Elucidation of the mechanism and site of action of quinuclidinyl benzilate (QNB) on the electrical excitability and chemosensitivity of the frog sartorius muscle

The effects of the muscarinic antagonist quinuclidinyl benzilate (QNB) on transmission at the frog sartorius neuromuscular junction have been examined. QNB decreases endplate potential (EPP) amplitude without affecting miniature endplate (MEPP) frequency or resting potential. QNB also increased the latency of the EPP and the nerve terminal spike in a frequency dependent fashion, suggesting the site of action is the unmyelinated nerve terminal. Since the rate of rise and amplitude of muscle action are potentials decreased it is likely that QNB causes a blockade of electrically excitable sodium channels; the agent also blocks ionic channels associated with nicotinic acetylcholine receptors. It is possible that these effects of QNB may explain some of the behavioral disturbances produced by its administration.     

Synaptic transmission: ion concentration changes in the synaptic cleft.

Currents flowing through the postsynaptic membrane of an active synapse will tend to change the concentrations of ions in the synaptic cleft. Published experimental data are used to predict (a) the sodium and potassium concentration changes in the cleft at the frog neuromuscular junction, and (b) the sodium depletion in the cleft under a Ia synaptic bouton on a cat motoneuron. Significant concentration changes are predicted at both synapses. These changes will contribute to the time dependence of the observed current and will cause the reversal potential of the current to be time dependent. At the frog neuromuscular junction, the time course of the endplate current has been shown previously to depend on the magnitude of the current flowing (at a given potential). We attribute this to changes of the cleft ion concentration. The time dependent changes of the endplate current reversal potential that we predict for the neuromuscular junction are probably too small to be detected. This is because the effects of sodium depletion and potassium accumulation on the reversal potential almost cancel. We predict that near the reversal potential small currents of complex time course will remain, i.e. no true reversl potential exists. Such currents have previously been experimentally. At the cat Ia synapse, the synaptic current is predicted to deplete a significant fraction of the available extracellular sodium ions. Consequently, the magnitude of the synaptic current should be relatively independent of the number of postsynaptic channels activated, and of the membrane potental, as has previously been found experimentally.     

Modification of electroplax excitability by veratridine

Veratridine influences membrane-potential changes arising both from the action potential and from the application of external cholinergic agonists in the isolated monocellular electroplax preparation. The action potential shows a long depolarizing after-potential in the presence of veratridine. The effects of various pharmacological agents and of external ion changes on this after-potential are similar to those reported for other nerve and muscle fibers and are consistent with the view that veratridine acts chiefly to increase the Na⁺ conductance.Membrane depolarizations by cholinergic agonists are inhibited by veratridine at pH 7 but strikingly amplified at pH 9. The former effect appears to involve interaction with the cholinergic receptor at the surface of the membrane, while the latter potentiation parallels the increase in the spike after-potential at pH 9 and presumably arises from a Na⁺ conductance increase.Veratridine appears to interact with the component involved in the Na⁺ conductance in the interior membrane phase. The possible localization of this component in both the conducting and synaptic membrane is discussed.     

Effects of 4-aminopyridine on the action potential and the after-hyperpolarization of cat spinal motoneurons

In cats under pentobarbital anaesthesia, intramotoneuronal administrations of 4-aminopyridine significantly prolong the falling phase of the antidromic action potential but have much less effect on the orthodromic action potential. 4-aminopyridine probably blocks the fast K channels involved in the repolarization of the membrane and indirectly activates ionic channels through enhancement of synaptic transmission, also suggested by the potentiation of excitatory postsynaptic potentials. In many cells, 4-aminopyridine depresses the amplitude and prolongs the time course of the after-hyperpolarization; therefore 4-aminopyridine may also partly block Ca2+-activated K+ channels.     

Halothane-induced changes in acetylcholine receptor channel kinetics are attenuated by cholesterol

The single-channel recording technique was used to investigate the role of membrane lipids in the action of general anesthetics on ion channels. We examined the effects of halothane on acetylcholine receptor channels in Xenopus laevis myocytes in which the plasma membrane cholesterol level had been changed by pretreatment with cholesterol-rich or cholesterol-free liposomes. We found that the alteration in acetylcholine receptor channel kinetics, elicited in the presence of clinically-relevant concentrations of halothane, is attenuated when membrane cholesterol is increased and enhanced when membrane cholesterol concentration is decreased. These findings support the idea that general anesthetics interact with synaptic receptor channels indirectly through the lipid domains in which these synaptic proteins are embedded.     

