Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt

Summary Electrical membrane properties of solitary spiking cells during newt (Cynops pyrrhogaster) retinal regeneration were studied with whole-cell patch-clamp methods in comparison with those in the normal retina.The membrane currents of normal spiking cells consisted of 5 components: inward Na⁺ and Ca⁺⁺ currents and 3 outward K⁺ currents of tetraethylammonium (TEA)-sensitive, 4-aminopyridine (4-AP)-sensitive, and Ca⁺⁺-activated varieties. The resting potential was about -40mV. The activation voltage for Na⁺ and Ca⁺⁺ currents was about -30 and -17 mV, respectively. The maximum Na⁺ and Ca⁺⁺ currents were about 1057 and 179 pA, respectively.In regenerating retinae after 19–20 days of surgery, solitary cells with depigmented cytoplasm showed slowrising action potentials of long duration. The ionic dependence of this activity displayed two voltage-dependent components: slow inward Na⁺ and TEA-sensitive outward K⁺ currents. The maximum inward current (about 156 pA) was much smaller than that of the control. There was no indication of an inward Ca⁺⁺ current.During subsequent regeneration, the inward Ca⁺⁺ current appeared in most spiking cells, and the magnitude of the inward Na⁺, Ca⁺⁺, and outward K⁺ currents all increased. By 30 days of regeneration, the electrical activities of spiking cells became identical to those in the normal retina. No significant difference in the resting potential and the activation voltage for Na⁺ and Ca⁺⁺ currents was found during the regenerating period examined.     

Postactivation inhibition of spontaneously active neurosecretory neurons in the medicinal leech

Spontaneously active neurosecretory neurons in vertebrate and invertebrate nervous systems share similarities in firing frequencies, spike shapes, inhibition by the transmitters they themselves release and postactivation inhibition, an intensity-dependent period of suppressed spontaneous generation of action potentials following phases of high-frequency activity. High-frequency activation of spontaneously active serotonin-containing Retzius cells in isolated ganglia of the leech Hirudo medicinalis induced prolonged membrane hyperpolarisations causing periods of postactivation inhibition of up to 33 s. The duration of the inhibitory periods was directly related to both the number and rate of spikes during activation and was inversely proportional to a cell’s spontaneous firing frequency. The periods of postactivation inhibition remained unaffected by both serotonin depletion through repeated injections of 5,7-dihydroxytryptamine and suppressing the afterhyperpolarisation following each action potential with tetraethylammonium (TEA), iberiotoxin or charybdotoxin, suggesting that neither autoinhibition by synaptic release of serotonin nor calcium-activated potassium channels contribute to the underlying mechanism. In contrast, the postactivation inhibitory period was significantly affected both by differential electrical stimulation of the same Retzius cells via microelectrodes filled with molar concentrations of either Na⁺-acetate or K⁺-acetate, and by partial inhibition of Na⁺/K⁺-ATPase with ouabain. Thus, postactivation inhibition in Retzius cells results from prolonged hyperpolarising activity of Na⁺/K⁺-ATPase stimulated by the accumulation of cytosolic Na⁺ during phases of high-frequency spike activity.     

The Sodium-Potassium Pump Controls the Intrinsic Firing of the Cerebellar Purkinje Neuron

In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na⁺/K⁺ pump activity sets the Purkinje cell''s operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na⁺/K⁺ pump. The model can replicate these recordings. We propose that Na⁺/K⁺ pump activity controls the intrinsic firing mode of cerbellar Purkinje cells.     

Measurement of plasma membrane potentials of yeast cells with glass microelectrodes

Glass microelectrodes were used to measure the electrical potential difference (Δψ) across plasma membrane of the yeast Pichia humboldtii. The cells were captured in the neck of a glass microfunnel and impaled with a glass microelectrode. The measurements were reproducible and stable for several minutes. The highest Δψ values were obtained in cells metabolizing glucose at pH 6. Δψ in cells deenergized by uncouplers or in dead cells was reduced to about one third of the maximal value. This residual Δψ probably represented Donnan potential. Δψ also was reduced by increasing concentrations of K⁺ in the medium. Other monovalent cations were distinctly less effective: Li⁺ ⪡ Na⁺ < K⁺, and Ca²⁺ was without effect. These experiments prove the applicability of the electrophysiological technique on yeast cells and thus open the way for direct determination of the electrical component of the plasma membrane electrochemical proton gradient.     

