The actions of phencyclidine [1-(1-phenylcyclohexyl)piperidine, PCP] and its morpholine analog [1-(1-phenylcyclohexyl)morpholine, PCM] on ionic currents of nicotinic acetylcholine receptors were studied at the neuromuscular junction of frog skeletal muscle and on embryonic rat muscle cells in tissue culture. PCP and PCM reduced the peak amplitude and the decay time constant of the endplate current (EPC). PCP produced a voltage-dependent curvature and a time-dependent hysteresis loop at negative potentials (at potentials from -50 to -150 mV). In contrast, PCM caused a depression of EPC peak amplitude, but the current-voltage relationship (+60 to -150 mV) remained linear. When PCP-modified EPCs were elicited in trains at hyperpolarized potentials the amplitudes of successive events were progressively decreased and the magnitude of the decrease was dependent on the level of hyperpolarization. At positive potentials the process was reversed; the amplitude increased with successive stimulations. The EPC decayed exponentially in the presence of PCP and PCM, with a shortened time constant of decay that was less dependent on membrane potential than control. PCP and PCM caused only a 20% decrease of the amplitude of the iontophoretically evoked acetylcholine potential, which was significantly different from that induced by the desensitizing alkaloid perhydrohistrionicotoxin. Both PCP and PCM reduced by 50% the mean channel open time obtained from rat myoballs, giving a potency ratio for PCP to PCM of 2.5. This relative potency was correlated with that obtained for the reduction in the decay time constant of the EPC (ratio = 2.2). The effects of PCP on the peak amplitude of the EPC seem to be related to a conformational change of the acetylcholine receptor occurring before channel activation and not to a receptor desensitization.

Acetylcholine↗

PubMed1984

A study of the novel synthetic analog (+/-)-depentylperhydrohistrionicotoxin on the nicotinic receptor-ion channel complex.

(+/-)-Depentylperhydrohistrionicotoxin [(+/-)-depentyl-H12-HTX] is a synthetic analog of perhydrohistrionicotoxin (H12-HTX) that lacks the 5-carbon side chain. Recent studies with N-benzylazaspiro analogs of histrionicotoxin (HTX) in which both side chains are removed revealed that this alteration restricted the action of the compounds on the acetylcholine receptor-ionic channel complex (AChR) to an open channel blockade. Thus, an important question was raised as to the role of the side chains in affecting the interaction of these inhibitors with the AChR. In addition, the effect of (+/-)-depentyl-H12-HTX on membrane excitability was investigated. (+/-)-Depentyl-H12-HTX blocked the indirectly elicited twitch without affecting the directly elicited twitch. It decreased the amplitude and rate of rise and prolonged the falling phase of the action potential and blocked delayed rectification suggestive of blockade of sodium and potassium conductances. However, its effects on sodium and potassium conductances were less marked than those of HTX. It decreased the peak amplitude of the end-plate currents (EPCs) and accelerated the decay time constant of EPCs (tau EPC) in a concentration-dependent manner. The analog also induced voltage- and time-dependent nonlinearity in the current-voltage relationship of EPCs. Despite marked shortening of tau EPC, the decay phase of the EPC remained a single exponential function of time. Single channel conductance was unaffected by the analog, but the single channel lifetime was shortened. The voltage- and time-dependent effects of the analog that occurred without prior activation of AChR suggest reaction with the ionic channel in its closed conformation.

Acetylcholine↗

PubMed1984

Interaction of analogs of histrionicotoxin with the acetylcholine receptor ionic channel complex and membrane excitability.

