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Hydroxide, bicarbonate and buffer anion permeabilities in semitendinosus muscle fibers of Rana pipiens were measured. In all experiments, the fibers were initially equilibrated in isotonic, high KSO solutions at pH=7.2 buffered with phosphate. Two different methods were used to estimate permeabilities: (i) membrane potential changes were recorded in response to changes in external ion concentrations, and (ii) intracellular pH changes were recorded in response to changes in external concentrations of ions that alter intracellular pH. Constant field equations were used to calculate relative or absolute permeabilities. In the first method, to increase the size of the membrane potential change produced by a sudden change in anion entry, external K was replaced by Cs prior to changes of the anion under study. At constant external Cs activity, a hyperpolarization results from increasing external pH from 7.2 to 10.0 or higher, using either CAPS (3-[cyclohexylamino]-1-propanesulfonic acid) or CHES (2-[N-cyclohexylamino]-ethanesulfonic acid) as buffer. For each buffer, the protonated form is a zwitterion of zero net charge and the nonprotonated form is an anion. Using reported values of H permeability, calculations show that the reduction in [H] cannot account for the hyperpolarizations produced by alkaline solutions. Membrane hyperpolarization increases with increasing total external buffer concentration at constant external pH, and with increasing external pH at constant external buffer anion concentration. Taken together, these observations indicate that both OH and buffer anions permeate the surface membrane. The following relative permeabilities were obtained at pH, 10.0± 0.3: (P/P) = 890 ± 150, (P/P) = 12 ± 2 (P/P) = 5.3 ± 0.9, and (P/P) = 4.7 ± 0.5 P/P was independent of pH up to 10.75. At pH = 9.6, (P/P) = 0.49 ± 0.03; at pH = 8.9, (P/P) = 18± 2 and at pH = 7.1, (P/P) = 20 ± 2. In the second method, on increasing external pH from 7.2 to 10.0, using 2.5 m m CAPS (total buffer concentration), the internal pH increases linearly with time over the next 10 min. This alkalinization is due to the entry of OH and the absorption of internal H by entering CAPS anion. The rate of CAPS entry was determined in experiments in which the external CAPS concentration was increased at constant external pH. Such increases invariably produced an increase in the rate of internal alkalinization, which was reversed when the CAPS concentration was reduced to its initial value. From the internal buffer power, the diameter of the fiber under study and the rates of change of internal pH, the absolute permeability for both OH and CAPS were calculated. At external pH = 10.0, the average (± sem) permeabilities were: P=1.68±0.19×10 cm/sec and P=2.10±0.74×10cm/sec. We conclude that OH is about 50 times more permeable than Cl at alkaline pH and that the anionic forms of commonly used buffers have significant permeabilities. [ABSTRACT FROM AUTHOR]

Efflux of 36Cl- 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 degrees 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 relation mu = 1/(1 + 10n(pK alpha-pH], where mu 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 150 mM KCl plus 120 mM NaCl (internal potential about -15 mV), the apparent pK alpha is 6.5 at both 0 and 20 degrees C, and n = 2.5 for 0 degrees C and 1.5 for 20 degrees C. For muscles equilibrated in solutions containing 7.5 mM KCl plus 120 mM NaCl (internal potential about -65 mV), the apparent pK alpha at 0 degrees C is 6.9 and n is 1.5. The voltage dependence of the apparent pK alpha 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 that n 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), where t is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mM). At 17 degrees C, k-1 = 188 mM . min. For temperatures between 10 and 23 degrees C, k has an apparent Q10 of 2.5. The Cl- efflux inhibitor SCN- at a concentration of 20 mM substantially retards the reduction of the pH-dependent Cl- efflux by DEPC. The findings that the apparent pK alpha 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. [ABSTRACT FROM AUTHOR]