Effect of external ATP on the plasma membrane permeability and (Na++K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na⁺ relative to K⁺, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K⁺ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na⁺. Under these conditions the pumping activity of the Neuro-2A (Na⁺+K⁺)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na⁺+K⁺)-ATPase for ouabain, as measured by direct [³H]ouabain-binding studies and by inhibition studies of active K⁺ uptake. In the presence of 3.5 mM ATP and the absence of external K⁺ both techniques indicate an apparent dissociation constant for ouabain of 2·10^?6 M. Neuro-2A cells contain (3.5±0.7)·10⁵ ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10^?20 mol K⁺/min per copy of (Na⁺+K⁺)-ATPase at room temperature.     

Physiological evidence for a proton pump and sodium exclusion mechanisms at the plasma membrane of the marine angiosperm Zostera marina L

The basic electrical plasma membrane characteristics of leaf cells from the seagrass Zostera marina L. have been investigated with respect to its primary transport system and its Na⁺/K⁺ selectivity. In natural seawater Z. marina exhibits a membrane potential of -15610 mV. The phytotoxin fusicoccin stimulates H⁺ extrusion and hyperpolarizes the plasma membrane. Ouabain, an inhibitor of the mammalian Na⁺K⁺-ATPase did not depolarize the plasma membrane of Z.marina. Both flushing the leaves with CO2 and 'light off' acidified the cytoplasm and hyperpolarized the cells. It is suggested that a H⁺-ATPase rather than a Na⁺-ATPase is the primary pump in Z.marina. In the presence of cyanide plus salicylhydroxamic acid the membrane potential changed to -6411 mV. This so-called diffusion potential was sensitive to external [K⁺] from 0.05 to 0.5 mM in the presence of 0.5 M Na⁺ and revealed a relative permeability PK⁺/PNa⁺ of 303. We suggest that this high ratio is the basic adaptation which permits Z. marina to grow in high [Na⁺] conditions and to exhibit a rather negative resting potential. Since amiloride, an inhibitor of the nH⁺/Na⁺ antiporter, hyperpolarized the plasma membrane, it is suggested that this transporter could be present in the plasma membrane of Z. marina acting as an overflow valve for Na⁺ which leaks into the cell.     

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon

Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na⁺ or K⁺ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na⁺ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K⁺ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f _c=15.6±0.91 Hz and a mean plateau valueS _o=(7.04±2.94)×10^–20 A² sec/cm². The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K⁺ entry across the apical membrane. Elimination of the K⁺ gradient by bathing the colon on both sides with K⁺ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs⁺ or tetraethylammonium (TEA) and by serosal addition of Ba²⁺. The one-sided action of these K⁺ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K⁺ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na⁺ transport pathway.     

Volume changes of mammalian cells subjected to hypotonic solutions in vitro: evidence for the requirement of a sodium pump for the shrinking phase

A Coulter-orifice pulse-height analyzer system was used to measure volume spectra of mammalian cells in suspension at different times after the addition of an equal volume of water. In appropriate hypotonic medium, cultured mammalian cells rapidly increase in volume and then shrink, more slowly, approaching their initial volumes within 20 to 30 minutes at 37.5°C. The shrinking phase was found to be reversibly inhibited by ouabain and inhibited in both K⁺-free and Na⁺-free solutions; neither choline⁺ nor Li⁺ could substitute for extracellular Na⁺ in supporting the shrinking phenomenon but Rb⁺ and Cs⁺ were fairly good substitutes for K⁺. Under conditions similar to those with which the shrinking phenomenon was observed with cultured cells, it was not found with either human or mouse red blood cells. Two methods were used to determine intracellular Na⁺ and K⁺ content in osmotically shocked cells and in unshocked controls. An isotope equilibration method was employed with L5178-Y mouse lymphoblasts and a chemical determination by flame photometry was used with Ehrlich ascites tumor cells. The K⁺ content was significantly reduced and the Na⁺ content was unchanged or somewhat increased in cells which had returned to their original volumes in hypotonic medium. The K⁺ content was even more reduced but the Na⁺ content was greatly increased in cells which were osmotically shocked in the presence of ouabain.     

Kinetics of charge translocation in the passive downhill uptake mode of the Na/H antiporter NhaA of Escherichia coli

The Na⁺/H⁺ antiporter NhaA is the main Na⁺ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li⁺ and Na⁺ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na⁺ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K_m values were 11 mM for Na⁺ and 7.3 mM for Li⁺ at basic pH and 180 mM for Na⁺ and 50 mM for Li⁺ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K_m as well as turnover (V_max).     

