Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation

Opioid receptors were found to activate two different types of membrane potassium conductance in acutely dissociated neurons from the CA1/subiculum regions of the adult rat hippocampal formation. Opioid-responsive neurons were distinguished based on their morphology and electrophysiological responses. In one population of neurons having a multipolar, nonpyramidal cell shape, mu-selective opioid agonists increased an inward rectifying potassium current. Opioid activation of the inward rectifying conductance resulted in small outward potassium currents at resting membrane potentials and increased inward currents at hyperpolarized potentials. In a second population of nonpyramidal neurons, mu opioid agonists increased a novel voltage-gated potassium current. This current was blocked by internal CsCl2, unaffected by external BaCl2 or CdCl2, irreversibly activated by intracellular GTP-gamma-S, and inactivated by sustained depolarization. In contrast to the inward rectifying conductance, the voltage-gated conductance was not activated at resting membrane potentials or hyperpolarized potentials. The opioid-activated, voltage-gated conductance represents a new class of G protein-regulated potassium current in the brain.     

Evidence for existence of two acid groups controlling the conductance of sodium channel

The inhibition of the sodium current in nodal membrane at low pH external solutions was studied under voltage clamp conditions. Analysis of the data for membrane potentials from +10 to +150 mV shows that the inhibition of the Na+ currents at high positive potentials cannot be described by a titration curve of a single acid group. The data can be explained on assumption that the conductance of each sodium channel is controlled by two acid groups: one is located within the pore, the other just near the outer mouth of the pore. The affinity of both groups for H+ is estimated.     

Hemagglutinin-induced fusion of HAb2 and PLC cells: dynamics of fusion pore conductance

Infection of cells with influenza virus is mediated by the virus envelope protein hemagglutinin (HA) which induces fusion of viral and target membranes. Earlier we showed using fluorescent microscopy that HAb2 cells expressing HA on their plasma membranes fused with PLC cells when pH of the external medium was decreased to -5. In the present work we used double whole-cell recording to monitor the intercellular conductance in HAb2/PLC cell pairs during fusion. In approximately 40% of cell pairs the pH drop induced the intercellular conductance, which we interpret as the formation of a fusion pore. The following stages of the conductance growth were distinguished: initial fluctuations near zero (flicker), a subsequent slow increase up to 1-4 nS and a final rapid increase up to 10-100 nS (complete fusion). The first detectable intercellular conductance change (opening of a fusion pore) was accompanied by an increase in the conductances of both HAb2 and PLC cell membrane. This observation suggests that the early pore complex should be leaky. The dynamics of the intercellular conductance appeared to depend upon the voltage difference between the fusing HAb2 and PLC cells: voltages higher than 40 mV facilitated the conductance growth.     

Current-Voltage Curves of Porous Membranes in the Presence of Pore-Blocking Ions: I. Narrow Pores Containing No More Than One Moving Ion

We propose a physical model for voltage-dependent conductance changes of excitable cell membranes. It is based on competition of uni- and bivalent ions for chains of stable sites extending through the membrane. These one-dimensional pathways (pores) have different profiles of chemical potential for the two ionic species so that bivalent ions can block the passage of univalent ions at large membrane potentials. We treat the special case that each pore is either empty or, because of electrostatic repulsion, contains no more than one uni- or bivalent ion at a time. A system of linear differential equations describes the time-dependent probabilities of the various possible pore states. The states are limited by transition rate constants involving the profile of the chemical potential, the membrane voltage, the ionic concentrations in the adjacent baths, and electrostatic interactions between the ions. The steady-state solutions (Kirchhoff-Hill theorem) yield expressions for the relationship between the small signal conductance of univalent ions and the concentration of these ions in the external bathing medium (a saturation curve) and for the ionic currents and the steady-state current-voltage curve (N-shaped). From the latter curve we compute the shift of theshold potential caused by concentration changes of the external bathing medium. The model yields a number of predictions which can be tested experimentally.     

Pore formation induced by an antimicrobial peptide: electrostatic effects

We investigate the mode of action of Cateslytin, an antimicrobial peptide, on zwitterionic biomembranes by performing numerical simulations and electrophysiological measurements on membrane vesicles. Using this natural β-sheet antimicrobial peptide secreted during stress as a model we show that a single peptide is able to form a stable membrane pore of 1 nm diameter of 0.25 nS conductance found both from calculation and electrical measurements. The resulting structure does not resemble the barrel-stave or carpet models earlier predicted, but is very close to that found in the simulation of α-helical peptides. Based on the simulation of a mutated peptide and the effects of small external electric fields, we conclude that electrostatic forces play a crucial role in the process of pore formation.     

A functional view of the entrances of L-type Ca2+ channels: estimates of the size and surface potential at the pore mouths.

At extreme membrane potentials, the unitary inward and outward currents through L-type Ca2+ channels become diffusion controlled and saturate. The magnitudes of these currents indicate that the pore entrances are asymmetric, with the external mouth being much larger than the internal one. On the other hand, negative surface potentials at the two ends of the pore are rather similar. Both would be significant only when the ambient ionic strength is 110 mM or less. We conclude that the surface charges will not help much in concentrating the channel's favorite divalent cations in the physiological condition. However, the pore does possess an external mouth large enough to make the important inward Ca2+ flow not limited by diffusion, even with only 1 mM external Ca2+.     

Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".     

Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).     

Kinetics of erythrocyte swelling and membrane hole formation in hypotonic media.

Red blood cell (RBC) swelling and membrane hole formation in hypotonic external media were studied by measuring the time-dependent capacitance, C, and the conductance, G, in the beginning of the beta-dispersion range. At high and moderate osmolarities of the external solution the capacitance reaches a steady-state whereas at low osmolarities it reveals a biphasic kinetics. Examination of RBC suspensions exposed to different concentrations of HgCl(2) demonstrates that water transport through mercury-sensitive water channel controls RBC swelling. Unlike the capacitance, an increase in the conductance to a stationary level is observed after a certain delay. A comparison of G(t) curves recorded for the suspensions of the intact cells and those treated with cytochalasin B or glutaraldehyde demonstrates the significant effect of the membrane viscoelasticity on the pore formation. It is shown that the stretched membrane of completely swollen RBC retains its integrity for a certain time, termed as the membrane lifetime, t(memb). Therefore, the resistivity of RBCs to a certain osmotic shock may be quantified by the distribution function of RBC(t(memb)).     

Ionic mechanism for the generation of horizontal cell potentials in isolated axolotl retina

The ionic mechanism of horizontal cell potentials was investigated in the isolated retina of the axolotl Ambystoma mexicanum. The membrane potentials of both receptors and horizontal cells were recorded intracellularly while the ionic composition of the medium flowing over the receptor side of the retina was changed. The membrane potential of the horizontal cell is highly depender side of the retina was changed. The membrane potential of the horizontal cell is highly dependent on the extracellular concentration of sodium. When the external ion concentration of either chloride or potassium was changed independently of the other, there were shifts in the membrane potential of the horizontal cell which could not be explained by changes in the equilibrium potential of these ions. If the external concentrations of both potassium and chloride ions were varied so that the product of their external concentrations did not change, the shift in the membrane potential of the horizontal cell was in the direction predicted by the Nernst equation. The results are consistent with the suggestion that in the dark the receptors release a synaptic transmitter which increases primarily the sodium conductance of the horizontal cell postsynaptic membrane.     

Localization of a voltage gate in connexin46 gap junction hemichannels.

Cysteine replacement mutagenesis has identified positions in the first transmembrane domain of connexins as contributors to the pore lining of gap junction hemichannels (Zhou et al. 1997. Biophys. J. 72:1946-1953). Oocytes expressing a mutant cx46 with a cysteine in position 35 exhibited a membrane conductance sensitive to the thiol reagent maleimidobutyryl biocytin (MBB). MBB irreversibly reduced the single-channel conductance by 80%. This reactive cysteine was used to probe the localization of a voltage gate that closes cx46 gap junction hemichannels at negative potentials. MBB was applied to the closed channel either from outside (whole cell) or from inside (excised membrane patches). After washout of the thiol reagent the channels were tested at potentials at which the channels open. After extracellular application of MBB to intact oocytes, the membrane conductance was unaffected. In contrast, channels treated with intracellular MBB were blocked. Thus the cysteine in position 35 of cx46 is accessible from inside but not from the outside while the channel is closed. These results suggest that the voltage gate, which may be identical to the "loop gate" (Trexler et al. 1996. Proc. Natl. Acad. Sci. USA. 93:5836-5841), is located extracellular to the 35 position. The voltage gate results in regional closure of the pore rather than closure along the entire pore length.     

Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion

We have investigated the mechanism of cell fusion mediated by HA, the fusogenic hemagglutinin of the Influenza viral envelope. Single erythrocytes (RBCs) were attached to fibroblasts expressing the HA on their cell surface, and fusion of the paired cells was triggered by rapid acidification. The RBC membrane was stained with fluorescent lipid, and the fusion-induced escape of lipid into the fibroblast was observed by quantitative image analysis. At the same time, the formation of an aqueous connection (i.e., the fusion pore) between the two cells was monitored electrically. Within minutes after acidification, an electrical conductance between the two cells appeared abruptly as the fusion pore opened, and then increased gradually as the pore dilated. Later, fluorescent lipid diffused into the fibroblast, approaching equilibrium over the next 5-20 min. No lipid flux was seen while the pore conductance remained 0.5 nS or less. Evidently lipid flux requires a threshold pore size. Our finding suggests that the smallest and earliest fusion pores are surrounded by a ring of protein. A fusion pore expands by breaking this ring and recruiting lipid into its circumference.     

Kinetics of pore size during irreversible electrical breakdown of lipid bilayer membranes.

