Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes II. Extrinsic influence of cations

Use-dependent declines of Na⁺ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na⁺), divalent (Ca²⁺, Mg²⁺) and trivalent (La²⁺) cations on use dependence were found: Increasing the Ca²⁺ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca²⁺. Doubling the external Na⁺ concentration in 0.2 mM Ca²⁺ solutions did not initiate phasic block. External Mg²⁺ ions induced a smaller, and La²⁺ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La²⁺. The static block of Na⁺ currents by La³⁺ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na⁺ channel whose affinity for TTX or STX depends oni) the gating conformation of the channel, probably the inactivation andii) the occupancy of a blocking site by di- or trivalent external cations.     

Voltage-dependent sodium channels in human small-cell lung cancer cells: Role in action potentials and inhibition by lambert-eaton syndrome IgG

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na⁺ channels are only weakly tetrodotoxin (TTX)-sensitive (K _d =215 mm). With the membrane potential maintained at –60 to –80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (>20 pA). Similar all-ornothing spikes were also observed with anodal break excitation. Removal of external Ca²⁺ did not affect the action potential production, whereas 5 m TTX or substitution of Na⁺ with choline abolished it. Action potentials elicited in the Ca²⁺-free condition were reversibly blocked by 4 mm MnCl₂ due to the Mn²⁺-induced inhibition of voltage-dependent sodium currents (I _Na). Therefore, Na⁺ channels, not Ca²⁺ channels, underlie the excitability of SCLC cells. Whole-cell I _Na was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na⁺-selective channel. I _Na evoked by depolarizing test potentials (–60 to +40 mV) exhibited a transient time course and activation/ inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m and h, also revealed biophysical similarity between SCLC and neuronal Na⁺ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at –20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit I _Ca and I _Na in bovine adrenal chromaffin cells, also significantly inhibited I _Na in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na⁺ channel conductance; (ii) fundamental characteristics of SCLC Na⁺ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na⁺ channels are an additional target of the pathological IgG present in the patients' sera.Department of Biomedical EngineeringThis study was supported by National Institutes of Health grant NS18607 and a research grant from the Muscular Dystrophy Association. Dr. Y.I. Kim is the recipient of a Javits Neuroscience Investigator Award from the National Institute of Neurological Disorder and Stroke.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

Effect of extracellular pH on sodium current in isolated,single rat ventricular cells

Summary Effects of extracellular pH on the sodium current (I _Na) of single rat ventricular cells were examined under conditions of voltage clamp and internal perfusion. In this way, pH _i was controlled while pH _o was changed. The combined suction pipette-microelectrode method was used. The suction pipette passed current and perfused the cell's interior; the microelectrode measured membrane potential. Increasing extracellular H⁺ depressedI _Na and slowed inactivation. The current-voltage curves forI _Na and Slowed inactivation. The current-voltage curves forI _Na were shifted to positive and negative potentials at low and high pH _o , respectively. Similar potential shifts were observed in both the conductance voltage curve and the steadystate inactivation voltage curve (h ). Conduction was also depressed at low pH _o . The shifts were probably due to surface charge effects, while the impaired conduction was probably due to protonation of a site in the Na channel.     

Na+ channel activity in cultured renal (A6) epithelium: Regulation by solution osmolarity

Summary Solution osmolarity is known to affect Na⁺ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na⁺ currents (I _sc).I _sc for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25±2, 9±2, 3±0.4, and 0.6±0.5 A/cm², respectively. Similar results were obtained following changes in the serosal tonicity; mucosal changes did not significantly affectI _sc. The changes inI _sc were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na⁺ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na⁺ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na⁺ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na⁺ transport rates for A6 epithelia reported in the literature.     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

A study on Na+-coupled oxidative phosphorylation: ATP formation supported by artificially imposed ΔpNa and ΔpK inVibrio alginolyticus cells

Addition of Na⁺ to the K⁺-loadedVibrio alginolyticus cells, creating a 250-fold Na⁺ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na⁺/H⁺ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K⁺ gradient (high [K⁺] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na⁺ pulse-induced ATP formation. The data agree with the assumption that Na⁺-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H⁺ and Na⁺ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H⁺ and Na⁺, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H⁺, Na⁺, and K⁺, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Na+-translocating NADH-quinone reductase of marine and halophilic bacteria

The respiratory chain of marine and moderately halophilic bacteria requires Na⁺ for maximum activity, and the site of Na⁺-dependent activation is located in the NADH-quinone reductase segment. The Na⁺-dependent NADH-quinone reductase purified from marine bacteriumVibrio alginolyticus is composed of three subunits, , , and , with apparentM _r of 52, 46, and 32kDa, respectively. The FAD-containing -subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones. In the presence of the FMN-containing -subunit and the -subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals. The reaction catalyzed by the -subunit is strictly dependent on Na⁺ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na⁺. A similar type of Na⁺-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria. The respiratory chain ofV. alginolyticus contains another NADH-quinone reductase which is Na⁺ independent and has no energy-transducing capacity. These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.     

