The N-Shaped Current-Potential Characteristic in Frog Skin: II. Kinetic Behavior during Ramp Voltage Clamp

Previous step voltage-clamp measurements on frog skin showed the presence of an N-shaped current-potential (I-V) relation in excitable skin. However, the collection and reconstruction of I-V data using discrete step changes of skin potential was tedious because of the long refractory period (up to 1 min) in frog skin. A direct and rapid (5 msec) method for recording the N-shaped I-V characteristic in real time is presented. Ramp functions are used as the command to the clamp system instead of a step function. Consequently the skin potential is forced to change in a linear manner (as commanded) and the skin current can be recorded as a continuous function of the controlled change of skin potential. With the ramp clamp, a low-resistance membrane state ( 10 Omega . cm(2)) resembling a breakdown phenomenon was observed at high skin potential ( 300 mv). Entry into the low resistance state resulted in a collapse of the N-shaped I-V relation to a nearly linear function. The utility of the ramp measurement is demonstrated by predicting (1) that the maximum rate of rise of the spike occurs at a voltage corresponding to the valley (local minimum) in the N-shaped I-V curve, (2) that the rate of rise of the spike increases with increasing clamp currents, (3) the voltage peak of the spike, and (4) the time course of the rising phase of the spike.     

The N-Shaped Current-Potential Characteristic in Frog Skin: III. Ionic Dependence

A ramp voltage clamp measurement described previously is used to detect alterations in the frog skin current-potential (I-V) characteristic following removal or replacement of various ions in the solutions bathing the skin. The ionic requirements for the maintenance of a negative-slope I-V property are the following: Ca⁺⁺, Na⁺, and Cl^- must be in the outside solution; K⁺ and Cl^- must be in the inside solution. Removal of any one of these ions from its respective solution results in the decay and eventual disappearance of the negative slope.

The similarity between the I-V characteristic following Ca⁺⁺ removal with EDTA from the outside solution and the I-V relation in a refractory skin suggests that the loss (refractory state) and recovery of the negative slope is a consequence of unbinding and subsequent rebinding of Ca⁺⁺ to membrane sites. The role of the univalent ions is not clear—presumably some or all of these ions constitute the current through the skin; however, some of these ions may also be involved in maintaining a membrane condition necessary for the existence of a negative slope I-V relation. Further, excitation does not appear to be a direct consequence of the Na⁺ pump.

     

Voltage jump/capacitance relaxation studies of bilayer structure and dynamics. Studies on oxidized cholesterol membranes.

A voltage-jump technique for the study of the time course of small, voltage-induced changes in the structure of single bilayers is presented, and a method is introduced whereby electromechanical (electrostrictive) phenomena can be separated from dielectric relaxations. As no foreign molecules need be introduced into the bilayers, the question about probe artifacts is eliminated. The time constants and amplitudes of dielectric relaxations in oxidized cholesterol bilayers at 21 degrees C, along with their tentative identification are: (a) tau = 3.3 msec, deltaC/Co = 0.8% and tau = 0.7 msec, deltaC/Co = 0.6%: reorientation in the plane of the membrane of domains or clusters of dipoles. (b) tau = 155 musec, deltaC/Co = 1.5-3%: rotational reorientation of individual molecules. (c) tau = 18 musec, deltaC/Co - 1.4%: small amplitude reorientations individual dipoles about an axis lying in the plane of the membrane. Electrostictive effects with time constants between about 2 and 50 msec were also detected. A temperature study of both the dielectric and electrostrictive phenomena is reported. The application of the technique to other membrane compositions and to a variety of BLM problems is discussed.     

