Membrane Potentials of the Lobster Giant Axon Obtained by Use of the Sucrose-Gap Technique

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.     

The changes in CiNa of sheep cardiac Purkinje fibres in hyperosmotic solutions

1. The changes in intracellular sodium ion concentration (CiNa) of sheep cardiac Purkinje fibres in hyperosmotic solutions were studied using Na-sensitive liquid ion-exchanger microelectrodes. 2. CiNa was increased in hyperosmotic solutions containing different concentrations of sucrose from 0 to 300 mM. 3. The changes in resting membrane potential (RMP) in hyperosmotic solutions had no regularity. In most of the experiments there was hyperpolarization of the membrane but in a few cases a depolarization or no change of RMP were also observed. 4. The N-shape of I-V relations of the fibres became more pronounced in hyperosomotic solutions.     

Double Sucrose-Gap Method Applied to Single Muscle Fiber of Xenopus laevis

Passive electrical properties (internal conductance, membrane conductance, low frequency capacity, and high frequency capacity obtained from the foot of the action potential) of normal and glycerol-treated muscle of Xenopus were determined with the intracellular microelectrode technique. The results show that the electrical properties of Xenopus muscle are essentially the same as those of frog muscle. Characteristics of the action potential of Xenopus muscle were also similar to those of frog muscle. Twitch tension of glycerol-treated muscle fibers of Xenopus recovered partially when left in normal Ringer for a long time (more than 6 h). Along with the twitch recovery, the membrane capacity increased. Single isolated muscle fibers of Xenopus were subjected to the double sucrose-gap technique. Action potentials under the sucrose gap were not very different from those obtained with the intracellular electrode, except for the sucrose-gap hyperpolarization and a slight tendency toward prolongation of the shape of action potential. Twitch contraction of the artificial node was recorded as a change of force from one end of the fiber under the sucrose gap. From the time-course of the recorded force and the sinusoidal stress-strain relationship at varying frequencies of the resting muscle fiber, the time-course of isotonic shortening of the node was recovered by using Fourier analysis. It was revealed that the recorded twitch force can approximately be regarded as isotonic shortening of the node.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

The correction factors for sucrose gap measurements and their practical applications.

The distribution of extracellular and intracellular potential in the sucrose gap apparatus, previously established for a single fiber using the cable equations for a core conductor model (Jirounek and Straub, Biophys. J., 11:1, 1971), is obtained for a multifiber preparation. The exact equation is derived relating the true membrane potential change to the measured potential differences across the sucrose gap, the junction potentials between sucrose and physiological solution, the membrane potential in the sucrose region, and the electrical parameters of the preparation in each region of the sucrose gap. The extracellular potential distribution has been measured using a modified sucrose gap apparatus for the frog sciatic nerve and the rabbit vagus nerve. The results indicate a hyperpolarization of the preparations in the sucrose region, of 60--75 mV. The hyperpolarization is independent of the presence of junction potentials. The calculation of the correction terms in the equation relating the actual to the measured potential change is illustrated for the case of complete depolarization by KC1 on one side of the sucrose gap. The correction terms in the equation are given for various experimental conditions, and a number of nomographic charts are presented, by means of which the correction factors can be rapidly evaluated.     

Depression of glutamate-mediated synaptic transmission by benzyl alcohol.

The data obtained from this study suggest that the nonionizable anesthetic benzyl alcohol has two prominent actions on GABA- and glutamate-mediated synaptic transmission at the lobster neuromuscular junction. They are as follows: (1) depression of the excitatory end-plate potential and the postsynaptic membrane response to applied glutamate, and (2) a hyperpolarization of the postsynaptic resting membrane potential associated with a decrease in effective membrane resistance. No change in amplitude of the inhibitory end-plate potential or inhibitory reversal potential was seen. Excitatory miniature end-plate potential frequency was also unaffected. The depression of excitatory synaptic transmission appears to be due to a decreased responsiveness of the postsynaptic receptor-ionophore complex.     

Variations in the hyperpolarization potential of ethanol on muscle during reinnervation

Ethanol causes the hyperpolarization of the excitable membranes. In the Extensor Digitorum Longus (EDL) muscle of the rat the increase of resting membrane potential is 2-5% and is independent of the concentration of alcohol between 0.2 and 0.4 M, while at higher concentrations the membrane potential falls to levels equal or inferior to the normal potential. We have studied the hyperpolarization action of ethanol on the denervated muscle by crushing the sciatic nerve. Also under these conditions in which, as is known, there is a drop in the resting potential, ethanol causes hyperpolarization, however it is in general greater and it is dependent upon the concentration between 0.2 and 0.8 M.     