Inhibitory effect of menaquinone-7 (vitamin K2) on the bone-resorbing factors-induced bone resorption in elderly female rat femoral tissues in vitro

A number of data are consistent with the hypothesis that increases in intracellular Na⁺ concentration (Na⁺ _i) during ischemia and early reperfusion lead to calcium overload and exacerbation of myocardial injury. However, the mechanisms underlying the increased Na⁺ _i remain unclear. ²³Na nuclear magnetic resonance spectroscopy was used to monitor Na⁺ _i in isolated rat hearts perfused with a high concentration of fatty acid as can occur under some pathological conditions. Whole-cell patch-clamp experiments were also performed on isolated cardiomyocytes in order to investigate the role of voltage-gated sodium channels. Na⁺ _i increased to substantially above control levels during no-flow ischemia. The results show that a pharmacological reduction of Na⁺ _i increase by cariporide (1 mol/L, a Na⁺/H⁺ exchange blocker) is not the only protection against ischemia-reperfusion damage, but that such protection may also be brought about by metabolic action aimed at reducing fatty acid utilization by myocardial cells. This action was obtained in the presence of etomoxir (0.1 mol/L), an inhibitor of carnitine palmitoyltransferase-1 (the key enzyme involved in fatty acid uptake by the mitochondria) which also decreases long-chain acyl carnitine accumulation. The possibility of Na⁺ channels participating in Na⁺ _i increase as a consequence of alterations in cardiac metabolism was studied in isolated cells. Sustained I_Na was stimulated by the presence of lysophosphatidylcholine (LPC, 10 mol/L) whose accumulation during ischemia is, at least partly, dependent on increased long-chain acyl carnitine. Current activation was particularly significant in the range of potentials between –60 and –20 mV. This may have particular relevance in ischemia. The quantity of charge carried by sustained I_Na was reduced by 24% in the presence of 1 mol/L cariporide. Therefore, limitation of long-chain fatty acid metabolism, and consequent limitation of ischemia-induced long-chain acyl carnitine accumulation, may contribute to reducing intracellular Na⁺ increase during ischemia-reperfusion.     

Ultraviolet Photoalteration of Late Na+ Current in Guinea-pig Ventricular Myocytes

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca²⁺ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K⁺-, Na⁺-free pipette solution, and bathed with K⁺-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca²⁺ current. Trials with depolarized holding potential, Ca²⁺ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na⁺ current (I_Na). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late I_Na may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I_Na.     

Do neurons have a voltage or a current threshold for action potential initiation?

The majority of neural network models consider the output of single neurons to be a continuous, positive, and saturating firing ratef(t), while a minority treat neuronal output as a series of delta pulses (t — t _i ). We here argue that the issue of the proper output representation relates to the biophysics of the cells in question and, in particular, to whether initiation of somatic action potentials occurs when a certain thresholdvoltage or a thresholdcurrent is exceeded. We approach this issue using numerical simulations of the electrical behavior of a layer 5 pyramidal cell from cat visual cortex. The dendritic tree is passive while the cell body includes eight voltage- and calcium-dependent membrane conductances.We compute both the steady-state (I ^static (V _m )) and the instantaneous (I _o (V_m)) I–V relationships and argue that the amplitude of the local maximum inI ^static (V _m ) corresponds to the current thresholdI _th for sustained inputs, while the location of the middle zero-crossing ofI _o corresponds to a fixed voltage thresholdV _th for rapid inputs. We confirm this using numerical simulations: for rapid synaptic inputs, spikes are initiated if the somatic potential exceedsV _th, while for slowly varying inputI _th must be exceeded. Due to the presence of the large dendritic tree, no charge thresholdQ _th exists for physiological input.Introducing the temporal average of the somatic membrane potential (V _m) while the cell is spiking repetitively, allows us to define a dynamic I-V relationship ^dynamic ((V _m)). We find an exponential relationship between (V _m) and the net current sunk by the somatic membrane during spiking (diode-like behavior). The slope ofI/dynamic((V _m)) allows us to define a dynamic input conductance and a time constant that characterizes how rapidly the cell changes its output firing frequency in response to a change in its input.     