Putative synaptic mechanisms of inhibition in Limulus lateral eye

Serotonin (5-HT) perfusion of a thin section of Limulus lateral eye hyperpolarizes retinular and eccentric cell membrane potential, and blocks spike action potentials fired by the eccenteric cell. The indoleamine does not directly affect retinular cell receptor potential or eccenteric cell generator potential in response to light stimuli. LSD perfusion blocks both this inhibitory action of 5-HT and light-evoked, synaptically mediated, lateral inhibition. Iontophoretic application of 5-HT to the synaptic neuropil produces shorter latency and duration and larger amplitude of inhibition than does the perfusion technique. This inhibition is dose dependent; the accompanying inhibitory postsynaptic potential (IPSP) appears to have an equilibrium potential more hyperpolarized than normal resting potential levels of ca. -50 mV. IPSP amplitude is sensitive to extracellular potassium ion concentration: it increases with decreased [K+]0 and decreases with increased [K+]0. LSD blocks the inhibition produced by iontophoretic application of 5-HT. Interaction between light-evoked, natural synaptic transmitter-mediated IPSP's and 5-HT IPSP's suggests a common postsynaptic receptor or transmitter-receptor-permeability change mechanism.     

Effect of electrolyte composition of various solutions on potential-sensitive ion channels, formed by syringomycin E in lipid bilayers]

Using the planar lipid bilayer technique, the dependence of properties of ion channels formed by syringomycin E on bath ion composition (electrolyte concentration and pH) and on the applied potential was studied. Time courses of the membrane current in field reversal experiments with 1M NaCl, pH 6 in the bathing solution were similar to the time courses observed with a bathing solution of 0.1 M NaCl, pH 2: there was no increase (decrease) of the membrane current at positive (negative) values of the transmembrane potential. However, kinetic curves were different at 0.1 M. NaCl, pH 6 and pH 9. At pH 6 there was an abrupt increase (decrease) of the membrane current over the time at the positive (negative) values of the applied potential. At pH 9 there was a reversed current response. The results indicate that charged groups are likely to be responsible for opening or closing the channel facing the water phase. Based on these data, a model is proposed describing the potential dependent opening and closing of ion channels formed by syringomycin E.     

A network model for biofilm development in Escherichia coli K-12

Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl^--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts. In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl^-. Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl^- ions needs to be >20 times higher outside the cell. When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.     

Fine‐scale spatial variability in anatoxin‐a and homoanatoxin‐a concentrations in benthic cyanobacterial mats: implication for monitoring and management

Aims: The purpose of this study was to determine the variability in anatoxin‐a (ATX) and homoanatoxin‐a (HTX) concentrations in benthic cyanobacterial mats within sampling sites and to assess the applicability of using a PCR‐based approach to determine ATX‐ and HTX‐production potential. Methods and Results: ATX and HTX variability was investigated by collecting 15 samples from 10 × 10 m grids in seven rivers. ATX and HTX concentrations were determined using liquid chromatography–mass spectrometry (LC–MS). Samples from two sites contained no ATX or HTX and at one site ATX and HTX were detected in all samples. At four sites, both toxic and nontoxic samples co‐occurred and these samples were sometimes spaced less than 1 m apart. PCR amplification of a region of a polyketide synthase (ks2, putatively involved in the biosynthetic pathway of ATX and HTX) successfully distinguished ATX‐and‐HTX‐ and non‐ATX‐and‐HTX‐producing cultured Phormidium strains. Results from environmental samples were more variable, and the results were in congruence with the LC–MS data in only 58% of samples. Conclusions: Fine‐scale spatial variability in ATX and HTX concentrations occurs among benthic cyanobacterial mats. Significance and Impact of the Study: Multiple benthic cyanobacterial mat samples must be collected at a sampling site to provide an accurate assessment of ATX and HTX concentrations at that location. The PCR‐based technique offers the potential to be a useful early warning technique.     

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

Mechanism of pyrithione-induced membrane depolarization in Neurospora crassa.

