine. The new test compounds showed a profile of action like naloxone.