The effects of the four N-benzylazaspiro analogs of histrionicotoxin, which are without the two side-chains typical of histrionicotoxin, were studied on the ionic channels of electrically excitable membrane and the nicotinic acetylcholine receptors in frog sartorius muscles. Each analog reversibly blocked the indirectly elicited twitch and potentiated the directly elicited twitch in a concentration-dependent manner. The analogs decreased the amplitude and rate of rise and prolonged the falling phase of the directly elicited action potential and blocked delayed rectification suggesting blockade of sodium and potassium conductances. All of the analogs caused a concentration- and voltage-dependent depression of the peak end-plate current amplitude and induced nonlinearity but no hysteresis or time dependency in the current-voltage relationship. The marked shortening of the time constant of end-plate current decay produced by the analogs was concentration-dependent. The relationship between the time constant of end-plate current decay and membrane potential remained a single exponential function of time despite the marked shortening of the decay phase and loss of voltage dependence. The effect of the analogs on miniature end-plate current was identical to that on end-plate current. Single channel conductance was unaffected by the analogs, but the single channel lifetime was shortened. The marked shortening of the time constant of the end-plate current decay and single channel lifetime plus linear relationship between reciprocal of the time constant of decay and analog concentrations strongly suggest that the analogs interact with the ionic channels of the nicotinic acetylcholine receptor in their open conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed1983

Electrophysiological and biochemical studies on enhancement of desensitization by phenothiazine neuroleptics.

The actions of the phenothiazines chlorpromazine, prochlorperazine, and trifluoperazine were studied on the acetylcholine receptor-ionic channel complex of frog and rat skeletal muscle and of Torpedo californica to determine their role in pharmacological desensitization and their interactions with different states of the receptor-ionic channel complex. The phenothiazines depressed the peak amplitude of spontaneous and evoked endplate currents while having negligible effect on the decay time constants. Mean channel lifetime and single channel conductance were not altered by these drugs. They also produced a frequency-dependent depression of the peak amplitude of endplate potentials evoked by repetitive microiontophoresis at the extrajunctional region. In addition, these drugs enhanced the ability of carbamoylcholine to displace 125I-labeled alpha-bungarotoxin from receptor-rich membrane preparations of T. californica when used in concentrations that had no effect on 125I-labeled alpha-bungarotoxin binding alone (10 microM). Similarly, the phenothiazines inhibited the binding of tritiated ionic channel ligands, such as phencyclidine and perhydrohistrionicotoxin, a process also enhanced by the presence of carbamoylcholine. These data suggest that the phenothiazines augment agonist-induced desensitization primarily by interacting with the receptor-ionic channel complex prior to channel opening.

Acetylcholine↗

PubMed1983

Potencies and channel properties induced by semirigid agonists at frog nicotinic acetylcholine receptors.

Structure-activity relationships were investigated in a series of semirigid nicotinic agonists. Three of the agonists, (-)-ferruginine methiodide, arecoline methiodide, and its ketonic analogue arecolone methiodide, were cyclic analogues of anatoxin-a, a potent, naturally occurring, bicyclic alkaloid. Two other cyclic agonists, (-)-cytisine and (+/-)-muscarone, and the simplest agonist, the tetramethylammonium ion, were also tested. Arecolone methiodide and (-)-ferruginine methiodide have been tested as nicotinic agonists for the first time. Relative potency was assayed by contracture on the rectus abdominis muscle of the frog Rana pipiens. Natural, (+)-anatoxin-a, the most active of all of the agonists, was more than twice as potent as racemic anatoxin-a. Arecolone methiodide ranked after anatoxin-a in potency, being 8.6 times more potent than carbamycholine. A correlation between nicotinic potency and steric requirements probably involves the position of positively charged groups out of the plane defined by the carbonyl group and its two substituents. Channel properties induced by the agonists were evaluated by Fourier analysis of the end-plate current noise that resulted when the agonists were iontophoretically applied to frog sartorius muscle fibers. Average channel lifetimes were exponential functions of membrane potential, but the voltage sensitivity of channel lifetime seemed to vary among the agonists. Channel conductance, which was independent of membrane potential, also varied significantly among the agonists. The average charge traversing the membrane through each open channel, calculated from the product of average channel lifetime and current, did not correlate with potency. Therefore, the dominant component of potency is the frequency of channel opening. No clear relationship between the structure of the agonist and channel lifetime or conductance was evident.

Alkaloids↗

PubMed1982

(+/-)-2-Depentylperhydrohistrionicotoxin: a new probe for a regulatory site on the nicotinic acetylcholine receptor-channel.