Pyrithione is a general inhibitor of membrane transport in fungi and is widely used in antidandruff shampoos as an antifungal agent. An electrophysiological approach has been used to determine the mode of action of pyrithione on the plasma membrane of the model ascomycete, Neurospora crassa. At pH 5.8, pyrithione induces a dramatic dose-dependent electrical depolarization of the membrane which is complete within 4 min, amounts to 110 mV at saturating pyrithione concentrations, and is half maximal between 0.6 and 0.8 mM pyrithione. Zinc pyrithione induces a similar response but exerts a half-maximal effect at around 0.3 mM. The depolarization is strongly dependent on external pH, being almost absent at pH 8.2, at which the concentration of the uncharged form of pyrithione--which might be expected to permeate the membrane freely--is markedly lowered. However, quantitative considerations based on cytosolic buffer capacity, the pKa of pyrithione, and the submillimolar concentration at which it is active appear to preclude significant cytosolic acidification on dissociation of the thiol proton from the uncharged form of pyrithione. Current-voltage analysis demonstrates that the depolarization is accompanied by a decrease in membrane electrical conductance in a manner consistent with inhibition of the primary proton pump and inconsistent with a mode of action of pyrithione on plasma membrane ion channels. We conclude that pyrithione inhibits membrane transport via a direct or indirect effect on the primary proton pump which energizes transport and that the site of action of pyrithione is likely to be intra- rather than extracellular.     

Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide

A COOH-terminal tryptic fragment (Mr approximately equal to 20,000) of colicin E1 has been proposed to contain the membrane channel-forming domain of the colicin molecule. A comparison is made of the conductance properties of colicin E1 and its COOH-terminal fragment in planar bilayer membranes. The macroscopic and single channel properties of colicin E1 and its COOH-terminal tryptic fragment are very similar, if not indistinguishable, implying that the NH2-terminal, two-thirds of the colicin E1 molecule, does not significantly influence its channel properties. The channel-forming activity of both polypeptides is dependent upon the presence of a membrane potential, negative on the trans side of the membrane. The average single channel conductance of colicin E1 and the COOH-terminal fragment is 20.9 +/- 3.9 and 19.1 +/- 2.9 picosiemens, respectively. The rate at which both proteins form conducting channels increases as the pH is lowered from 7 to 5. Both molecules require negatively charged lipids for activity to be expressed, exhibit the same ion selectivity, and rectify the current to the same extent. Both polypeptides associate irreversibly with the membrane in the absence of voltage, but subsequent formation of conducting channels requires a negative membrane potential.     

Small iminium ions block gramicidin channels in lipid bilayers.

Guanidinium and acetamidinium, when added to the bathing solution in concentrations of approximately 0.1M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.     

Kinetics of the slow variation of peak sodium current in the membrane of myelinated nerve following changes of holding potential or extracellular pH.

(1) Changes of the holding potential applied to the membrane of myelinated nerve fibres induced slow variations of the peak sodium current, which are super-imposed on the effect of sodium inactivation. (2) These slow variations are transitions between various steady levels of available sodium conductance. Their time course can be described by the function erfc (square root t/tau) where tau is the time and erfc the error function complement. The characteristic time tau lies in the range 2-4 min and depends on the membrane potential. (3) Changes of extracellular pH cause a rapid change of the peak sodium current followed by a slow variation as observed after changes of the holding potential. This slow variation can be prevented by applying simultaneously an appropriate change of the holding potential, e.g. the effect of changing pH from 7.3 to 5.3 is balanced by changing the potential from --70 to --55 mV. (4) The results are interpreted by postulating charged components diffusion slowly within the nodal membrane. Their transverse distribution controls the number of sodium channels available at a given membrane potential. The equivalence between change of pH and voltage is explained by assuming negative fixed charges at the outer surface of the membrane, which are protonated at low pH and thus affect the intrinsic membrane potential. (5) It is concluded that effects which are ascribed to the action of agents on individual sodium channels have to be corrected for variations in the number of available channels if these agents influence the intrinsic membrane potential, e.g. changes of extracellular pH.     