ACTION POTENTIAL OF NITELLA INTERNODES

The ionic current during a non-propagating action potentialis analysed from the voltage clamp experiments. The shape ofthe action potential of the Nitella internode can be reconstructedfrom the data of the voltage clamp experiments. The N-shapedcurrent-voltage characteristics (I-V curve) of the Nitella membraneis not constant with time as it is in the tunnel diode, butdecays with time, converging finally into a delayed rectificationcurve. The temporal locus of the potential at which each I-Vcurve crosses the voltage axis coincides almost exactly withthe action potential. The membrane resistance which is calculatedfrom the slope of the I-V curve at each intersection with thevoltage axis also changes in parallel to the action potential.Such correlations are found in the Nitella not only in the pondwater, but also in high Na, high Ca or high Mg medium, wherethe shape of the action potential is modified in various ways.It is highly probable that the action potential is a locus ofthe change of the membrane potential so that the net membranecurrent may be maintained at zero after the transient modificationof the membrane structure by stimulation. (Received June 30, 1966; )     

Influence of intercellular clefts on potential and current distribution in a multifiber preparation.

A theoretical model is presented for current and voltage clamp of multifiber bundles in a double sucrose gap. Attention is focused on methodological errors introduced by the intercellular cleft resistance. The bundle is approximated by a continuous geometry. Voltage distribution, as a function of radial distance and time, is defined by a parabolic partial differential equation which is specified for different membrane characteristics. Assuming a linear membrane, analytical solutions are given for current step and voltage step conditions. The theoretical relations (based on Bessel functions) may be used to calculate membrane conductance and capacity from experimental clamp data. The case of a nonlinear membrane with standard Hodgkin-Huxley kinetics for excitatory Na current is treated assuming maximum Na conductances (gNa) of 120, 10, and 1 mmho/cm2. Numerical simulations are presented for potential and current distribution in a bundle of 60 microns diameter during depolarizing voltage steps. Adequate voltage control is restricted to the peripheral fibers of the bundle whereas the membrane potential of the inner fibers deviates from the command level during early inward current, tending to the Na equilibrium potential. In the peak current-voltage diagram the loss of voltage control is reflected by an increased steepness of the negative region and a decreased slope conductance of the positive region. With gNa = 120 mmho/cm2, the positive slope conductance is approximately 25% of the slope expected from ideal space clamping. With the lower values of gNa, the slope conductance ratio is in the order of 50%. Implications of the results for an experimental voltage clamp analysis of early inward current on multifiber preparations are discussed.     

The voltage-dependent proton pumping in bacteriorhodopsin is characterized by optoelectric behavior

The light-driven proton pump bacteriorhodopsin (bR) was functionally expressed in Xenopus laevis oocytes and in HEK-293 cells. The latter expression system allowed high time resolution of light-induced current signals. A detailed voltage clamp and patch clamp study was performed to investigate the DeltapH versus Deltapsi dependence of the pump current. The following results were obtained. The current voltage behavior of bR is linear in the measurable range between -160 mV and +60 mV. The pH dependence is less than expected from thermodynamic principles, i.e., one DeltapH unit produces a shift of the apparent reversal potential of 34 mV (and not 58 mV). The M(2)-BR decay shows a significant voltage dependence with time constants changing from 20 ms at +60 mV to 80 ms at -160 mV. The linear I-V curve can be reconstructed by this behavior. However, the slope of the decay rate shows a weaker voltage dependence than the stationary photocurrent, indicating that an additional process must be involved in the voltage dependence of the pump. A slowly decaying M intermediate (decay time > 100 ms) could already be detected at zero voltage by electrical and spectroscopic means. In effect, bR shows optoelectric behavior. The long-lived M can be transferred into the active photocycle by depolarizing voltage pulses. This is experimentally demonstrated by a distinct charge displacement. From the results we conclude that the transport cycle of bR branches via a long-lived M(1)* in a voltage-dependent manner into a nontransporting cycle, where the proton release and uptake occur on the extracellular side.     

Changes in the electrical properties of atrial trabecula induced by an electric stimulus]

Electrical activity, excitation threshold and "steady--state" current-voltage, relationship (estimated under voltage clamp conditions) in the membrane of chicken embryo atrial trabecula were investigated by means of double succrose-gap technique. Application of short (10 msec) electrical stimuli with magnitudes up to 6.10(-5) a (1-150 threshold) resulted in increased excitability and appearance of spontaneous activity. Release of neurotransmitters and polarity of the applied sitmulus are shown not to play a significant role in the changes in membrane excitability observed, the main cause of the latter being the increased sodium permeability of the membrane.     