Ca-sensitive K channels in phagocytic cell membranes

In phagocytic cells evidence for properties of Ca²⁺-sensitive K⁺-selective channels comes mostly from electrophysiological studies. Macrophages and macrophage-like cells are compared with fibroblasts (L-cells) where the Ca⁺-dependent K⁺ conductance is better understood. This model shares a mesenchymal origin and an accessory phagocytic capacity with the professional phagocytes. In macrophages several values of transmembrane potentials have been measured by different groups, using various techniques. Microelectrode measurements have demonstrated a voltage-dependent K⁺ conductance involved in transition from low to high membrane potentials. Current-voltage relationships in mouse peritoneal exudate cells have revealed a region of negative slope resistance. Slow calcium spikes were found in a subpopulation of cells from human dialysis fluid that appear to be distinct from typical macrophages. Action potentials have been recorded from human monocyte-derived macrophages. Their ionic mechanism has not yet been established. Spontaneous and electrically elicited slow membrane hyperpolarizations have been described in macrophages and macrophage-like cells. Similar activity is well known in L-cells and in both cases it is possible to identify a Ca²⁺-sensitive K⁺ conductance as the underlying mechanism. Phagocytosis is a cell function that has been related to membrane hyperpolarization and to slow hyperpolarizing activity. In some cases no changes of electrical activity have been observed during the phagocytic process. Chemotactic factors induce membrane hyperpolarizations in macrophages, but the relation between electrical change and cell motility has not been established. Exocytosis, a is another Ca²⁺ sensitive cell function that awaits correlation with electrochemical changes. The evidences accumulated to date are compatible with several models for gating and modulation of the voltage-independent K⁺ conductance by Ca²⁺. The use of higher resolution techniques, such as patch-clamp, with well defined subpopulations of phagocytic cells may produce the missing link in the transduction of membrane signals into the specifically targeted cell functions.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Characterization of the cardiac Na/K pump by development of a comprehensive and mechanistic model

A large amount of experimental data on the characteristics of the cardiac Na⁺/K⁺ pump have been accumulated, but it remains difficult to predict the quantitative contribution of the pump in an intact cell because most measurements have been made under non-physiological conditions. To extrapolate the experimental findings to intact cells, we have developed a comprehensive Na⁺/K⁺ pump model based on the thermodynamic framework (Smith and Crampin, 2004) of the Post-Albers reaction cycle combined with access channel mechanisms. The new model explains a variety of experimental results for the Na⁺/K⁺ pump current (I_NaK), including the dependency on the concentrations of Na⁺ and K⁺, the membrane potential and the free energy of ATP hydrolysis. The model demonstrates that both the apparent affinity and the slope of the substrate-I_NaK relationship measured experimentally are affected by the composition of ions in the extra- and intracellular solutions, indirectly through alteration in the probability distribution of individual enzyme intermediates. By considering the voltage dependence in the Na⁺- and K⁺-binding steps, the experimental voltage-I_NaK relationship could be reconstructed with application of experimental ionic compositions in the model, and the view of voltage-dependent K⁺ binding was supported. Re-evaluation of charge movements accompanying Na⁺ and K⁺ translocations gave a reasonable number for the site density of the Na⁺/K⁺ pump on the membrane. The new model is relevant for simulation of cellular functions under various interventions, such as depression of energy metabolism.     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.     

Localization of Na+, K+-ATPase to the inside of the basolateral cell membranes of epithelial cells of proximal and distal tubules in rabbit kidney

Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na⁺, K⁺-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na⁺, K⁺-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na⁺, K⁺-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na⁺, K⁺-ATPase sites, which are involved in extrusion of Na⁺ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.     

Early changes in membrane potential of Saccharomyces cerevisiae induced by varying extracellular K+, Na+ or H+ concentrations