(+/-)-2-Depentylperhydrohistrionicotoxin (4), several of its analogues, and N- and O-substituted derivatives were prepared and tested for their effects on the neuromuscular transmission of the frog sartorius muscle. Compound 4, its N-methyl derivative 5, the O-acetyl derivative 9, and the quaternary methiodides 19 and 20 blocked the indirectly elicited twitch. The oxidation of 4 and 5 to ketones 12 and 14 and their reduction to the epimeric alcohols 17 and 18 afforded materials with substantially reduced activity. N-Acetylation of 4 to 11 changed the course of the activity to a transient potentiation of muscle twitch. Both 4 and 5 were not very toxic to mice after subcutaneous administration. (+/-)-7-n-Butyl-1-azaspiro[5,5]undecan-8-one (12) epimerized readily at room temperature to afford the epimer 13, and preparation of the hydrochloride of its N-methylated derivative 14 was accompanied by a retro-Michael reaction, affording the 2-n-butyl-3-[4-(methylamino)butyl]cyclohexene-2-one (22). The strongly hydrogen-bonded alcohol 4 was analyzed as the hydrobromide by a single-crystal X-ray analysis, confirming its structure.

Amphibian Venoms↗

PubMed1981

Anatoxin-a interactions with cholinergic synaptic molecules.

Anatoxin-a, a bicyclic amine isolated from blue-green alga, binds to the nicotinic acetylcholine receptor of Torpedo electric tissue, thereby inducing conformational changes in the postsynaptic receptor--ion channel complex as evidenced by alterations in the binding of radiolabeled ligands to the complex. Anatoxin-a binds to the acetylcholine recognition site (Kd = 0.1--0.2 microM) as indicated by its competitive inhibition of specific [3H]acetylcholine and d-[3H]tubocurarine binding, Anatoxin-a stimulates the binding of three physiologically identified "ion channel blockers," [3H]perhydrohistrionicotoxin, [3H]phencyclidine, and [3H]phencyclidine methiodide. The 50% effective doses for these effects range from 0.14 to 0.28 microM. Incubation of Torpedo membranes with anatoxin-a before addition of a radiolabeled channel probe produces a time- and concentration-dependent attenuation of the binding compared to the situation in which anatoxin-a and the probe are added simultaneously. The time course for the elaboration of this decrease corresponds to electrophysiological measurements of anatoxin-a-induced desensitization of neuromuscular junction responses. In these nicotinic actions, anatoxin-a is about as potent as acetylcholine. Anatoxin-a has relatively low affinity for the muscarinic acetylcholine receptors of rat brain, inhibiting 3-[3H]quinuclidinyl benzilate binding (10(-10) M) by 50% at concentrations between 10 and 20 microM. In contrast to classical muscarinic agonists, anatoxin-a displays little regional selectivity in its binding, and its receptor affinity is unaltered by alkylation of the neural membranes with N-ethylmaleimide.

Animals↗

PubMed1981

Differentiation of the open and closed states of the ionic channels of nicotinic acetylcholine receptors by tricyclic antidepressants.

The actions of two clinically important dibenzocycloheptane antidepressant drugs, amitriptyline and nortriptyline, were studied on ionic channels of nicotinic acetylcholine (AcCho) receptors at the neuromuscular junction of frog skeletal muscle. Amitriptyline (5-10 microM) and nortriptyline (1-2 microM), like imipramine (5-10 microM), did not react with the nicotinic AcCho receptor but caused a voltage- and time-dependent decrease in the peak amplitude of the endplate current (epc). The time constant of epc decay, however, retained its voltage sensitivity. The voltage- and time-dependent effect of amitriptyline was nonlinear with regard to the current/voltage (I/V) relationship. Nortriptyline also had a more pronounced voltage- and time-dependent effect evidenced by a hysteresis loop in the I/V relationship of the epc was eliminated by the use of 50-msec stepwise changes of the membrane potential. The nonlinearity and hysteresis were due to a time-dependent phenomenon and did not involve previous AcCho receptor activation. The rate constant of the voltage- and time-dependent decrease in epc amplitude was sensitive to the membrane electric field and varied linearly with the membrane potential. Iontophoretically elicited epcs were much more depressed by both drugs than were spontaneous miniature epcs. There was no effect on the time constant of miniature epc decay, single-channel lifetime, or conductance. Thus (as we have pointed out in our histrionicotoxin studies) the primary site of action of these agents presumably is the activated but nonconducting species of the ionic channel of the nicotinic AcCho receptor. These agents, particularly nortriptyline, point to several different binding sites of the ionic channel and are suitable tools for the separation of the effects on peak current amplitude from its time constant of decay.