Response of delayed (K+) channels to the time-dependent clamping function in squid giant axon. I. Ascending ramps

Squid giant axons are voltage-clamped with ascending potential ramps whose slopes range from 0.5 mV/msec to 60 mV/msec and delayed (K+) currents are observed. The parametric current-voltage curves exhibit a delay period of minimal current followed by a rapid increase of current toward a final steady state. Both the initial delay and the slope of the subsequent rising phase increase with increasing ramp slope. When the Hodgkin-Huxley equations are used to generate theoretical current-voltage curves, the sharp difference between the delay and rising phases is muted and the ramp slope must be increased to produce an adequate representation of the data. A muted biphasic response is also observed when the current-voltage curves are generated using modified Hodgkin-Huxley parameters and a correction for K+ accumulation in the periaxonal space. These modified equations provide an accurate fit for step-potential clamp current data. Since the ramp experiments include all relevant clamping potentials, the experiments provide a sensitive test for kinetic models of K+ on flow in the delayed (K+) channels of squid giant axon.     

Two Light-Induced Processes in the Photoreceptor Cells of Limulus Ventral Eye

The dark-adapted current-voltage (I-V) curve of a ventral photoreceptor cell of Limulus, measured by a voltage-clamp technique, has a high slope-resistance region more negative than resting voltage, a lower slope-resistance region between resting voltage and zero, and a negative slope-resistance region more positive than 0 v. With illumination, we find no unique voltage at which there is no light-induced current. At the termination of illumination, the I-V curve changes quickly, then recovers very slowly to a dark-adapted configuration. The voltage-clamp currents during and after illumination can be interpreted to arise from two separate processes. One process (fast) changes quickly with change in illumination, has a reversal potential at +20 mv, and has an I-V curve with positive slope resistance at all voltages. These properties are consistent with a light-induced change in membrane conductance to sodium ions. The other process (slow) changes slowly with changes in illumination, generates light-activated current at +20 mv, and has an I-V curve with a large region of negative slope resistance. The mechanism of this process cannot as yet be identified.     

Short and middle latency vestibular evoked responses to acceleration in man

We have succeeded in recording short and middle latency vestibular evoked responses in human subjects. The head was held rigidly in a special, patented head holder, constructed individually for each subject, which gripped the teeth of the upper jaw. The stimulus consisted of 2/sec steps of angular acceleration impulses produced by a special motor with intensities of about 10,000°/sec² and with a rise time of 1–2 msec. The electrical activity was recorded as the potential difference between special forehead and mastoid electrodes having a large, secure contact area with the skin. The activity was digitally filtered and averaged in 2 separate channels by means of a Microshev 2000 evoked response system. The short latency responses, with peaks at about 3.5 msec (forehead positive), 6.0 msec (forehead negative) and 8.4 msec (forehead positive; bandpass: 200–2000 Hz; average of 1024 trials), had amplitudes of about 0.5 μV. The middle latency responses had peaks at about 8.8 msec (forehead positive), 18.8 msec (forehead negative) and 26.8 msec (forehead positive; 30–300 Hz; N = 128 trials), with larger amplitudes (about 15 μV). These responses were consistently recorded in the same subject at different times and were similar in different normal subjects. Strenuous control experiments were conducted in order to ensure that these responses are not artefacts due to the movement of conducting media (head, electrodes and leads) in the electromagnetic field of the motor and are elicited by activation of normal labyrinths. Among other controls, they were not present in a cadaver, in patients with bilateral absence of nystagmus to caloric stimuli and in conducting volumes the size of the human head. They were also not masked by white noise.     