The Potential Distribution and the Short-Circuiting Factor in the Sucrose Gap

The sucrose gap technique, though widely employed in many tissues, could not be used for quantitative measurements of the membrane potential, because the value of the short-circuiting factor and the influence of junction potential on the recorded potential difference were unknown. The formula that relates the recorded potential to the true resting membrane potential was found by application of the cable equations to a core conductor placed in a system with three different media, e.g. Ringer, sucrose, and KCl. The formula shows that the potential difference recorded over the sucrose insulator depends on the extracellular and the intracellular longitudinal resistances, the membrane resistance and the membrane potentials in each region, and on the junction potentials between the different media. The true membrane potential in the Ringer region can be calculated from the potential difference recorded after complete depolarization by KCl on one side of the preparation, if the longitudinal resistances, the membrane resistances, the extracellular potential in the sucrose, and the junction potential between Ringer and sucrose are determined by separate measurements.     

Cell type-specific regulation of ion channels within the maize stomatal complex

The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K(+)-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear. In order to bridge this gap, we performed patch-clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca(2+) and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2',7'-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined. Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.     

Influence of homologous n-alkanoic on functional properties of isolated skeletal muscles. II. Membrane resting potential and the osmotic effectiveness of alkanoic acid]

The influence of butyric, hexanoic, octanoic, and decanoic acid on the membrane resting potential of isolated frog skeletal muscles were studied and the osmotic effects of n-alkanoic acids tested. 1. n-alkanoic acids cause osmotic effects like impermeable non-electrolytes (sucrose). Therefore, the permeability to alkanoic acids of the resting muscle cell membrane seems to be small. There are no differences between the acids tested. 2. The membrane resting potential is differently affected. Butyric acid in high concentration effects a hyperpolarization of the membrane whereas higher homologues (C6--C10) cause a depolarization. The depolarizing action increases with increasing concentration, exposure, and with the length of the hydrocarbon chain of the alkanoic acids. 3. It is suggested that osmotic effects are the cause for hyperpolarization of the membrane by high concentrations of butyric acid. 4. The depolarizing action of hexanoic, octanoic, and decanoic acid is discussed with regard to alterations induced by alkanoic acids in the membrane permeability and/or in the metabolism of the cells.     

Electrical responses of supporting cells in the frog taste organ to chemical stimuli

1. The mean resting potential of supporting cells in the frog taste organ was -19.1 mV. The supporting cells responded to the four basic taste stimuli with a depolarization but responded to water with a depolarization or a hyperpolarization. 2. The membrane resistances of supporting cells decreased during stimulation with sucrose, NaCl and acetic acid, but increased during stimulation with Q-HCl and water. 3. Reversal potential of the depolarizing response for 0.5 M NaCl in supporting cells was +7.6 mV. The depolarizing responses for Q-HCl and acetic acid were independent of the membrane potential level. 4. These results suggest that the characteristics of taste responses in supporting cells are similar to those in taste cells.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

A theoretical study on the sucrose gap technique as applied to multicellular muscle preparations. II. Methodical errors in the determination of outward currents.

This paper presents a mathematical model for analyzing systematic errors associated with the membrane conductance of multicellular muscle preparations as determined in a sucrose gap apparatus. The errors arise because of the interdiffusion of sucrose and saline in the interstitial fluid spaces, which results (a) in spatial variations of equilibrium potentials, membrane conductance, and solution conductivity, and (b) in the existence of a liquid junction potential. The model was applied to simulate the measurement of outward currents predominantly carried by potassium ions; time variations were not considered. Output current/voltage (I/V) curves were computed and compared with the membrane I/V relationship used in the computation. The output curves look very much like experimental results but are distorted considerably from the membrane I/V relationship: (a) under favorable conditions (negligible shunt current), the membrane current is overestimated over the entire range of membrane potential, (b) regions with negative slope conductance of I/V relations with "anomalous rectifier" properties are found to be less pronounced or even absent, and (c) resting potentials may be either increased or reduced. The origin of these errors is related to currents emerging from the sucrose compartment (local circuit as well as externally applied currents). Their dependence on several experimental parameters is discussed.     

Transmission of Electric Signals in Sieve Tubes of Zucchini Plants

Membrane potentials of ?;160 to ?210 mV were recorded with microelectrodes inserted into meta-phloem sieve tubes of intact zucchini plants (Cucurbita pepo L. var. medullosa Alef.). The effects of darkness, white light and colored light on membrane potential were studied. Reference electrodes were in contact with the apoplast via fluid-filled cavities or “drinks”. Electrolyte solutions (100 mM) in the cavities could be quickly replaced by flushing with 100 mM solutions of sucrose, KCl, sorbitol, or EDTA without altering osmolarity. KCl and EDTA caused depolarization of the sieve tube membrane potential, while sucrose caused depolarization or hyperpolarization of the sieve tube membrane potential in mature or growing plant parts respectively. Recovery of the original voltage was recorded when rapid (sucrose) or slow (sorbitol) transients occurred. When two measuring circuits were installed, one in a growing fruit and the other in the petiole of the subtending mature leaf, the alteration of the sieve tube membrane potential at one site was accompanied by an alteration of the potential at the other site after a few seconds. The responses were opposite in the exporting leaf and importing fruit when sucrose was applied. The signal, transmitted via the sieve tubes, reached maximum velocities of 10 cm per second.     

