Membrane potential measurements of unfertilized and fertilized Xenopus laevis eggs are affected by damage caused by the electrode

Upon penetration in an unfertilized Xenopus egg bathed in 1/10 Ringer, the voltage recorded by a microelectrode shows an abrupt jump to a negative voltage (Ep) followed by a rapid depolarization to a steady value (Er) (Ep = -39.4 +/- 1.9 mV and Er = -11.5 +/- 0.5 SE, 54 eggs from 9 females). The same is true for fertilized eggs impaled 16-35 min after insemination (Ep = -29.5 +/- 2.1 mV, Er = -11.5 +/- 0.9 mV, SE, 18 eggs from 3 females). The voltage recorded by a second microelectrode inserted into the same egg does not show the transient initial negativity. The stationary level of the membrane potential is close to the diffusion potential calculated from the Goldman equation with equal permeabilities for all the relevant ions. It is concluded that the low resting potentials measured in Xenopus eggs before and after fertilization are largely due to damage caused by the electrode. Using an upper limit of -39 mV for the true membrane potential and correlating the input resistance with the stationary membrane potential, a lower limit of 22 M omega (about 1 M omega cm2) for the membrane resistance can be obtained. Insertion of a microelectrode during the first 3 min after insemination shows a steady positive potential while, at later times (3-16 min post-insemination), a positive peak followed by a repolarization can be observed. This indicates that the measurement of the peak of the fertilization potential is not seriously affected by the electrode penetration while its time course after the first 3 min may be deformed by the presence of a large leakage conductance.     

Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.     

Current noise parameters derived from voltage noise and impedance in embryonic heart cell aggregates.

We have recorded membrane impedance and voltage noise in the pacemaker range of potentials (-70 to -59 mV) from spheroidal aggregates of 7-d embryonic chick ventricle cells made quiescent by exposure to tetrodotoxin in medium containing 4.5 mM K+. The input capacitance is proportional to aggregate volume and therefore to total membrane area. The specific membrane capacitance is 1.24 microF/cm2. The input resistance at constant potential is inversely proportional to aggregate volume and therefore to total membrane area. The specific membrane resistance in 18 k omega . cm2 at -70 mV and increases to 81 k omega . cm2 at -59 mV. The RC time constant is 22 ms at -70 mV and increases to 146 ms at -59 mV. The aggregate transmembrane small-signal impedance can be represented by a parallel RC circuit itself in parallel with an inductive branch consisting of a resistor (rL) and an inductor (L) in series. The time constant of the inductive branch (L/rL) is 340 ms, and is only weakly dependent on potential. Correlation functions of aggregate voltage noise and the impedance data were modeled by a population of channels with simple open-close kinetics. The time constant of a channel (tau s) derived from the noise analysis is 300 ms. The low frequency limit of the pacemaker current noise (SI[0]), derived from the voltage noise and impedance, increases from 10(-20) A2/Hz . cm2 at -67 mV to 10(-19) A2/Hz . cm2 at -61 mV.     

The magnetic field of a single axon. A comparison of theory and experiment.

The magnetic field and the transmembrane action potential of a single nerve axon were measured simultaneously. The volume conductor model was used to calculate the magnetic field from the measured action potential, allowing comparison of the model predictions with the experimental data. After analyzing the experiment for all systematic errors, we conclude that the shape of the magnetic field can be accurately predicted from the transmembrane potential and, more importantly, the shape of the transmembrane potential can be calculated from the magnetic field. The data are used to determine ri, the internal resistance per unit length of the axon, to be 19.3 +/- 1.9 k omega mm-1, implying a value for the internal conductivity of 1.44 +/- 0.33 omega -1 m-1. Magnetic measurements are compared with standard bioelectric techniques for studying nerve axons.     

Effect of glycerin removal on the electrical resistance of isolated muscle fiber in the frog

Fibres isolated from iliofibularis muscles of the frog Rana esculenta were studied under current-clamp conditions with a double sucrose-gap technique. An increase of membrane resistance in muscle fibres (Rm) was demonstrated during the first 10-15 min of glycerol removal in K2SO4 solution. After a 30 min treatment in glycerol-containing K2SO4 Rm was 1.40 +/- 0.12 k omega X X cm2. The transfer of muscles from the glycerol-K2SO4 solution to an isotonic K2SO4 solution resulted in a progressive increase in the Rm which after a 15 minutes removal of glycerol reached the mean value of 2.02 +/- 0.22 k omega X cm2. It is suggested that this increase may reflect detubulation of the fibres caused by glycerol removal in muscles.     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Improved electrical coupling in uterine smooth muscle is associated with increased numbers of gap junctions at parturition

