Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes

A voltage-gated hydrogen ion-selective conductance has been previously described in the immature oocyte of the urodele amphibian Ambystoma. The present study was prompted by reports that changes in membrane voltage and internal pH, as well as in internal sodium ion concentration, occur during the hormone-induced maturation of oocytes from other amphibians. As activation of membrane currents might mediate changes in internal ion concentrations in addition to altering the membrane voltage, microelectrode recording techniques have been employed to examine changes in membrane conductances which occur during maturation of Ambystoma oocytes. It was observed that during the first 5 hr of maturation the magnitude of the hydrogen ion conductance gradually decreased, and that subsequently there was an increase in the amplitude of a voltage-dependent noninactivating sodium conductance. After 6 to 7 hr, after the loss of the hydrogen conductance and at about the time of germinal vesicle breakdown, the resting potential of the oocyte spontaneously shifted from approximately -10 mV to approximately +30 mV, where it remained until at least 24 hr after the initiation of maturation. This voltage transition was due to the appearance of mechanisms generating inward current in the oocyte membrane; part of this inward current was due to the tonic activation of the sodium conductance. Changes in internal pH and internal sodium ion concentration occurred during maturation, as judged from shifts in the reversal potentials of both hydrogen and sodium currents. A gradual decrease in internal hydrogen ion concentration was observed up until the time of disappearance of the hydrogen conductance (change in internal pH from about 7.15 in immature oocytes to about 7.40 by 3 hr after application of progesterone). This was followed, as sodium conductance increased, by an apparent rise in the internal sodium ion concentration (from about 6 mM to about 17 mM by 10 hr postprogesterone).     

Membrane capacity of squid giant axon during hyper- and depolarizations

Summary The change in membrane capacitance and conductance of squid giant axons during hyper- and depolarizations was investigated. The measurements of capacitance and conductance were performed using an admittance bridge with resting, hyperpolarized and depolarized membranes. The duration of DC pulses is 20–40 msec and is long enough to permit the admittance measurements between 1 and 50 kHz. The amplitudes of DC pulses were varied between 0 and 40mV for both depolarization and hyperpolarization. Within these limited experimental conditions, we found a substantial increase in membrane capacitance with depolarization and a decrease with hyperpolarization. Our results indicate that the change in membrane capacitance will increase further if low frequencies are used with larger depolarizing pulses. The change in membrane capacitance is frequency dependent and it increases with decreasing frequencies. The analyses based on an equivalent circuit (vide infra) gives rise to a time constant of active membrane capacitance close to that of sodium currents. This result indicates that the observed capacitance changes may arise from sodium channels. A brief discussion is given on the nature of frequency-dependent membrane capacitance of nerve axons.     

Admittance change of squid axon during action potentials. Change in capacitive component due to sodium currents.

Since the discovery of Cole and Curtis (1938. Nature (Lond.). 142:209 and 1939. J. Gen. Physiol. 22:649) that the imaginary components, i.e., capacitive and inductive components, of the admittance of squid axon membrane remained unchanged during the action potential, there have been numerous studies on impedance and admittance characteristics of nerves. First of all, it is now known that the dielectric capacitance of the membrane is frequency dependent. Second, the recent observation of gating currents indicates that dipolar molecules may be involved in the onset of ionic currents. Under these circumstances, the author felt it necessary to reinvestigate the membrane admittance characteristics of nerve axons. The measurements by Cole and Curtis were performed mainly at 20 kHz, indicating that their observation was limited only to the passive membrane capacitance. To detect the change in the capacitive component during the action potential, we performed transient admittance measurements at lower frequencies. However, the frequency range of the measurements was restricted because of the short duration of the normal action potential. In addition, a change in the inductive component obscured the low frequency behavior of the capacitance. To use wider frequency range and simplify the system by eliminating the inductive component, the potassium current was blocked by tetraethyl ammonium, and the increase in the capacitive component was reinvestigated during the long action potential. The admittance change under this condition was found to be mostly capacitive, and conductance change was very small. The increase in the capacitive component was from 1.0 to 1.23 muF/cm2.     