Amitriptyline↗

PubMed1980

Phencyclidine interactions with the ionic channel of the acetylcholine receptor and electrogenic membrane.

The effects of phencyclidine (PCP) were studied on the electrogenic and chemosensitive properties of the neuromuscular junction of skeletal muscle as well as on the binding sites on the acetylcholine (AcCho) receptor and its ionic channel in the electric organ membranes of the electric ray. The directly elicited muscle twitch was markedly potentiated by prolonging the falling phase of the muscle action potential and blocking delayed rectification. The indirectly elicited muscle twitch was transiently potentiated and then blocked by PCP at concentrations below 60 muM. PCP blocked miniature endplate potentials and AcCho sensitivities at the junctional region of innervated muscle, blocked the extrajunctional sensitivity of the chronically denervated muscle, and significantly depressed the peak amplitude of the endplate current (EPC) in a voltage- and time-dependent manner. PCP also caused acceleration of the time course of EPC decay and shortening of the mean life-time of the open ionic channel. The effects of PCP were not due to inhibition of AcCho receptor sites because PCP did not protect against the quasi-irreversible inhibition of receptor sites by alpha-bungarotoxin, nor did it inhibit binding of [(3)H]AcCho or [(125)I-labeled alpha-bungarotoxin to the receptor sites. On the other hand, PCP blocked the binding of [(3)H]perhydrohistrionicotoxin to the sites of the ionic channel of the AcCho receptor. The data suggest that PCP reacts with the electrogenic K(+) channel and the ionic channel associated with the AcCho receptor in the open as well as the closed conformation.

Action Potentials↗

PubMed1980

A novel chiral microenvironmental probe at the active site of trypsin. Extrinsic cotton effects of acyl-trypsin possessing an enantiomeric pair of chromophores.

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made it possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-trypsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD spectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments.

Acylation↗

PubMed1980

Anatoxin-a: a novel, potent agonist at the nicotinic receptor.

Explore the source record for details and available documents.

Action Potentials↗

PubMed1978

Acetylcholine receptor and ionic channel of Torpedo electroplax: binding of perhydrohistrionicotoxin to membrane and solubilized preparations.

The electric organ of the ray, Torpedo ocellata, can serve as a source for both the acetylcholine (ACh) receptor and its ionic channel. The two entities were identified by their specific binding of [3H]ACh and [3H]perhydrohistrionicotoxin ([3H]H12-HTX), respectively. Binding of [3H]H12-HTX was inhibited by certain drugs and toxins, e.g., histrionicotoxin (HTX), amantadine, and tetraethylammonium (TEA) ions at concentrations that did not inhibit [3H]ACh binding. However, the specific carbamoylcholine-induced 22Na efflux from microsacs from the electric organ membranes was blocked by inhibitors of either the receptor or its ionic channel. The ionic channel had the properties of a protein as judged by heat sensitivity and the inhibition of [3H]H12-HTX binding, after incubation of the electric organ membranes with protein reagents such as p-chloromercuribenzenesulfonic acid (PCMBS) or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The "binding" of [3H]H12-HTX at 4 X 10(-8) M to lipids in the microsacs was 12% of the total binding to intact microsacs and was nonsaturable and insensitive to heat or specific drugs. After solubilization with cholate, the [3H]H12-HTX binding subunits retained the same affinities for toxins and drugs. The Kd for [3H]H12-HTX was 3 X 10(-7) M. The majority of the ionic channel could be separated from the ACh receptors in the cholate extract by incubation with ACh-receptor affinity gel and ACh-receptor antibodies. The ACh receptor purified by this affinity gel contained only a few active ionic channel units as judged by low levels of high affinity binding of [3H]H12-HTX. On the other hand, after solubilization with Triton X-100, all the ionic channel molecules were either separated or denatured so that the purified ACh receptor did not exhibit high affinity binding for [3H]H12-HTX.

Amphibian Venoms↗