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

Voltage-gated Na⁺ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca²⁺ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca²⁺ or Na⁺ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca²⁺ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

Na,K-ATPase and the development of Na+ transport in rat distal colon

Summary Na, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na⁺ transport during early postnatal development. The maximum Na⁺-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (I _sc ^N ) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.I _sc ^N was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na⁺ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (K _I ) was not changed during development (0.1–0.3mm). Specific [³H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (K _D ) was 8 and 21 m in young and adult rats, respectively. The Na⁺ turnover rate per single Na, K pump, which was calculated fromI _sc ^N and estimated density of binding sites per cm² of tissue was 500 in adult and 6400 Na⁺/min·site in young rats. These data indicate that the very high Na⁺ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na⁺ of a single isoform of the Na, K pump. The development of Na⁺ extrusion across the basolateral membrane is not directly regulated by corticosteroids.     

Opposite effects of angiotensin II and the protein kinase C activator OAG on cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C in cell-attached and inside-out patch-clamp experiments to study the influence of the vasoactive peptide angiotensin II (A II) and of the diacylglycerol analogue OAG (1-oleoyl-2-acetyl-snglycerol) on open probability and gating properties of single cardiac Na⁺ channels from cultured neonatal rat cardiocytes. Treating the cardiocytes with A II caused Na⁺ channel activation: reconstructed peak I_Na increased to 137 ± 17.5% of control at 3 mol/liters and to 176 ± 42% at 30 mol/liter. This NP_o increase developed without major changes in open state and burst activity, even at 30 mol/liter. OAG (6 mol/liter) did not mimic this A II action. By contrast, OAG treatment of the cardiocytes had the opposite effect on NP_o and diminished reconstructed peak I_Na to 67 ± 4.9% of the control. The putative protein kinase C inhibitor staurosporine (0.2 mol/liter) abolished this I_Na depression and led to a normalization of NP_o. OAG had the same effect on isolated Na⁺ channels. Exposure of the cytoplasmic surface of inside-out patches to 1 mol/liter OAG reversibly depressed, in the simultaneous presence of 50 mol/liter Mg-ATP, the reconstructed peak I_Na to 40 ± 9.7% of the control but left i _unit, _open and burst activity unaffected. No NP_o depression was obtained in the absence of Mg-ATP indicating that Mg-ATP may serve as phosphate donor. Obviously, after phosphorylation by protein kinase C, cardiac Na⁺ channels attain a reduced open probability but appear to preserve their kinetic properties. It is also concluded that activation of protein kinase C is not the mechanism underlying the A II induced channel activation.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-3), Bonn.     

Na+ and K+ fluxes stimulated by Na+-coupled glucose transport: Evidence for a Ba2+-insensitive K+ efflux pathway in rabbit proximal tubules

Summary Addition of glucose or the nonmetabolizable analogue -methyl-d-glucoside to rabbit proximal tubules suspended in a glucoseand alanine-free buffer caused a sustained increase in intracellular Na⁺ content (+43±7 nmol · (mg protein)^–1) and a concomitant but larger decrease in K⁺ content (–72±11 nmol· (mg protein)^–1). A component of the net K⁺ efflux was Ba²⁺ insensitive, and was inhibited by high (1mm) but not low (10 m) concentrations of the diuretics, furosemide and bumetanide. The increase in intracellular Na⁺ content is consistent with the view that the increased rates of Na⁺ and water transport seen in the proximal tubule in the presence of glucose can be attributed (at least in part) to a stimulation of basolateral pump activity by an increased [Na⁺] _i .     

Ionic channels and nerve membrane constituents. Tetrodotoxin-like interaction of saxitoxin with cholesterol monolayers

Saxitoxin (STX) and tetrodotoxin (TTX) have the same striking property of blocking the Na⁺ channels in the axolemma. Experiments with nerve plasma membrane components of the squid Dosidicus gigas have shown that TTX interacts with cholesterol monolayers. Similar experiments were carried out with STX. The effect of STX on the surface pressure-area diagrams of lipid monolayers and on the fluorescence emission spectra of sonicated nerve membranes was studied. The results indicate a TTX-like interaction of STX with cholesterol monolayers. The expansion of the monolayers caused by 10^-6 M STX was 2.2 A²/cholesterol molecule at 25°C. From surface pressure measurements at constant cholesterol area (39 A²/molecule) in media with various STX concentrations, it was calculated that the STX/cholesterol surface concentration ratio is 0.54. The apparent dissociation constant of the STX-cholesterol monolayer complex is 4.0 x 10^-7 M. The STX/cholesterol ratio and the apparent dissociation constant are similar to those determined for TTX. The presence of other lipids in the monolayers affects the STX-cholesterol association. The interactions of STX and TTX with cholesterol monolayers suggest (a) that cholesterol molecules may be part of the nerve membrane Na⁺ channels, or (b) that the toxin receptor at the nerve membrane shares similar chemical features with the cholesterol monolayers.     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.