We have studied some passive electrical properties of uterine smooth muscle to determine whether a change in electrical parameters accompanies gap junction formation at delivery. The length constant of the longitudinal myometrium increased from 2.6 +/- 0.8 mm (X +/- SD) before term to 3.7 +/- 1 mm in tissues from delivering animals. The basis of the change was a 33% decrease in internal resistance and a 46% increase in membrane resistance. Axial current flow in an electrical syncytium such as myometrium is impeded by the cytoplasm of individual cells plus the junctions between cells. Measurement of the longitudinal impedance indicated that the specific resistance of the myoplasmic component was constant at 319 +/- 113 omega . cm before term and 340 +/- 93 omega . cm at delivery. However, a decrease in junctional resistance was apparent from 323 +/- 161 omega . cm to 134 +/- 64 omega . cm at delivery. 1.5-2 d after delivery, the junctional resistance was increased, as was the myoplasmic resistance. Thin-section electron microscopy of some of the same muscle samples showed that gap junctions were present in significantly greater numbers in the delivering tissues. Therefore, our results support the hypothesis that gap junction formation at delivery is associated with improved electrical coupling of uterine smooth muscle.     

Electrophysiological properties of isolated rat liver cells

The electrophysiological properties of isolated rat liver cells were studied using the patch clamp method in whole-cell configuration. The membrane potential in isolated hepatocytes was -42 +/- 7 mV (n = 20). The input resistance (Rin) and the time constant (tau m) were 51 +/- 17 M (the range of 34 to 180 M omega) (n = 20) and 4.2 +/- 1.0 msec (the range of 3 to 16.5 ms) (n = 20). Assuming that the specific membrane capacitance is 1 microF/cm2, the membrane resistance and membrane capacitance were 42. +/- 9.0 K omega cm2 and 87 +/- 27 pF. These values indicate that isolated rat hepatocytes are not abnormally permeable or leaky. The current-voltage relationship was linear with no rectification. The depolarizing pulse from the resting potential did not induce fast or slow inward currents even when norepinephrine or high Ca2 (3.6 mM) were applied. This indicates that there is no voltage-sensitive Ca2+ channel in the isolated hepatocytes.     

Compensation for resistance in series with excitable membranes.

Extracellular resistance in series (Rs) with excitable membranes can give rise to significant voltage errors that distort the current records in voltage-clamped membranes. Electrical methods for measurement of and compensation for such resistances are described and evaluated. Measurement of Rs by the conventional voltage jump in response to a current step is accurate but the measurement of sine-wave admittance under voltage-clamp conditions is better, having about a fivefold improvement in resolution (+/- 0.1 omega cm2) over the conventional method. Conventional feedback of the membrane current signal to correct the Rs error signal leads to instability of the voltage clamp when approximately two-thirds of the error is corrected. We describe an active electronic bridge circuit that subtracts membrane capacitance from the total membrane current and allows full, yet stable, compensation for the voltage error due to ionic currents. Furthermore, this method provides not only fast and accurate control of the membrane potential in response to a command step, but also fast recovery following an abrupt change in the membrane conductance. Marked changes in the kinetics and amplitude of ionic currents resulting from full compensation for Rs are shown for several typical potential patterns.     

Diffusion of 133Xe through frog skins, toad bladders, and water boundary layers.

We have measured the total permeability coefficients P as a function of stirring frequency omega for 133Xe through frog skins and toad bladders. The permeability coefficients for the frog skins and toad bladders proper are, respectively, Pm = (3.9 +/- 0.8) X 10(-4) cm/s and (7.4 +/- 4.2) X 10(-4) cm/s. "Unstirred" water layer thickness delta is determined concurrently, from the frequency dependence of P(omega); the result for frog skin is delta = (0.060 +/- 0.016) square root of omega(rad/s) cm. The stirring frequency range is from omega = 7.5 rad/s (72 rpm) to 55 rad/s (530 rpm). The results support the conclusions that the principal barrier to Xe diffusion in these epithelia is inter- and intracellular water, and that the diffusion is passive and rapid. The experimental method may be straightforwardly adapted to the measurement of diffusion or counterdiffusion of any gamma-radioactive soluble or partly soluble solute through any flat membrane or through a solvent. We estimate the amount of total body-absorbed radioactivity due to environmental 133Xe to be 50 fCi for an ambient concentration of 2.6 pCi/m3 of air.     