Inferences about membrane properties from electrical noise measurements

Four sources of electrical noise in biological membranes, each with a different physical basis, are discussed; the analysis of each type of noise potentially yields a different sort of information about membrane properties. (a) From the thermal noise spectrum, the passive membrane impedance may be obtained, so that thermal noise measurements are essentially equivalent to the type of since wave analysis carried out by Cole and Curtis. (b) If adequately high frequency measurements could be made, the shot noise spectrum should give information about the average motion of a single ion within the membrane. (c) The number of charge carriers and single ion mobilities within the membrane can possibly be inferred from measurements of noise with a 1/f spectrum. Available data indicate, for example, that increases in axon membrane conductance are not achieved by modulations in the mobility of ions within the membrane. (d) Fluctuations arising from the mechanisms normally responsible for membrane conductance changes can produce a type of electrical noise. Analysis of such conductance fluctuations provides a way to assess the validity of various microscopic models for the behavior of individual channels. Two different probabilistic interpretations of the Hodgkin-Huxley equations are investigated here and shown to yield different predictions about the spectrum of conductance fluctuations; thus, appropriate noise measurements may serve to eliminate certain classes of microscopic models for membrane conductance changes. Further, it is shown how the analysis of conductance fluctuations can, in some circumstances, provide an estimate of the conductance of a single channel.     

Activation of mechanosensitive ion channels in plasma membrane of K562 cells

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.     

Nonselective voltage-gated ionic channels in type II taste cells

In cells of different types outward voltage-gated (VG) ion currents are generally carried by potassium ions. However, in mouse type II taste cells these currents persist when K⁺-selective ion channels are inhibited. In this study, we examined the ion channels that provide a pathway for atypical VG outward currents in type II taste cells. These channels are found to be weakly selective and permeabile to large molecules such as NMDG, gluconate, and ATP. According to non-stationary fluctuation analysis, single channel conductance is about 200 pS. The data obtained suggest that the nonselective ion channels are similar to hemichannels formed by connexins, the gap-junction proteins, in the plasma membrane of vertebrate cells.     

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.     

The effect of ion channels on cell membrane motion

Cell membranes possess ion channels and pumps which allow them to pass electrical currents. In this paper it is shown that the ion channels and pumps should induce electric fields in the area surrounding the membrane. The magnitude of this field is computed. It is shown that when the channels and pumps are separated by a sufficient distance, then the electric fields which they induce will cause the solution which surrounds the membrane to move. This movement of the fluid should cause motion of the membrane. Thus it is demonstrated that under certain conditions the conductance of ions across cell membranes will give rise to cell membrane motion. It is suggested that this phenomenon may play a role in processes such as chemotaxis, cell division, and pinocytosis.     

Photogating of ionic currents across lipid bilayers. Hydrophobic ion conductance by an ion chain mechanism.

The photogating of hydrophobic ion currents across the lipid bilayer membrane allows the direct study of their kinetics by symmetrically forming charge within the membrane and across each interface, rather than across the membrane. We find that the photoinduced conductance continues to increase beyond the region where the tetraphenylboride charge density in the membrane exceeds the estimated porphyrin cation density. This photoconductance is proportional to the tetraphenylboride charge density raised to the second to third power. The risetime of the photogating effect increases with increasing concentration of tetraphenyl boride. The porphyrin cation mobility is increased when the tetraphenylboride anion is present, and low concentrations of tetraphenylphosphonium cation increase the dark conductivity while inhibiting the photoconductivity. The activation energy for both the porphyrin and phosphonium cation induced conductance is more positive than that of the tetraphenylboride conductance. From these results we conclude that in addition to some cancellation of space charge within the membrane, the mechanism of increased conductance involves the transport of these hydrophobic anions via an alternating anion-cation chain, analogous to the Grotthuss mechanism for excess proton conduction in water. This ion chain conductance can be viewed as an evolutionary prototype of an ion channel across the membrane. It also underscores the importance of the counter ion in the transport of large ions such as peptides across the lipid bilayer.     

Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20- associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.     

Frequency domain analysis of asymmetry current in squid axon membrane.

The change in capacity of squid axon membrane during hyper- and depolarizations was investigated in the absence of ionic currents after the membrane was treated with pronase. In the presence of the inactivation process (h parameter), failure to observe the gating current in the frequency domain was attributed to the rapid attenuation of the possible capacity change during depolarizations, which is likely to be due to the sodium activation process. Elimination of the h process would therefore enable us to observe the gating current in the frequency domain as the change in the capacitance component of membrane admittance. However, even after the inactivation process was abolished by pronase, the capacity of the axon membrane remained constant when ionic currents were blocked by external tetrodotoxin and internal Cs+ ion. Actually capacity was observed to decrease slightly with depolarization, contrary to the prediction based on the magnitude of gating currents.     

Influence of conductance changes on patch clamp capacitance measurements using a lock-in amplifier and limitations of the phase tracking technique.