The pacemaker current in cardiac Purkinje myocytes

It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time- dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [K+]o to 10.8 mM almost abolished iKdd. Cs+ (2 mM) markedly reduced or blocked the time-dependent current at potentials positive and negative to EK. Ba2+ (4 mM) abolished the time-dependent current in its usual range of potentials and unmasked another time-dependent current (presumably i(f)) with a threshold of approximately -90 mV (> 20 mV negative to that of the time-dependent current in Tyrode solution). During more negative steps, i(f) increased in size and did not reverse. During i(f) the slope conductance measured with small (8-10 mV) superimposed clamp steps increased. High [K+]o (10.8 mM) markedly increased and Cs+ (2 mM) blocked i(f). We conclude that: (a) in the absence of Ba2+, a time-dependent current does reverse near EK and its reversal is unrelated to K+ depletion; (b) the slope conductance of that time-dependent current decreases in the absence of K+ depletion at potentials positive to EK where inactivation of iK1 is unlikely to occur. (c) Ba2+ blocks this time-dependent current and unmasks another time-dependent current (i(f)) with a more negative (> 20 mV) threshold and no reversal at more negative values; (d) Cs+ blocks both time-dependent currents recorded in the absence and presence of Ba2+. The data suggest that in the diastolic range of potentials in Purkinje myocytes there is a voltage- and time-dependent K+ current (iKdd) that can be separated from the hyperpolarization- activated inward current i(f).     

Procaine effects on the sodium transport in frog skin

A study on the influence of procaine on the sodium transport properties in frog skin was carried out. The application of procaine hydrochloride on either the mucosal or the serosal sides of the isolated frog skin has opposite effects. When added to the mucosal compartment, the procaine (as well as two procaine based drugs: Gerovital H3 and Aslavital) biphasically increase the short-circuit current (Isc) with a noticeable "recline" phenomenon, and decrease the slope resistance, as given by the I-V curves. When applied in the serosal compartment, Isc is decreased and the slope resistance of the epithelium is increased. The procaine effect on the apical membranes shows a pronounced dependence on the external sodium concentration. The shift of the E2 inflection point (which indicates the critical intensity of the electric field at which the epithelial conductance changes), with respect to the transepithelial open-circuit potential, shows a rapid and quasi-exponential increase following the application of 25 mM procaine in addition to the different mucosal Na concentrations.     

Sodium currents in the giant axon of the crab Carcinus maenas

Measurements were made of the kinetics and steady-state properties of the sodium conductance changes in the giant axon of the crab Carcinus maenas. The conductance measurements were made in the presence of small concentrations of tetrodotoxin and as much electrical compensation as possible in order to minimize errors caused by the series resistance. After an initial delay of 10-150 microsec, the conductance increase during depolarizing voltage clamp pulses followed the Hodgkin-Huxley kinetics. Values of the time constant for the activation of the sodium conductance lay on a bell-shaped curve with a maximum under 180 microsec at -40 mV (at 18 degrees C). Values of the time constant for the inactivation of the sodium conductance were also fitted using a bell-shaped curve with a maximum under 7 msec at -70 mV. The effects of membrane potential on the fraction of Na channels available for activation studied using double pulse protocols suggest that hyperpolarizing potentials more negative than -100 mV lock a fraction of the Na channels in a closed conformation.     

Background K+ current in isolated canine cardiac Purkinje myocytes.

The current-voltage (I-V) relation of the background current, IK1, was studied in isolated canine cardiac Purkinje myocytes using the whole-cell, patch-clamp technique. Since Ba2+ and Cs+ block IK1, these cations were used to separate the I-V relation of IK1 from that of the whole cell. The I-V relation of IK1 was measured as the difference between the I-V relations of the cell in normal Tyrode (control solution) and in the presence of either Ba2+ (1 mM) or Cs+ (10 mM). Our results indicate that IK1 is an inwardly rectifying K+ current whose conductance depends on extracellular potassium concentration. In different [K+]0's the I-V relations of IK1 exhibit crossover. In addition the I-V relation of IK1 contains a region of negative slope (even when that of the whole cell does not). We also examined the relationship between the resting potential of the myocyte, Vm, and [K+]0 and found that it exhibits the characteristic anomalous behavior first reported in Purkinje strands (Weidmann, S., 1956, Elektrophysiologie der Herzmuskelfaser, Med. Verlag H. Huber), where lowering [K+]0 below 4 mM results in a depolarization.     