Recently we introduced a fluorescent probe technique that makes possible to convert changes of equilibrium fluorescence spectra of 3,3’-dipropylthiadicarbocyanine, diS-C₃(3), measured in yeast cell suspensions under defined conditions into underlying membrane potential differences, scaled in millivolts (Plasek et al. in J Bioenerg Biomembr 44: 559–569, 2012). The results presented in this paper disclose measurements of real early changes of plasma membrane potential induced by the increase of extracellular K⁺, Na⁺ and H⁺ concentration in S. cerevisiae with and without added glucose as energy source. Whereas the wild type and the ?tok1 mutant cells exhibited similar depolarization curves, mutant cells lacking the two Trk1,2 potassium transporters revealed a significantly decreased membrane depolarization by K⁺, particularly at lower extracellular potassium concentration [K⁺]_out. In the absence of external energy source plasma membrane depolarization by K⁺ was almost linear. In the presence of glucose the depolarization curves exhibited an exponential character with increasing [K⁺]_out. The plasma membrane depolarization by Na⁺ was independent from the presence of Trk1,2 transporters. Contrary to K⁺, Na⁺ depolarized the plasma membrane stronger in the presence of glucose than in its absence. The pH induced depolarization exhibited a fairly linear relationship between the membrane potential and the pH_o of cell suspensions, both in the wild type and the Δtrk1,2 mutant strains, when cells were energized by glucose. In the absence of glucose the depolarization curves showed a biphasic character with enhanced depolarization at lower pH_o values.     

The Sodium-Potassium Exchange Pump: II. Analysis of Na+-Loaded Frog Sartorius Muscle

A model for the Na-K exchange pump was applied to data on Na⁺-loaded frog sartorius muscle, and was used to relate the rate of adenosine triphosphate (ATP) hydrolysis to the electrical properties of the cell membrane. Membrane hyperpolarization was considered to arise from an electrical current which was produced by the hydrolysis reaction coupled to ion movements, and which flowed across the membrane. The reaction rate, as calculated from hyperpolarization, agreed with direct measurements of ATP hydrolysis and with the rate estimated from Na⁺ tracer efflux studies. Although Na⁺ is actively extruded, the model showed that K⁺ is inwardly transported if the potassium permeability of the membrane is less than about 6.6 × 10^-6 cm/sec, as is suggested by resistance data. Calculations indicated that the reaction conductance L_rr was relatively constant when compared with the reaction rate and reaction free energy for large changes in internal and external ionic concentrations. Its value agreed with the value obtained from the dependence of Na⁺ tracer efflux on external K⁺. A set of experiments was suggested which would provide a more complete interpretation of the data.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Generation of plasma membrane potential by the Na+-pump coupled to proton extrusion

Lettré cells maintain a plasma membrane potential near — 60mV, yet are scarcely depolarized by 80 mM Rb⁺ and are relatively impermeable to ⁸⁶Rb⁺. They are depolarized by ouabain without a concomitant change in intracellular cation content. Addition of K⁺ to cells suspended in a K⁺ free medium, or of Na⁺ to cells in a Na⁺ free medium, hyperpolarizes the cells. They contain electroneutral transport mechanisms for Na⁺, K⁺ and H⁺ which can function as Na⁺:K⁺ and Na⁺:H⁺ exchanges. It is concluded that plasma membrane potential of Lettré cells, in steady-state for Na⁺ and K⁺, is produced by an electrogenic Na⁺ pump sustained by electroneutral exchanges, and restricted by anion leakage.     

Nerve Growth Factor influences potassium movements in chick embryo dorsal root ganglionic cells

Recently we have shown that Nerve Growth Factor (NGF) influences the movement of Na⁺ across the membrane of chick embryo dorsal root ganglion (DRG) cells. When cell dissociates from 8-day embryonic chick DRG, equilibrated with ⁸⁶Rb⁺ (a K⁺ analog) in the presence of NGF, were transferred to NGF-free medium a marked loss of intracellular K⁺ occurred over several hours. The time course of K⁺ loss was similar to the time course of Na⁺ accumulation which occurs in the absence of NGF. NGF-deprived, K⁺-depleted DRG cells reaccumulated K⁺ within minutes of delayed NGF presentation, just as delayed NGF administration results in the rapid extrusion of Na⁺ from Na⁺-loaded cells. Restoration of K⁺ competence was dependent upon NGF concentration. The occurrence of this K⁺ response to exogenous NGF in other ganglionic preparations correlated with traditional responses to NGF in culture and previously observed Na⁺ responses. Neither the development nor the expression of the ionic defect (K⁺ depletion, Na⁺ filling) during NGF deprivation required the presence of both cations in the medium. NGF-dependent restoration of intracellular K⁺ in NGF-deprived chick DRG cells required the presence of intracellular Na⁺, and NGF-dependent extrusion of Na⁺ required extracellular K⁺. Thus NGF appears to influence the coupled (active) movements of Na⁺ and K⁺ across the membrane of its target cells, possibly by means of the classical Na⁺, K⁺-ATPase pump.