Active dendrites,potassium channels and synaptic plasticity

The dendrites of CA1 pyramidal neurons in the hippocampus express numerous types of voltage-gated ion channel, but the distributions or densities of many of these channels are very non-uniform. Sodium channels in the dendrites are responsible for action potential (AP) propagation from the axon into the dendrites (back-propagation); calcium channels are responsible for local changes in dendritic calcium concentrations following back-propagating APs and synaptic potentials; and potassium channels help regulate overall dendritic excitability. Several lines of evidence are presented here to suggest that back-propagating APs, when coincident with excitatory synaptic input, can lead to the induction of either long-term depression (LTD) or long-term potentiation (LTP). The induction of LTD or LTP is correlated with the magnitude of the rise in intracellular calcium. When brief bursts of synaptic potentials are paired with postsynaptic APs in a theta-burst pairing paradigm, the induction of LTP is dependent on the invasion of the AP into the dendritic tree. The amplitude of the AP in the dendrites is dependent, in part, on the activity of a transient, A-type potassium channel that is expressed at high density in the dendrites and correlates with the induction of the LTP. Furthermore, during the expression phase of the LTP, there are local changes in dendritic excitability that may result from modulation of the functioning of this transient potassium channel. The results support the view that the active properties of dendrites play important roles in synaptic integration and synaptic plasticity of these neurons.     

Assessment of uncoupling by amiloride analogs.

The amiloride analogs N5-methyl-N5-isobutylamiloride, N5-ethyl-N5-isopropylamiloride, and N5,N5-hexamethyleneamiloride are frequently used to investigate NaH exchange on the premise that they are highly specific inhibitors of the NaH-antiporters. We assessed the relative protonophoric activity of these compounds in reconstituted and native membrane vesicles, using acridine orange fluorescence to measure intravesicular pH. All the compounds tested were found to be potent protonophores at concentrations which are normally used to demonstrate inhibition of NaH exchange. Uncoupling was dependent on both the pH of the assay system and the total amount of lipid present. At the pH optima, which lay in a range from 7.5 to 8.5, these amiloride analogs were more potent uncouplers than the classical protonophore carbonyl cyanide m-chlorophenylhydrazone. Therefore, extreme care must be taken in the interpretation of results obtained using these or similar derivatives of amiloride.     

Barbiturate effects on hippocampal excitatory synaptic responses are selective and pathway specific

Barbiturate actions on excitatory synaptic responses in CA 1 and dentate regions of hippocampal slices were studied to determine whether different effects occur on anatomically distinct synaptic pathways. Pentobarbital facilitated transmission between stratum radiatum inputs and CA 1 neurons at low concentrations (0.02-0.08 mM) and produced postsynaptic depression at higher concentrations. Only depression was observed for stratum oriens inputs to CA 1 and perforant path inputs to dentate granulae neurons. The (+) isomer of pentobarbital was approximately four times more potent than the (-) isomer of racemic mixture. Phenobarbital (0.04-0.12 mM) produced only depression of synaptic responses in CA 1 and dentate pathways. Comparison of effect on field excitatory postsynaptic potentials and population spike responses indicated that the barbiturates act at selective and pathway-specific sites. The results provide further evidence for specific cellular and membrane recognition sites for barbiturate action.     

A study on the potential-dependence of proton block of sodium channels

Ionic currents through sodium channels in nodal membranes were measured under voltage clamp conditions both at normal and at low (4.8–4.9) external solution pH. The measurements of so-called ‘instantaneous’ currents were used to distinguish between the proton blockage in open channels and the influence of low pH on channel gating processes. It is shown that the amount of the proton blockage in open channels decreases as membrane potential becomes more positive. This result suggests that at least one of the acid groups accessible from the outside is located within the conducting pore. The influence of the other group(s) on the degree of potential-dependence of proton blockage is discussed.     

Cholesterol ester turnover in isolated liver cells. Effects of cholesterol feeding

Ionic currents through sodium channels in nodal membranes were measured under voltage clamp conditions both at normal and at low (4.8-4.9) external solution pH. The measurements of so-called 'instantaneous' currents were used to distinguish between the proton blockage in open channels and the influence of low pH on channel gating processes. It is shown that the amount of the proton blockage in open channels decreases as membrane potential becomes more positive. This result suggests that at least one of the acid groups accessible from the outside is located within the conducting pore. The influence of the other group(s) on the degree of potential-dependence of proton blockage is discussed.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.