We characterized the influence of conductance changes on whole-cell patch clamp capacitance measurements with a lock-in amplifier and the limitations of the phase-tracking method by numerical computer simulations, error formulas, and experimental tests. At correct phase setting, the artifacts in the capacitance measurement due to activation of linear conductances are small. The cross talk into the capacitance trace is well approximately by the second-order term in the Taylor expansion of the admittance. In the case of nonlinear current-voltage relationships, the measured conductance corresponds to the slope conductance in the range of the sine wave amplitude, and the cross talk into the capacitance trace corresponds to the second-order effect of the slope conductance. The finite gating kinetics of voltage-dependent channels generate phase-shifted currents. These lead to major artifacts in the capacitance measurements when the angular frequency of the sine wave is close to the kinetic rate constant of the channel. However, when the channel kinetics are sufficiently slow, or sufficiently fast, the cross talk is still close to the second-order effect of the measured conductance. The effects of activation of voltage-dependent currents on the capacitance measurements may be estimated, provided a detailed characterization of the kinetics and voltage dependence is available. A phase error of the lock-in amplifier of a few degrees leads to significant projections. The phase-tracking method can be used to keep the phase aligned only during periods of low membrane conductance. However, nonideal properties of the equivalent circuit, in particular the fast capacitance between the pipette and the bath solutions, may lead to large phase errors when the phase-tracking method is used, depending on the electrical properties of the cell. In this article we provide practical values, setting the range where possible artifacts are below defined limits. For proper evaluation of capacitance measurements, the capacitance and conductance traces should always be displayed together.     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

Study of the conductivity of single supernarrow pores (1–15 nm in diameter) formed in thin membranes (10–12 μm in thickness) from polyethylene terephthalate (PETP) has revealed discrete changes in the currents passing through such pores when applied from an external source of potential difference of 200–1000 mV. Based on several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical to the so-called currents of single ionic channels in cell membranes. The supernarrow pores whose properties are described in the present work were obtained by alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). On the walls of the pores, carboxyl groups, i.e., negative fixed charges, and their compensating counterion (cation) layer are formed. Upon setting the potential difference onto the PETP membrane, this cation layer is able to transfer current, through a process called surface conductance. In the case of nanometer-sized diameters of the pores, such surface conductance can turn out to be dominating. We have shown that these discrete changes of currents passing through the nanometer pores are associated with metastability of their surface conductance. In the highly cation-selective channels in the cell membranes, there should inevitably exist an area with dominating cation surface conductance and, hence, conductance metastability. Therefore, a new explanation is proposed of the characteristic discreteness of the currents of single cation-specific ionic channels in cell membranes. Such an explanation does not rule out the existence of any other traditional explanation of the discreteness of ion channel currents.     

Monitoring voltage-dependent charge displacement of Shaker B-IR K+ ion channels using radio frequency interrogation

Here we introduce a new technique that probes voltage-dependent charge displacements of excitable membrane-bound proteins using extracellularly applied radio frequency (RF, 500 kHz) electric fields. Xenopus oocytes were used as a model cell for these experiments, and were injected with cRNA encoding Shaker B-IR (ShB-IR) K(+) ion channels to express large densities of this protein in the oocyte membranes. Two-electrode voltage clamp (TEVC) was applied to command whole-cell membrane potential and to measure channel-dependent membrane currents. Simultaneously, RF electric fields were applied to perturb the membrane potential about the TEVC level and to measure voltage-dependent RF displacement currents. ShB-IR expressing oocytes showed significantly larger changes in RF displacement currents upon membrane depolarization than control oocytes. Voltage-dependent changes in RF displacement currents further increased in ShB-IR expressing oocytes after ～120 μM Cu(2+) addition to the external bath. Cu(2+) is known to bind to the ShB-IR ion channel and inhibit Shaker K(+) conductance, indicating that changes in the RF displacement current reported here were associated with RF vibration of the Cu(2+)-linked mobile domain of the ShB-IR protein. Results demonstrate the use of extracellular RF electrodes to interrogate voltage-dependent movement of charged mobile protein domains--capabilities that might enable detection of small changes in charge distribution associated with integral membrane protein conformation and/or drug-protein interactions.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

The interactive effects of fatigue and pH on the ionic conductance of frog sartorius muscle fibers

The effects of fatigue on the membrane conductance of frog sartorius muscle at the resting potential and during an action potential were studied. When muscles were exposed to an extracellular pH of 8.0 the membrane conductance at the resting potential increased during fatigue by about 20% and returned to prefatigue level in about 20 min. The membrane conductance of muscle fibers exposed to pH 6.4 was about three times less than that of pH 8.0 and decreased further during fatigue. Furthermore, the recovery of a normal membrane conductance was slow at pH 6.4. Both the inward, depolarizing and the outward, repolarizing currents during the action potential are reduced in fatigue. In each case the effect is greater at pH 6.4 than at 8.0 and recovery towards normal values is slower at pH 6.4. It is concluded that the ionic conductance of the sarcolemmal membrane at the resting potential and during an action potential are modified by fatigue and that these changes are modulated by pHo.     