The kinetics of pore formation followed by mechanical rupture of lipid bilayer membranes were investigated in detail by using the charge-pulse method. Membranes of various compositions were charged to a sufficiently high voltage to induce mechanical breakdown. The subsequent decrease of membrane voltage was used to calculate the conductance. During mechanical breakdown, which was probably caused by the widening of one single pore, the membrane conductance was a linear and not exponential function of time after the initial starting process. In a large number of experiments using various lipids and electrolytes, the characteristic opening process of the pore turned out to be independent of the actual membrane potential and electrolyte concentration. Our theoretical analysis of the pore formation suggested that the voltage-induced irreversible breakdown is due to a decrease in edge energy when the pore had formed. After initiation of the pore, the electrical contribution to surface tension is negligible. The time course of the increase of pore size shows that our model of the irreversible breakdown is in good agreement with mechanical properties of membranes reported elsewhere.     

The cationically selective state of the mitochondrial outer membrane pore: a study with intact mitochondria and reconstituted mitochondrial porin.

The outer mitochondrial membrane pore at a voltage above 20 to 30 mV can adopt a state of low conductance which may restrict free permeability of mitochondrial substrates. In order to obtain insight into the physiological meaning of this property we took advantage of the fact that the low conductance pore state could be induced by a polyanion in lipid bilayer membranes as well as in intact mitochondria. Upon reconstitution in artificial bilayers the pore in this substate became exclusively cation selective when the polarity of the applied voltage was negative on the cis-side. This behaviour of the pore would explain why induction of the low conductance pore state in intact mitochondria led to a complete inhibition of mitochondrial intermembranous kinases, such as creatine kinase and adenylate kinase, but not of peripheral kinases, for example hexokinase, when utilizing external ATP. The possibility that the inner membrane potential might be transduced to the outer membrane in the contact sites, suggests the existence of cation selective pores in these sites. This aspect may be important in the regulation of peripheral kinases like creatine kinase, nucleoside diphosphate kinase and adenylate kinase which are located behind the outer mitochondrial membrane.     

The nature of the voltage-dependent conductance induced by alamethicin in black lipid membranes

Summary Alamethicin induces a conductance in black lipid films which increases exponentially with voltage. At low conductance the increase occurs in discrete steps which form a pattern of five levels, the second and third being most likely. The conductance of each level is directly proportional to salt concentration, inversely proportional to solution viscosity, and nearly independent of voltage.The probability distribution of the five steps is not a function of voltage, but as the voltage is increased, more levels begin to appear. These can be explained as super-positions of the original five, both in position and relative probability.This suggests that the five levels are associated with a physical entity which we call a pore. This point of view is confirmed by the following measurements. The kinetic response of the current to a voltage step is first order, and shows an exponential increase in rate of pore formation and an exponential decrease in rate of pore disappearance with voltage. If these rates are statistical, the number of pores should fluctuate about a voltage-dependent mean. High conductance current fluctuations are too large to be explained by fluctuation in the number of pores alone. But if fluctuations among the five levels are included, the magnitude of the fluctuations at high conductance is accurately predicted.Alamethicin adsorbs reversibly to the membrane surface, and the conductance at a fixed voltage depends on the ninth power of alamethicin concentration and on the fourth power of salt concentration, in the aqueous phase. In our bacterial phosphatidyl ethanolamine membranes, alamethicin added to one side of the membrane produces elevated conductance only when the voltage on that side is increased.On leave of absence from the Facultad de Ciencias, Universidad de Chile, Santiago de Chile.     

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells.

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.     

Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells.

Receptor-dependent translocation of diphtheria toxin across the surface membrane of Vero cells was studied using patch clamp techniques. Translocation was induced by exposing cells with surface-bound toxin to low pH. Whole cell current and voltage clamp recordings showed that toxin translocation was associated with membrane depolarization and increased membrane conductance. The conductance increase was voltage independent, with a reversal potential of approximately 15 mV. This value was unaffected by changing the Cl- gradient across the membrane and microfluorometric measurements showed that the cytosolic Ca2+ concentration was only marginally elevated by the translocation. The conductance increase is thus mainly due to monovalent cations. Exposing outside-out and cell-attached patches with bound toxin to low pH induced a new type of ion channel in the membrane. The channel current was inward at negative membrane potentials and the single channel conductance was approximately 30 pS. This value is about three times larger than for receptor-independent channels induced by diphtheria toxin or toxin fragments in artificial lipid membranes.     

Changes in the conductance of the neuronal nicotinic acetylcholine receptor channel induced by magnesium

We have investigated the effect of magnesium on the single-channel conductance of neuronal nicotinic acetylcholine receptors (nAChRs) in nerve growth-factor treated rat pheochromocytoma (PC12) cells. The patch-clamp technique was used to record single-channel currents from cell attached and excised, outside-out patches in the presence of various internal and external Mg2+ concentrations. Mg2+ reduced the single-channel conductance in a concentration-dependent manner with an IC50 of 9.2 mM for external Mg2+ (inward conductance) and 0.69 mM for external Mg2+ (outward conductance). Both estimated and measured conductances for divalent cation-free CsCl solutions were around 60 pS. We also find that divalent cations are not involved in the inward rectification of whole-cell ACh-induced currents in these cells. Our results imply that the amino acids screened by divalent cations sense electric fields only weakly and are presumably outside the lipid bilayer. They also suggest that the density and the number of charges (or both) differ on either side of the ion pore.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. I. A study of the "cell-attached" configuration

A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials.