Effect of amiloride on the apical cell membrane cation channels of a sodium-absorbing,potassium-secreting renal epithelium

Summary The effect of the K-sparing diuretic amiloride was assessed electrophysiologically in the isolated cortical collecting tubule of the rabbit, a segment which absorbs Na and secretes K. Low concentrations of amiloride in the perfusate caused a rapid, reversible, decrease in the magnitude of the lumen negative transepithelial potential difference,V _te, transepithelial conductanceG _te, and equivalent short-circuit current,I _sc, with an apparentK _1/2 of approximately 7×10^–8 m. The effects of a maximum inhibitory concentration of amiloride (10^–5 m) were identical to those observed upon Na removal from lumen and bath (Na removal from the bath alone has no effect). Removal of Na in the presence of 10^–5 m amiloride had no affect onV _te,G _te, orI _sc, and is consistent with the view that amiloride blocks the Na conductive pathways of the apical cell membrane. Further, in the absence of Na, the subsequent addition of amiloride had no influence. In tubules where active Na absorption was either spontaneously low, or abolished by removal of Na from lumen and bath, the elevation of K from 5 to 155 meq/liter in the perfusate caused a marked change of theV _te in the negative direction and an increase in theG _te. These effects could be attributed to a high K permeability of the apical cell membrane and not of the tight junctions. Amiloride (10^–5 m) had no effect on these responses to K. It is concluded that amiloride selectively blocks the apical cell membrane Na channels but has no effect on the K conductive pathways(s). This selective nature of amiloride may indicate that Na and K are transported across the apical cell membrane via separate conductive pathways.     

Apical membrane K conductance in the toad urinary bladder

Summary The conductance of the apical membrane of the toad urinary bladder was studied under voltage-clamp conditions at hyperpolarizing potentials (mucosa negative to serosa). The serosal medium contained high KCl concentrations to reduce the voltage and electrical resistance across the basal-lateral membrane, and the mucosal solution was Na free, or contained amiloride, to eliminate the conductance of the apical Na channels. As the mucosal potential (V _m) was made more negative the slope conductance of the epithelium increased, reaching a maximum at conductance of the epithelium increased, reaching a maximum atV _m=–100 mV. This rectifying conductance activated with a time constant of 2 msec whenV _m was changed abruptly from 0 to –100 mV, and remained elevated for at least 10 min, although some decrease of current was observed. ReturningV _m to+100 mV deactivated the conductance within 1 msec. Ion substitution experiments showed that the rectified current was carried mostly by cations moving from cell to mucosa. Measurement of K flux showed that the current could be accounted for by net movement of K across the apical membrane, implying a voltage-dependent conductance to K (G _K). Mucosal addition of the K channel blockers TEA and Cs had no effect onG _K, while 29mm Ba diminished it slightly. Mucosal Mg (29mm) also reducedG _K, while Ca (29mm) stimulated it.G _K was blocked by lowering the mucosal pH with an apparent pK₁ of 4.5. Quinidine (0.5mm in the serosal bath) reducedG _K by 80%.G _K was stimulated by ADH (20 mU/ml), 8-Br-cAMP (1mm), carbachol (100 m), aldosterone (5×10^–7 m for 18 hr), intracellular Li and extracellular CO₂.     

Effect of poly-L-lysine on potassium fluxes in red beet tissue

Summary Poly-L-lysine concentrations (10^–6 m) which cause slight leakage of pigment from beet cells completely disrupt the kinetics of^*K (labeled) absorption at 25°C in the range 0.01 to 50mm KCl. Lower concentrations of polylysine (10^–7 to 10^–9 m) interfere with potassium fluxes at both cell membranes, initially increasing efflux across the plasma membrane and decreasing the capacity of the cytoplasm to retain ions during flux experiments at 2°C. At 25°C, these concentrations of polylysine increase^*K (labeled) absorption from 0.2mm KCl, but not from 10mm KCl. These responses are discussed in relation to ion transport via the three-compartment in-series model proposed for plant cells. Particular emphasis is placed on the role of the plasma membrane in K transport from solutions of low concentration.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

Influence of lead ions on cation permeability in human red cell ghosts

Summary Intracellular Pb²⁺ ions can replace Ca²⁺ ions in stimulating the Ca-dependent K permeability of human red blood cells. In metabolically depleted resealed ghosts, the threshold for stimulation of⁸⁶Rb efflux by internal Pb²⁺ is around 5×10^–10 m, and stimulation is half-maximal at about 2×10^–9 m, and maximal at 10^–8 m Pb²⁺. There is no effect on²²Na efflux in this concentration range.⁸⁶Rb efflux is antagonized by internal Mg²⁺ ions, and by the channel-blocking drugs quinidine and diS-C₂(5), as observed for the Ca-dependent K permeability in red cells. In ghosts containing EDTA, which prevents any internal effects of Pb²⁺ ions, external Pb²⁺ increases both²²Na and⁸⁶Rb permeability when its concentration exceeds 6×10^–7 m. This effect is seemingly unrelated to the Ca-dependent K permeability. This work makes extensive use of Pb²⁺ ion buffers, and gives information about their preparation and properties.     