A novel method for measuring membrane conductance changes by a voltage-sensitive optical probe.

This study presents a method whose principles enable using a voltage-sensitive optical probe, to quantitatively measure conductivity changes elicited in membrane vesicles and cells. The procedure is based on the fact that the amplitude of the transmembrane potential difference, established across a membrane by an external electric field, is decreased when membrane conductivity is increased upon incorporation of ionophores into the membrane. The method was applied to osmotically swollen thylakoid membranes whose membrane conductivity was changed by the addition of gramicidin or ionomycin. The electric field induced stimulated luminescence from photosystem I (electrophotoluminescence-EPL) was used as a voltage-sensitive optical probe. We calculated the induced conductance changes by using a calibrated EPL vs external electric field response curve and measuring the ionophore-mediated attenuation of the EPL signal. The calculated ionophore-unmodified conductance of the thylakoid membrane yields a value of 171 +/- 56 nS/cm. The value of the membrane conductance, modified by 10 nM gramicidin was found to be 190 +/- 56 nS/cm. The modified membrane conductance and the membrane conductance changes induced by 1 microM ionomycin in the presence of CaCl2 were found to be 186 +/- 3 nS/cm and 15 +/- 3 nS/cm, respectively.     

Permeability properties of the subepithelial tissues of Necturus gallbladder

The permeability properties of the subepithelial connective tissue of Necturus gallbladder were evaluated by measurement of electrical resistance, dilution potentials and hydraulic water permeability. The gallbladder epithelial cells were removed by scraping and the underlying connective tissue placed in an Ussing chamber. The electrical resistance was 2.2 +/- 0.8 omega X cm2; the tissue was slightly cation selective relative to free solution. The subepithelial tissues restricted the rate of diffusion of small solutes to 50% of the free solution value. The hydraulic water permeability averaged 2.1 X 10(-2) cm/s per atm. We conclude that limitations of the area of subepithelium available for fluid movement are the most important factors in determining the restrictions to solute and water flow offered by the subepithelial tissues.     

Electrical properties of rabbit corneal endothelium as determined from impedance measurements.

Alternating- and direct-current electrical characteristics of rabbit corneal endothelium were studied under varying experimental conditions. The measurements were performed by sending a 10-microA current (AC or DC) across the tissue layer. Maximal values of transendothelial potential difference and resistance were 1.3 +/- 0.1 mV and 73 +/- 6 omega . cm2, respectively. The short-circuit current was estimated from the potential and resistance values. Impedance loci were obtained for the frequency range 0.5-100 kHz. A capacitive reactance (C = 0.63 +/- 0.02 microF/cm2) was observed in the 100 Hz-100 kHz range. To relate the impedance data to the electrical parameters of the cell membranes, the voltage-divider ratio was determined by sending square pulse across the tissue and measuring voltage responses across the apical and basal membranes with an intracellular microelectrode. The intracellular potential difference was on the average -61 +/- 1 mV, and the voltage-divider ratio was found to be between 0.33 and 4. Impedance data were fit by a computer to an equivalent circuit representing a "lumped" model, and the agreement between the model and the data was satisfactory. The results are discussed in terms of both the morphological characteristics and properties of the fluid transport mechanism across the preparation.     

Alterations of the apparent area expansivity modulus of red blood cell membrane by electric fields.

Red blood cell membrane exhibits a large resistance to changes in surface area. This resistance is characterized by the area expansivity modulus K, which relates the isotropic membrane force resultant, T, to the fractional change in membrane surface area delta A/Ao. The experimental technique commonly used to determine K is micropipette aspiration. Using this method, E. A. Evans and R. Waugh (1977, Biophys. J. 20:307-313) obtained a value of 450 dyn/cm for the modulus. In the present report, it is shown that the value of K, as determined using this method, is affected by electric potential differences applied across the tip of the pipette. Using Ag-AgCl electrodes and current clamping electronics, we obtained values for K ranging from 150 dyn/cm with -1.0 V applied, to 1,500 dyn/cm with 1.0 V applied. At 0.0 V the modulus obtained was approximately 500 dyn/cm. A reversible, voltage- and pressure-dependent change in the cell volume probably accounts for the effect of the voltage on the calculated value of the modulus. The use of lanthanum chloride or increasing the extra- and intracellular solute concentrations reduced the voltage dependence of the measurements. It was also found that when dissimilar metals were used to "ground" the pipette to the chamber to prevent lysis of cells by static charge, values for K ranged from 121 to 608 dyn/cm. Based on measurements made at zero applied volts, in the presence of 0.4 mM lanthanum and at high solute concentration, we conclude that the true value of the modulus is approximately 500 dyn/cm.     