Properties of Mg(2+)-dependent cation channels in human leukemia K562 cells

The endogenous Mg(2+)-inhibited cation (MIC) current was recently described in different cells of hematopoietic lineage and was implicated in the regulation of Mg2+ homeostasis. Here we present a single channel study of endogenously expressed Mg(2+)-dependent cation channels in the human myeloid leukemia K562 cells. Inwardly directed unitary currents were activated in cell-attached experiments in the absence of Ca2+ and Mg2+ in the pipette solution. The current-voltage (I-V) relationships displayed strong inward rectification and yielded a single channel slope conductance of approximately 30 pS at negative potentials. The I-V relationships were not altered by patch excision into divalent-free solution. Channel open probability (P(o)) and mean closed time constant (tau(C)) were strongly voltage-dependent, indicating that gating mechanisms may underlie current inward rectification. Millimolar concentrations of Ca2+ or Mg2+ applied to the cytoplasmic side of the membrane produced slow irreversible inhibition of channel activity. The Mg(2+)-dependent cation channels described in this study differ from the MIC channels described in human T-cells, Jurkat, and rat basophilic leukemia (RBL) cells in their I-V relationships, kinetic parameters and dependence on intracellular divalent cations. Our results suggested that endogenously expressed Mg(2+)-dependent cation channels in K562 cells and the MIC channels in other hematopoietic cells might be formed by different channel proteins.     

Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases

Modulation of the current generated by the Na+/K+ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I-V) relationship of the Na+/K+ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent Km value for pump stimulation by external [K+] (Rakowski et al., 1991). To study this feature in more detail, Xenopus oocytes were used for comparative analysis of the negative slope of the I-V relationship of the endogenous Na+/K+ pump and of the Na+/K+ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na(+)-free solution and in the presence of high concentrations of Ba2+ and tetraethylammonium, to block all nonpump related K(+)-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K+. The voltage dependence of the Km value for stimulation of the Na+/K+ pump by external [K+] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the Km value for the Na+/K+ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K+] and can be interpreted by voltage-dependent K+ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge. Application of 1,2-dioctanoyl-sn-glycerol (diC8, 10-50 microM), which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K+ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent Km at 0 mV increased from 0.46 to 0.83 mM. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impulse Activity Evokes Precocious Sprouting of Nociceptive Nerves into Denervated Skin

We have studied the sprouting of intact high-threshold mechanosensory nerves into adjacent denervated trunk skin in adult rats behaviorally, histologically, and electrophysiologically. In the anesthetized animal, stimulation of high-threshold endings in back skin by localized pinching elicits a bilateral reflex excitation of the underlying skeletal muscle, the cutaneous trunci muscle (CTM), visible as a twitch-like puckering of the skin. The reflex was also evoked by electrical excitation of Aδ and of C fibers in the dorsal cutaneous nerves (DCNs), with characteristic latencies of 7–20 msec and 40–60 msec, respectively; excitation of low-threshold (Aα) fibers was ineffective. After cutting selected DCNs, the deprived skin became insensible, but pinch responsiveness gradually recovered over the following 2 weeks. Regeneration of cut axons was not responsible for this recovery; when neighboring intact DCNs were cut, however, all responses were abolished in the recovered skin that had been initially denervated. By 3–5 days after denervation, axons in the dermis were all histologically absent or degenerating; when pinch sensitivity was restored to such skin, silver-stainable axons reappeared in the formerly empty Schwann tubes. During the work we noticed that the periodic examination by pinching, used to follow the time course of recovery of function in individual animals, led to an earlier development of this recovery than in animals that were examined only once at a specified time after denervation, This apparent acceleration in the redevelopment of pinch sensitivity was correlated with the appearance of axons in the recovered skin, and was shown to be due to the impulse activity evoked in the remaining nerves by the periodic pinching; it did not occur when the nerves were blocked by tetrodotoxin (TTX), and it was mimicked by a brief (10-min) period of electrical excitation of the Aδ fibers in a remaining nerve carried out at the time when the denervation of skin was done. The time course of the phenomenon suggested that the principal effect of the impulses was to shorten the latency to the onset of sprouting in the activated Aδ axons; that is, they induced precocious sprouting. The impulses needed to be conducted centrally for the effect to occur, and precocious sprouting failed to occur if the impulses were allowed to proceed only distally toward the skin.     

Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes

Whole-cell currents were recorded in guinea pig ventricular myocytes at approximately 36 degrees C before, during, and after exposure to maximally effective concentrations of strophanthidin, a cardiotonic steroid and specific inhibitor of the Na/K pump. Wide-tipped pipettes, in combination with a device for exchanging the solution inside the pipette, afforded reasonable control of the ionic composition of the intracellular solution and of the membrane potential. Internal and external solutions were designed to minimize channel currents and Na/Ca exchange current while sustaining vigorous forward Na/K transport, monitored as strophanthidin-sensitive current. 100-ms voltage pulses from the -40 mV holding potential were used to determine steady-state levels of membrane current between -140 and +60 mV. Control experiments demonstrated that if the Na/K pump cycle were first arrested, e.g., by withdrawal of external K, or of both internal and external Na, then neither strophanthidin nor its vehicle, dimethylsulfoxide, had any discernible effect on steady-state membrane current. Further controls showed that, with the Na/K pump inhibited by strophanthidin, membrane current was insensitive to changes of external [K] between 5.4 and 0 mM and was little altered by changing the pipette [Na] from 0 to 50 mM. Strophanthidin-sensitive current therefore closely approximated Na/K pump current, and was virtually free of contamination by current components altered by the changes in extracellular [K] and intracellular [Na] expected to accompany pump inhibition. The steady-state Na/K pump current-voltage (I-V) relationship, with the pump strongly activated by 5.4 mM external K and 50 mM internal Na (and 10 mM ATP), was sigmoid in shape with a steep positive slope between about 0 and -100 mV, a less steep slope at more negative potentials, and an extremely shallow slope at positive potentials; no region of negative slope was found. That shape of I-V relationship can be generated by a two-state cycle with one pair of voltage-sensitive rate constants and one pair of voltage-insensitive rate constants: such a two-state scheme is a valid steady-state representation of a multi-state cycle that includes only a single voltage-sensitive step.     

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.     

Sodium ions as blocking agents and charge carriers in the potassium channel of the squid giant axon

Instantaneous K channel current-voltage (I-V) relations were determined by using internally perfused squid axons. When K was the only internal cation, the I-V relation was linear for outward currents at membrane potentials up to +240 mV inside. With 25-200 mM Na plus 300 mM K in the internal solution, an N-shaped I-V curve was seen. Voltage-dependent blocking of the K channels by Na produces a region of negative slope in the I-V plot (F. Bezanilla and C. M. Armstrong. 1972. J. Gen Physiol, 60: 588). At higher voltages (greater than or equal to 160 mV) we observed a second region of increasing current and a decrease in the fraction of the K conductance blocked by Na. Internal tetraethylammonium (TEA) ions blocked currents over the whole voltage range. In a second series of experiments with K-free, Na-containing internal solutions, the I-V curve turned sharply upward about +160 mV. The current at high voltages increased with increasing internal Na concentration was largely blocked by internal TEA. These data suggest that the K channel becomes substantially more permeable to Na at high voltages. This change is apparently responsible for the relief, at high transmembrane voltages, of the blocking effect seen in axons perfused with Na plus K mixtures. Each time a Na ion passed through, vacating the blocking site, the channel would transiently allow K ions to pass through freely.