Potassium transport in the rabbit renal proximal tubule: Effects of barium,ouabain, valinomycin,and other ionophores

Summary Potassium fluxes in a suspension of rabbit proximal tubules were monitored using a potassium-sensitive extracellular electrode. Ouabain (10^–4 m) and barium (5mm) were used to selectively quantitate the potassium efflux pathway (105±5 nmol K⁺·mg protein^–1·min^–1) and the sodium pump-related potassium influx (108±7), respectively. These equal and opposite fluxes suggest that potassium accumulation in the cell occurs mainly through the sodium pump and that potassium efflux occurs mainly through barium-sensitive potassium channels. Thus the activity of the sodium pump (Na, K-ATPase) in the basolateral membrane of the proximal tubule is balanced by the efflux of potassium, presumably across the basolateral membrane, which has a high potassium permeability. In addition, the effect of valinomycin and other ionophores was examined on potassium fluxes and several metabolic parameters [oxygen consumption (QO₂), ATP content]. The addition of valinomycin to the tubules produced a net efflux of potassium which was quantitatively equivalent to the efflux produced by the addition of ouabain. The valinomycin-induced efflux was mainly due to the activity of valinomycin as a mitochondrial uncoupler, which indirectly inhibited the sodium pump by allowing a rapid reduction of the intracellular ATP. Amphotericin, nystatin, and monensin all produced large net releases of intracellular potassium. The action of the ionophores could be localized to the plasma or mitochondrial membrane and classified into three groups, as follows: (a) those which demonstrated full mitochondrial uncoupler activity (FCCP, valinomycin), (b) those which had no uncoupler activity (amphotericin B, nystatin); and (c) those which displayed partial uncoupler activity (monensin, nigericin).     

Evidence for a Na+/K+/Cl− cotransport system in basolateral membrane vesicles from the rabbit parotid

Summary Sodium (²²Na) transport was studied in a basolateral membrane vesicle preparation from rabbit parotid. Sodium uptake was markedly dependent on the presence of both K⁺ and Cl^– in the extravesicular medium, being reduced 5 times when K⁺ was replaced by a nonphysiologic cation and 10 times when Cl^– was replaced by a nonphysiologic anion. Sodium uptake was stimulated by gradients of either K⁺ or Cl^– (relative to nongradient conditions) and could be driven against a sodium concentration gradient by a KCl gradient. No effect of membrane potentials on KCl-dependent sodium flux could be detected, indicating that this is an electroneutral process. A KCl-dependent component of sodium flux could also be demonstrated under equuilibrium exchange conditions, indicating a direct effect of K⁺ and Cl^– on the sodium transport pathway. KCl-dependent sodium uptake exhibited a hyperbolic dependence on sodium concentration consistent with the existence of a single-transport system withK _m =3.2mm at 80mm KCl and 23°C. Furosemide inhibited this transporter withK _0.5=2×10^–4 m (23°C). When sodium uptake was measured as a function of potassium and chloride concentrations a hyperbolic dependence on [K] (Hill coefficient =1.31±0.07) were observed, consistent with a Na/K/Cl stoichiometry of 112. Taken together these data provide strong evidence for the electroneutral coupling of sodium and KCl movements in this preparation and strongly support the hypothesis that a Na⁺/K⁺/Cl^– cotransport system thought to be associated with transepithelial chloride and water movements in many exocrine glands is present in the parotid acinar basolateral membrane.     

Transient potassium fluxes in toad skin

Summary Experiments were carried out in the isolated short-circuited skin of the toadBufo marinus ictericus.⁴²K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 ±0.36 nmol cm^–2 hr^–1 (n=7) and efflux 3.62±0.38 nmol cm^– hr^–1 (n=7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of⁴²K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10^–4 m) to the outer solution, a sharp decline is observed in the rate of⁴²K discharge into the bathing solution,J ₂₁ ^K , which falls from 3.62±0.38 nmol cm^–2 hr^–1 to 2.02±0.04 nmol cm^–2 hr^–1 (n=7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline inJ ₂₁ ^K which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase inJ ₂₁ ^K and a linear relationship was observed betweenJ ₂₁ ^K and short-circuit current in the range of 0 to 115mm external Na concentration. The decline inJ ₂₁ ^K induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of⁴²K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of⁴²K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10^–3 m) to the inner bathing solution induces a transient rise in the rate of⁴²K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior ofJ ₂₁ ^K upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 11 Na–K pump.     

