Monoclonal antibodies modify acetylcholine-induced ionic channel properties in cultured chick myoballs

Summary Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick myoballs. Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time.     

Entropy effects on the ion-diffusion rate in transmembrane protein channels

We treat the transport of univalent cations through pore-like protein channels in biological membranes analytically, using two models (A + B) for the channel and the ion-channel interaction. A Lennard-Jones-type repulsion between the ions and the pore wall is introduced. We also include Van der Waals- and coulomb-type interactions between polar ligands of the pore-forming protein (e.g., carbonyl groups directed towards the axis of the channel) and the migrating particles. In model A, the polar groups are assumed to occur in pairs of dipoles pointing in opposite directions (as in the gramicidin A channel), while in model B the channel is treated as a pore with a radially isotropic charge distribution. In both models the ion-channel interaction leads to the occurrence of periodic potentials, corresponding to quasi-equilibrium and transition state sites of the ion in the pore. The diffusion rate can be calculated employing rate-theoretical concepts on the basis of microscopic parameters. It is demonstrated that the anomaly (inversion of the normal mass effect) for the transport rates of different ions can be related to differences in the activation entropy. The latter quantity is estimated analytically for both models. As a test, we performed numerical calculations with parameters based on the gramicidin A model. The results are in good agreement with experimental data and data from computer simulations. This shows that simple analytic expressions are well suited for predicting trends in the ionic conductivity of protein channels on the basis of microscopic interactions.     

Ion-specific diffusion rates through transmembrane protein channels. A molecular dynamics study

The migration of different alkali metal cations through a transmembrane model channel is simulated by means of the molecular dynamics technique. The parameters of the model are chosen in close relation to the gramicidin A channel. Coulomb- and van der Waals-type potentials between the ions and flexible carbonyl groups of the pore-forming molecule are used to describe the ion channel interaction. The diffusion properties of the ions are obtained from three-dimensional trajectory calculations. The diffusion rates for the different ions Li+, Na+, K+ and Rb+ are affected not only by the mass of the particles but also very strongly by their size. The latter effect is more pronounced for rigid channels, i.e., for binding vibrational frequencies of the CO groups with v greater than 400 cm-1. In this range the selectivity sequence for the diffusion rates is the inverse of that expected from normal rate theory but agrees with that found in experiments for gramicidin A.     

Knockdown resistance to dichlorodiphenyl-trichloroethane and pyrethroid insecticides in the napts mutant of Drosophila melanogaster is correlated with reduced neuronal sensitivity

Resistance to pyrethroid insecticides and dichlorodiphenyltrichloroethane (DDT) was investigated in the nap^ts (no action potential, temperature sensitive) mutant of Drosophila melanogaster. In surface contact bioassays, the nap^ts strain showed threefold resistance to deltamethrin at the LC₅₀ level when compared to susceptible Canton-S flies. Cross-resistance was also observed to DDT and the pyrethroids NRDC 157 [3-phenoxybenzyl [1R,cis]-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate], fenfluthrin, and MTI-800 [1-(3-phenoxy-4-fluorophenyl)-4-(4-ethoxyphenyl)-4-methylpentane]. The onset of intoxication by pyrethroids in nap^ts flies was markedly delayed, a finding that is consistent with the existence of a resistance mechanism involving reduced neuronal sensitivity. Resistance at the level of the nerve was confirmed by electrophysiological recordings of spontaneous and evoked activity in the dorsolongitudinal flight muscles of poisoned flies. Preparations from nap^ts insects treated with fenfluthrin displayed longer latencies to the appearance of spontaneous activity and also an absence or reduction in burst discharges compared to equivalent preparations from susceptible individuals. These results are discussed in light of competing hypotheses concerning the mechanism underlying knockdown resistance and reduced nerve sensitivity in insects.     

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Summary The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1–3 and 5–8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are –37, –36, and –23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are –139, –75 and –23 mV for the FN, FA and S currents, respectively.The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1–3 day-old rats the precent of cells with S, FA, and mixed S+FN currents is 22, 18, and 60% of the cells, respectively. In the 5–8 day-old, the percent of cells with S, FA, and FN+S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current.Antibodies directed against a part of the S₄ region of internal repeat I of the sodium channel (C ₁ ⁺ , amino acids 210–223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C ₁ ⁺ region in the membrane.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: II. Inductive property of membrane and effects of pH o and impermeable monovalent cations on breakdown phenomenon

Summary Changes in the chord conductanceG and the membrane electromotive forceE _m in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–V _m relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pH _o in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pH _o 7.In some cellsE _m shifted to a positive level as large as +50+70 mV during the breakdown phenomenon. Such a large positive shift ofE _m is caused mainly by the increase in conductance of Cl^– and partly Ca²⁺ and K⁺.     

Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties

Summary Two different³H-saxitoxin-binding proteins, with distinct biochemical and functional properties, were isolated from rat brain using a combination of anion exchange and lectin affinity chromatography as well as high resolution size exclusion and anion exchange HPLC. The alpha subunits of the binding proteins had different apparent molecular weights on SDS-PAGE (Type A: 235,000; Type B: 260,000). When reconstituted into planar lipid bilayers, the two saxitoxin-binding proteins formed sodium channels with different apparent single-channel conductances in the presence of batrachotoxin (Type A: 22 pS; Type B: 12 pS) and veratridine (Type A: 9 pS; Type B: 5 pS). The subtypes were further distinguished by scorpion (Leiurus quinquestriatus) venom which had different effects on single-channel conductance and gating of veratridine-activated Type A and Type B channels. Scorpion venom caused a 19% increase in single-channel conductance of Type A channels and a 35-mV hyperpolarizing shift in activation. Scropion venom double the single-channel conductance of Type B channels and shifted activation by at least 85 mV.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.