Voltage clamp studies on the effect of internal cesium ion on sodium and potassium currents in the squid giant axon

Isolated and cleaned giant axons of Loligo pealii were internally perfused with solutions containing cesium sulfate and potassium fluoride. Membrane currents obtained as a function of clamped membrane potentials indicated a severe depression of the delayed outward current component normally attributed to potassium ion movement. Steady-state currents showed a negative slope in the potential range from -45 to -5 mv which corresponded to the negative slope for the peak sodium current relation vs. membrane potential which suggested long duration sodium currents. Using sodium-free sea water externally, sodium currents were separated from total currents and these persisted for longer times than normal. This result suggested that internal cesium ion delays the sodium conductance turnoff. The separated nonsodium currents showed an abnormal rectification as compared with those predicted by the independence principle, such that while potassium permeability appeared normal at the resting potential, its value decreased progressively with increasing depolarization.     

Inward rectifier K+-channel kinetics from analysis of the complex conductance of Aplysia neuronal membrane.

Conduction in inward rectifier, K+-channels in Aplysia neuron and Ba++ blockade of these channels were studied by rapid measurement of the membrane complex admittance in the frequency range 0.05 to 200 Hz during voltage clamps to membrane potentials in the range -90 to -40 mV. Complex ionic conductances of K+ and Cl- rectifiers were extracted from complex admittances of other membrane conduction processes and capacitance by vector subtraction of the membrane complex admittance during suppressed inward K+ current (near zero-mean current and in zero [K+]0) from complex admittances determined at other [K+]0 and membrane potentials. The contribution of the K+ rectifier to the admittance is distinguishable in the frequency domain above 1 Hz from the contribution of the Cl- rectifier, which is only apparent at frequencies less than 0.1 Hz. The voltage dependence (-90 to -40 mV) of the chord conductance (0.2 to 0.05 microS) and the relaxation time (4-8 ms) of K+ rectifier channels at [K+]0 = 40 mM were determined by curve fits of admittance data by a membrane admittance model based on the linearized Hodgkin-Huxley equations. The conductance of inward rectifier, K+ channels at a membrane potential of -80 mV had a square-root dependence on external K+ concentration, and the relaxation time increased from 2 to 7.5 ms for [K+]0 = 20 and 100 mM, respectively. The complex conductance of the inward K+ rectifier, affected by Ba++, was obtained by complex vector subtraction of the membrane admittance during blockage of inward rectifier, K+ channels (at -35 mV and [Ba++]0 = 5 mM) from admittances determined at -80 mV and at other Ba++ concentrations. The relaxation time of the blockade process decreased with increases in Ba++ concentration. An open-closed channel state model produces the inductive-like kinetic behavior in the complex conductance of inward rectifier, K+ channels and the addition of a blocked channel state accounts for the capacitive-like kinetic behavior of the Ba++ blockade process.     

Calcium-dependent sodium currents inParamecium: Mutational manipulations and effects of hyper- and depolarization

Summary The membrane ofParamecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation,fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that theParamecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca²⁺ concentration. The function of the Ca-dependent Na conductance is discussed.     

Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance

Summary Potassium currents of various durations were obtained from squid giant axons voltage-clamped in artificial seawater solutions containing sufficient tetrodotoxin to block the sodium conductance completely. From instantaneous potassium current-voltage relations, the reversal potentials immediately at the end of these currents were determined. On the basis of these reversal potential measurements, the potassium ion concentration gradient across the membrane was shown to decrease as the potassium current duration increased. The kinetics of this change was shown to vary monotonically with the potassium ion efflux across the membrane estimated from the integral over time of the potassium current divided by the Faraday, and to be independent of both the external sodium ion concentration and the presence or absence of membrane series resistance compensation. It was assumed that during outward potassium current flow, potassium ions accumulated in a periaxonal space bounded by the membrane and an external diffusion barrier. A model system was used to describe this accumulation as a continuous function of the membrane currents. On this basis, the mean periaxonal space thickness and the permeability of the external barrier to K⁺ were found to be 357 Å and 3.21×10^–4 cm/sec, respectively. In hyperosmotic seawater, the value of the space thickness increased significantly even though the potassium currents were not changed significantly. Values of the resistance in series with the membrane were calculated from the values of the permeability of the external barrier and these values were shown to be roughly equivalent to series resistance values determined by current clamp measurements. Membrane potassium ion conductances were determined as a function of time and voltage. When these were determined from data corrected for the potassium current reversal potential changes, larger maximal potassium conductances were obtained than were obtained using a constant reversal potential. In addition, the potassium conductance turn-on with time at a variety of membrane potentials was shown to be slower when potassium conductance values were obtained using a variable reversal potential than when using a constant reversal potential.