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

Summary The ratio of valinomycin-mediated unidirectional K⁺ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K⁺ net efflux and⁴²K influx. The driving force for the net efflux (V _m –E _K ⁺) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K⁺ net efflux has been calculated from the K⁺ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K⁺ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 m and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.     

Aldosterone control of the density of sodium channels in the toad urinary bladder

Summary Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (P _Na), intraepithelial Na activity (Na _c ), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N₀), in the urinary bladder of the toad (Bufo marinus). To facilitate voltageclamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85mm) sucrose (50mm) medium for the conventional Na-Ringer's solution on the serosal side.Aldosterone (5×10^–7 m, serosal side only) elicited proportionate increases in the Na-specific current (I _Na and inP _Na, with no significant change in the dependence ofP _Na on mucosal Na (Na _o ).P _Na and the control ofP _Na by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5×10^–3 m) which evoked coordinate and equivalent increases inI _Na andP _Na.The aldosterone-dependent increase inP _Na was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Michaelis constant (K m ) of acid phospatase as affected by montmorillonite,illite, and kaolinite clay minerals

The influence of Ca homoionic clay minerals (montmorillonite, illite, and kaolinite) on the activity,K _m , andV _m values of acid phosphatase was examined in model experiments. At each substrate (p-nitrophenyl phosphate) level tested, the addition of increasing amounts of clays (50, 100, and 150 mg, respectively) decreased the activity and increased theK _m value from 1.43×10^–3 m PNP (in the soluble state) to 82.3×10^–3M (montmorillonite), 8.02×10^–3 m (kaolinite), and 7.65×10^–3 m (illite) at the 150 mg level. The maximum enzyme reaction velocity (V _m ) remained nearly constant at different amounts of kaolinite and illite, but increased remarkably with rising quantities of montmorillonite. Apparently, the substrate affinity of sorbed acid phosphatase is significantly lower with montmorillonite than with kaolinite or illite. This may be ascribed to an intensive sorption of both substrate and enzyme to the surface as well as to interlattice sites of montmorillonite.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Transepithelial transport in cell culture: Stoichiometry of Na/phlorizin binding and Na/d-glucose cotransport. A two-step,two-sodium model of binding and translocation

Summary The renal cell line LLC-PK₁ cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I _sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm^–2hr^–1, andI _sc, 0.50±0.50(8) eq cm^–2hr^–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 21 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI _sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI _sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation.     

Cation selectivity of the resting membrane of squid axon

Summary Permeability constant ratios among monovalent cations were studied in the resting membrane of a giant axon of a Pacific squid,Loligo opalescens, by observing the relationship between the membrane potential and the ion concentration.The average permeability ratios are: Tl, 1.8; K, 1.0; Rb, 0.72; Cs, 0.16; Na, <0.08; Li, <0.08. These permeability ratios suggest that neither valinomycin nor nonactin are adequate models for the sites producing the resting permeability in the axonal membrane.Cyclic polyetherbis(t-butyl cyclohexyl) 18-crown-6 does not increase the permeability ratioP _Cs/P _K except when applied at concentrations (5×10^–5 m) at which the surfactant properties of this molecule may become significant.     

Membrane potentials and intracellular Cl− activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl− conductance

Summary The potential dependence of unidirectional³⁶Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl^– or Br^– in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl^– ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV _a , and intracellular Cl^– activity,a _Cl ^c , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI _sc=27.0±2.0 A/cm², with NaCl-Ringer's bathing both surfaces,V _a was –67.9±3.8mV (mean ±se,n=24, six preparations) anda _Cl ^c was 18.0±0.9mM in skins from animals adapted to distilled water. BothV _a anda _Cl ^a were found to be positively correlated withI _sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV _a anda _Cl ^c were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl^– conductance (G _Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and –100 mV. AtV=40 mV; i.e. whenG _Cl was deactivated,V _a was –70.1±5.0 mV (n=15, eight preparations) anda _Cl ^c was 40.0±3.8mm. The fractional apical membrane resistance (fR _a) was 0.69±0.03. Clamping toV=–100 mV led to an instantaneous change ofV _a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera _Cl ^c norfR _a changed significantly within a 2 to 5-min period during whichG _Cl increased by 1.19±0.10 mS/cm². WhenV was stepped back to 40 mV,V _a instantaneously shifted to –67.8±3.9 mV whilea _Cl ^c andfR _a remained constant during deactivation ofG _Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D _MRC) cells was estimated and correlated with the Cl current (I _Cl though the fully activated (V=–100mV) Cl^– conductance). A highly significant correlation was revealed (r=–0.96) with a slope of –2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR _a anda _Cl ^a of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.     

Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium

Summary Internal perfusion ofHelix neurons with a solution containing potassium aspartate, MgCl₂, ATP, and HEPES causes the calcium-activated potassium current (I _K(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca⁺⁺ is strongly buffered to 10^–7 m by including 0.5mm EGTA and 0.225mm CaCl₂ in the internal solution,I _K(Ca) remains constant for up to 3 hours of perfusion. In cells whereI _K(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10^–7 m free Ca⁺⁺ solution produces increases inI _K(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10^–6 m free Ca⁺⁺,I _K(Ca) is low and does not change with perfusion. These results lead us to conclude thatI _K(Ca) is stable in perfusedHelix neurons and that the apparent loss ofI _K(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (I _Ca) provides the Ca⁺⁺ which activatesI _K(Ca) during a depolarizing pulse,I _Ca is also stable in perfused cells when free intracellular Ca⁺⁺ is buffered.Perfusion with 1 m calmodulin (CaM) produces no effect onI _K(Ca) with either 10^–7 or 10^–6 m free internal calcium. Inhibiting endogenous CaM by including 50 m trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect onI _K(Ca) with 10^–7 m free internal calciu. It is concluded that CaM plays no role inI _K(Ca) activation.     

Water and salt permeability of gastric vesicles

Summary Volume-dependent changes in light scatter have been shown to be a linear function of the osmotic gradient imposed upon gastric vesicles purified from hog gastric mucosa. Observation of the light scattered 90° to incident, using the Durrum stop flow system D-110, indicates that the vesicles exposed to hypertonic medium undergo rapid shrinkage due to water loss from the vesicle interior. The rate constant for this water movement is 1.1±0.09 sec^–1 (n=10) and is linearly dependent on temperature between 16 and 36°C. The activation energy of 13.93±0.60 kcal mole^–1 (n=3), calculated from an Arrhenius plot, is inconsistent with water movement facilitated by a large-pore aqueous channel. A slower reswell phase, dependent on solute entry into the intravesicular space, follows the water-dependent shrink phase. KCl entry, studied because of the intravesicular requirement for active K⁺/H⁺ transport, exhibits two entry stages. The faster, described by a single exponential imposed upon a constantly sloping background, has a rate constant of 7.75±0.48×10^–3 sec^–1 (n=15). The slower phase, which typically accounts for 90% of the reswell process, demonstrates a rate constant of 1.94±0.23×10^–4 sec^–1 (n=15). In the presence of valinomycin or nigericin, two fast rate constants and one slow rate constant of swelling are observed. The rate constant of the faster reswell phase is increased from 7.75±0.48×10^–3 sec^–1 (n=15) to 15.74±3.7×10^–3 sec^–1 (n=5) and 17.23±3.4×10^–3 (n=3) by the addition of nigericin (1 g ml^–1) and valinomycin (4.5 m), respectively. The second part of the faster reswell phase is approximately that seen in the control population. Transport-dependent volume changes of significant magnitude can be demonstrated following the addition of ATP to vesicles equilibrated with 150mm KCl. The volume change is a function of HCl leak rate and is abolished by ionophores which eliminate the transport-dependent pH gradient. So ₄ ^–- substitution, which eliminates the overshoot phenomena observed in KCl medium, also eliminates the shrinkage resulting from ATP addition.     

Effects of 2-deoxy-d-glucose,amiloride, vasopressin,and ouabain on active conductance andE Na in the toad bladder

Summary The effects of various agents on active sodium transport were studied in the toad bladder in terms of the equivalent circuit comprising an active conductanceK ^a, an electromotive forceE _Na, and a parallel passive conductanceK ^p. For agents which affectK ^a, but notE _Na orK ^p, the inverse slope of the plot of total conductance against short-circuit currentI ₀ evaluatesE _Na, and the intercept representsK ^p. Studies employing 5×10^–7 m amiloride to depressK ^a indicate a changingE _Na, invalidating the use of the slope technique with this agent. An alternative suitable technique employs 10^–5 m amiloride, which reducesI ₀ reversibly to near zero without effect onK ^p. Despite curvilinearity of the -I₀ plot under these conditions,K ^p may therefore be estimated fairly precisely from the residual conductance. It then becomes possible to follow the dynamic behavior ofK ^a andE _Na (in the absence of 10^–5 m amiloride) by frequent measurements of andI ₀, utilizing the relationshipsK ^a=K-K ^p, andK _Na=I _O/(K-K ^p). 2-deoxy-d-glucose (7.5×10^–3 m) depressedK ^a without affectingE _Na. Amiloride (5×10^–7 m) depressedK ^a and enhancedE _Na. Vasopressin (100 mU/ml) enhancedK ^a markedly and depressedE _Na slightly. Ouabain (10^–4 m) depressed bothK ^a andE _Na. All of the above effects were noted promptly;K ^p was unaffected. The electromotive force of Na transportE _Na appears not to be a pure energetic parameter, but to reflect kinetic factors as well, in accordance with thermodynamic considerations.