Membrane potential and bicarbonate secretion in isolated interlobular ducts from guinea-pig pancreas

The interlobular duct cells of the guinea-pig pancreas secrete HCO(3)(-) across their luminal membrane into a HCO(3)(-)-rich (125 mM) luminal fluid against a sixfold concentration gradient. Since HCO(3)(-) transport cannot be achieved by luminal Cl-/HCO(3)(-) exchange under these conditions, we have investigated the possibility that it is mediated by an anion conductance. To determine whether the electrochemical potential gradient across the luminal membrane would favor HCO(3)(-) efflux, we have measured the intracellular potential (V(m)) in microperfused, interlobular duct segments under various physiological conditions. When the lumen was perfused with a 124 mM Cl- -25 mM HCO(3)(-) solution, a condition similar to the basal state, the resting potential was approximately -60 mV. Stimulation with dbcAMP or secretin caused a transient hyperpolarization (approximately 5 mV) due to activation of electrogenic Na+-HCO(3)(-) cotransport at the basolateral membrane. This was followed by depolarization to a steady-state value of approximately -50 mV as a result of anion efflux across the luminal membrane. Raising the luminal HCO(3)(-) concentration to 125 mM caused a hyperpolarization (approximately 10 mV) in both stimulated and unstimulated ducts. These results can be explained by a model in which the depolarizing effect of Cl- efflux across the luminal membrane is minimized by the depletion of intracellular Cl- and offset by the hyperpolarizing effects of Na+-HCO(3)(-) cotransport at the basolateral membrane. The net effect is a luminally directed electrochemical potential gradient for HCO(3)(-) that is sustained during maximal stimulation. Our calculations indicate that the electrodiffusive efflux of HCO(3)(-) to the lumen via CFTR, driven by this gradient, would be sufficient to fully account for the observed secretory flux of HCO(3)(-).     

Role of Ba2+-resistant K+ channels in endothelium-dependent hyperpolarization of rat small mesenteric arteries

In rat small mesenteric arteries, the influence of modulation of basal smooth muscle K+ efflux on the mechanism of endothelium-dependent hyperpolarization was investigated. The membrane potentials of the vascular smooth muscle cells were measured using conventional microelectrode techniques. Incubation of resting arteries with the gap junction uncoupler carbenoxolone (20 micro M) decreased the endothelium-dependent hyperpolarization elicited by a submaximal concentration of acetylcholine (3 micro M) to about 65% of the control. In the presence of Ba2+ (200 micro M), which depolarized the membrane potential by 10 mV, the acetylcholine-induced membrane potential response was doubled in magnitude, reaching values not different from control. Moreover, the hyperpolarization was more resistant to carbenoxolone in these conditions. Finally, both in the absence and in the presence of carbenoxolone, the combined application of Ba2+ and ouabain (0.5 mM) did not abolish the acetylcholine response. These results suggest that gap junctional coupling plays a role in endothelium-dependent hyperpolarization of smooth muscle cells of resting rat small mesenteric arteries. Additionally, these findings show that the hyperpolarization does not rely on activation of inward rectifying K+ channels. Although a minor contribution of Na-K pumping cannot be excluded, the Ba2+ experiments show that the membrane electrical response is mediated by activation of a Ba2+-resistant K+ conductance.     