Electrical activity in white adipose tissue of rat

Intracellular recording of white adipocytes was performed in an in vitro preparation. Resting potential, input resistance and membrane time constant averaged: -34 +/- 9 mV, 295 +/- 161 M omega, and 58 +/- 19 ms respectively (mean +/- SD, n = 32). Intracellular injection of positive and negative square current pulses elicited membrane voltage responses, characterized by a rectification of the voltage change evoked by positive pulses, and a slow return to baseline at the offset of hyperpolarizing pulses. The amplitude and duration of the slow return to resting potential was dependent on membrane potential, pulse duration, and extracellular K+ concentration. This response was depressed when external Ca2+ was replaced by Co2+, and by external application of 4-aminopyridine. These results indicate that white adipocytes can generate membrane voltage responses which may mostly be a consequence of the activity of ionic channels. The properties of the slow return to baseline suggest that it may be due to a transient K+ current.     

Patch voltage clamp of squid axon membrane.

A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise.     

Electrophysiological studies on the cultured cells obtained from transplantable pancreatic carcinoma in Syrian golden hamsters

A pancreatic ductal carcinoma was established as a transplantable tumor line in an inbred strain of Syrian golden hamsters. Intracellular recordings of membrane potentials and input resistance were made from cultured cells obtained from the transplanted tumors using indwelling glass microelectrode. The mean value of the resting membrane potential was -46.5 +/- 1.8 mV (S.E.) (n = 13), while the mean resting input resistance was 21.2 +/- 4.3 M omega (S.E.) (N = 13). Dibutyryl cyclic AMP (2 X 10(-3)M) caused a marked hyperpolarization of about 30 mV accompanied by a reduction of input resistance. The transplantable tumor and its cultured cell line developed in this study have demonstrated their effectiveness as a reliable experimental model for use in pancreatic cancer research.     

The electrophysiological properties of the resting thyroid follicular cell

Glass microelectrodes have been useful in the study of the electrical properties of the resting thyroid follicular cell membrane. The resting transmembrane potential (RMP) has probably been underestimated in earlier work, possible as a result of leak artefacts, and it is clear that in most species the RMP is certainly greater than -60 mV. The ratio of membrane Na+ permeability to K+ permeability (PNa/PK) is of the order of 0.07 to 0.08, and Cl- is possibly (although not definitely) distributed in a passive fashion across the cell membrane, indicating that the transmembrane K+ gradient is the most important factor in the generation of the RMP. The existence of an electrogenic sodium pump in the follicular cell membrane has been demonstrated: the pump contributes about -2 mV to the RMP under control conditions. Follicular cells are completely electrically coupled, the basic coupled cellular unit probably being equivalent to the individual thyroid follicle, and the specific membrane resistance and specific membrane capacitance have been calculated to be 5 k omega. cm2 and 3.6 microF/cm2 respectively.     

Properties of internally perfused, voltage-clamped, isolated nerve cell bodies

The membrane properties of isolated neurons from Helix aspersa were examined by using a new suction pipette method. The method combines internal perfusion with voltage clamp of nerve cell bodies separated from their axons. Pretreatment with enzymes such as trypsin that alter membrane function is not required. A platinized platinum wire which ruptures the soma membrane allows low resistance access directly to the cell's interior improving the time resolution under voltage clamp by two orders of magnitude. The shunt resistance of the suction pipette was 10-50 times the neuronal membrane resistance, and the series resistance of the system, which was largely due to the tip diameter, was about 10(5) omega. However, the peak clamp currents were only about 20 nA for a 60-mV voltage step so that measurements of membrane voltage were accurate to within at least 3%. Spatial control of voltage was achieved only after somal separation, and nerve cell bodies isolated in this way do not generate all-or-none action potentials. Measurements of membrane potential, membrane resistance, and membrane time constant are equivalent to those obtained using intracellular micropipettes, the customary method. With the axon attached, comparable all-or-none action potentials were also measured by either method. Complete exchange of Cs+ for K+ was accomplished by internal perfusion and allowed K+ currents to be blocked. Na+ currents could then be blocked by TTX or suppressed by Tris-substituted snail Ringer solution. Ca2+ currents could be blocked using Ni2+ and other divalent cations as well as organic Ca2+ blockers. The most favorable intracellular anion was aspartate-, and the sequence of favorability was inverted from that found in squid axon.