Characterization of inhibitory innervation in porcine colonic circular muscle

Effects of stimulation of intramural nerves in the circular smooth muscle layer of the porcine colon (Sus scrofa domestica) were studied using the sucrose-gap technique. Electrical field stimulation of the preparation, superfused with Krebs solution at 21 degrees C, induced a transient hyperpolarization of the smooth muscle cell membrane. This hyperpolarization was an inhibitory junction potential (IJP). The responses obtained from circular muscle originating from either the centripetal or centrifugal gyri of the ascending colon did not differ significantly. The IJP was characterized as being mediated by intramural, nonadrenergic, noncholinergic (NANC) nerves. The amplitude and latency of the IJP changed linearly with temperature (15-25 degrees C: +1 mV and -0.1 s per degree Celsius, respectively) reflecting a temperature-dependent synchronization of transmitter release. The membrane resistance decreased during the IJP. The IJP amplitude decreased or increased during conditioning hyperpolarizations or depolarizations, respectively, and reversed at membrane potentials about 30 mV more negative than the resting membrane potential. Potassium conductance blocking agents, barium (1 mM), tetraethylammonium chloride (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM), apamin (1 microM), and aminacrine (10(-4) M) added to the superfusion medium increased the membrane resistance. Only barium, TEA, and apamin depolarized the smooth muscle cell membrane. The IJP amplitude decreased in the presence of aminacrine and apamin to 75 and 35%, respectively, suggesting that apamin-sensitive Ca2+-activated K+ channels are involved in this response. ATP, adenosine, and related adenine nucleotides in concentrations up to 10(-3) M did not mimic the IJP. Superfusion with ATP for 15 min revealed a gradually increasing attenuation by up to 20% of the IJP. This might suggest that the release of neurotransmitter from intramural NANC nerves is modulated presynaptically via purinoceptors. Exogenously applied vasoactive intestinal polypeptide (VIP) in concentrations of 10(-9) to 10(-4) M did not affect the preparation. Also at elevated temperatures (up to 35 degrees C), VIP (10(-7) to 10(-4) M) did not cause measurable effects. It is concluded that the inhibitory mediator of the intramural NANC nerves present in the circular muscle layers of the porcine colon is neither a purine nor VIP.     

The mouse taste cell response to five sugar stimuli

Intracellular recordings of mouse taste cell responses were made using glass microelectrodes filled with procion yellow dye solution. Only responses recorded from taste buds with fluorescent cells, as observed in subsequent histological preparations, were used in this study. The mouse taste cell depolarizes when stimulated with sucrose and is accompanied by either a resistance increase or no change. On the other hand, a NaCl stimulus produces a depolarization, hyperpolarization or null response and is accompanied by either a membrane resistance decrease or no change. Four sugars other than sucrose (maltose, fructose, glucose and lactose) produced the depolarization or null responses and were accompanied by an increase or no change in membrane resistance. From the above observations, it is suggested that each taste cell produces its own characteristic response profiles and membrane resistance changes for the five sugars and NaCl tested.     

Mechanism of active repolarization of inhibitory junction potential in murine colon

Enteric inhibitory responses in gastrointestinal (GI) smooth muscles involve membrane hyperpolarization that transiently reduce the excitability of GI muscles. We examined the possibility that an active repolarization mechanism participates in the restoration of resting membrane potential after fast inhibitory junction potentials (IJPs) in the murine colon. Previously, we showed these cells express a voltage-dependent nonselective cation conductance (NSCC) that might participate in active repolarization of IJPs. Colonic smooth muscle cells were impaled with micro-electrodes and voltage responses to nerve-evoked IJPs, and locally applied ATP were recorded. Ba2+ (500 muM), a blocker of the NSCC, slowed the rate of repolarization of IJPs. We also tested the effects of Ba2+, Ni2+, and mibefradil, all blockers of the NSCC, on responses to locally applied ATP. Spritzes of ATP caused transient hyperpolarization, and the durations of these responses were significantly increased by the blockers of the NSCC. We considered whether NSCC blockers might affect ATP metabolism and found that Ni2+ decreased ATP breakdown in colonic muscles. Mibefradil had no effect on ATP metabolism. Because both Ni2+ and mibefradil had similar effects on prolonging responses to ATP, it appears that restoration of resting membrane potential after ATP spritzes is not primarily due to ATP metabolism. Neurally released enteric inhibitory transmitter and locally applied ATP resulted in transient hyperpolarizations of murine colonic muscles. Recovery of membrane potential after these responses appears to involve an active repolarization mechanism due to activation of the voltage-dependent NSCC expressed by these cells.     

Measurement of the transmembrane electrical potential of Dunaliella acidophila by microelectrodes

A method was developed to determine electrical potential differences across the plasma membrane of the microalga Dunaliella by means of potential-sensitive microelectrodes. Special emphasis was put on the measurement of the membrane potential in the acidophilic Dunaliella acidophila (optimal growth at pH 1.0), but neutrophilic, halotolerant Dunaliella species were used as reference systems. For Dunaliella acidophila positive membrane potentials (cytoplasma relative to the medium), ranging from +30 to +65mV were measured. Illumination caused a decrease of the positive potential by about 10 mV. The ATPase inhibitor omeprazole caused an increase of the positive membrane potential ranging from +60 to +100 mV, whereas the ionophore gramicidin caused a decrease of the MP to +10 to +30 mV. The salt tolerant, neutrophilic Dunaliella parva and Dunaliella bardawil exhibited negative membrane potentials in the order of -40 to -60mV, and light caused a hyperpolarization of about 10 mV. A negative membrane potential was measured also in D. acidophila cells transferred to pH 7.0. The physiological significance of a positive membrane potential for acidophilic algae is discussed.Abbreviations E _m membrane potential - PM plasma membrane - TPB^– tetraphenylborone anion - TPP⁺ tetraphenyl-phosphonium cation - SCN^– isothiocyanate