Efflux of 36Cl- from frog sartorius muscles equilibrated in 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. In depolarized muscles from Rana pipiens, the pH-dependent Cl- efflux has an apparent pKa near 6.4. The reduction of Cl- efflux by external Zn2+ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl- efflux was also examined. At pH 6.5 and a membrane potential of -22 mV, increasing external Cl- activity from 0.108 to 0.28 M decreased inhibition of the pH-dependent Cl- efflux at all activities of Zn2+. The Zn2+ activity needed to reduce Cl- efflux by half increased from 0.39 x 10(-3) to 2.09 x 10(-3) M. By contrast, external Cl- activity had no measurable effect on the apparent pKa of the pH-dependent efflux. At constant Cl- activity less than 0.21 M, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn2+ activities with either a slight increase or no change in the Zn2+ activity producing half-inhibition. In other words, for relatively low Cl- activities, protection against inhibition of Cl- efflux by low Zn2+ activities was obtained by raising, not lowering, external pH; this is not what is expected if H+ and Zn2+ ions compete at the same site to produce inhibition of Cl- efflux. We conclude that Zn2+ and low pH inhibit Cl- efflux by separate and distinct mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]

The effects of pH on the permeability and conductance of the membranes to nitrate and to chloride of semitendinosus and lumbricalis muscle fibers were examined. Membrane potential responses to quick solution changes were recorded in semitendinosus fibers initially equilibrated in isotonic, high K2SO4 solutions. External solutions were first changed to ones in which either Rb+ or Cs+ replaced K+ and then to solutions containing either NO3- or Cl- to replace SO4(2-). The hyperpolarizations produced by Cl- depend on external pH, being smaller in acid than in alkaline solutions. By contrast, hyperpolarizations produced by NO3- were independent of external pH over a pH range from 5.5 to 9.0. In addition, voltage-clamp measurements were made on short lumbricalis muscle fibers. Initially they were equilibrated in isotonic solutions containing mainly K2SO4 plus Na2SO4. KCl or KNO3 were added to the sulfate solutions and the fibers were equilibrated in these new solutions. When finally equilibrated the fibers had the same volume they had in the sulfate solutions before the additions. Constant hyperpolarizing voltage pulses of 0.6-sec duration were applied when all external K+ was replaced by TEA+. For these conditions, inward currents flowing during the voltage pulses were largely carried by Cl- or NO3- depending on the final equilibrating solution. Cl- currents during voltage pulses were both external pH and time dependent. By contrast, NO3- currents were independent of both external pH and time. The voltage dependence of NO3- currents could be fit by constant field equations with a PNO3 of 3.7.10(-6) cm/sec. The voltage dependence of the initial or "instantaneous" Cl- currents at pH 7.5 and 9.0 could also be fit by constant field equations with PCl of 5.8 x 10(-6) and 7.9 x 10(-6) cm/sec, respectively. At pH 5.0, no measurable "instantaneous" Cl- currents were found. From these results we conclude that NO3- does not pass through the pH, time-dependent Cl- channels but rather passes through a distinct set of channels. Furthermore, Cl- ions do not appear to pass through the channels which allow NO3- through. Consequently, the measured ratio of PCl/PNO3 based on membrane potential changes to ionic changes made on intact skeletal muscle fibres is not a measure of the selectivity of a single anion channel but rather is a measure of the relative amounts of different channel types.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]

Efflux of 42K+ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K+ concentrations [( K+]0) between 75 and 300 mM and NaCl concentrations of 60, 120, or 240 mM. For several combinations of KCl and NaCl, steady-state internal potentials (Vi) were the same for different [K+]0. For the range of Vi examined, K+ efflux occurs principally through the K+ inward rectifier channels. When external K+ is removed Vi remains constant for 2 to 3 hr because of the high membrane conductance to Cl-, but K+ efflux drops by about one order of magnitude. External Ba2+ in the presence or absence of external K+ produces an inhibition of K+ efflux described by a relation of the form u = (u1/(1 + C [Ba2+]0] + u2, where u is the uninhibited fraction of K+ efflux; u1, u2 and C are constants; and u1 + u2 = 1. C depends both on [K+]0 and Vi. When [K+]0 greater than or equal to 75 mM, increasing [K+]0 at constant Vi reduces Ba2+ sensitivity. For constant Vi greater than or equal to -30 mV, Ba2+ sensitivity is less when [K+]0 = 0 than when [K+]0 greater than or equal to 75 mM. When [K+]0 = 0, Ba2+ sensitivity decreases as Vi is made more positive. The dependence of the Ba2+ sensitivity on Vi at constant [K+]0 is greater when [K+]0 = 0 than when [K+]0 greater than or equal to 75 mM. Both the activation of K+ efflux by external K+ and the Ba2+ inhibition of K+ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K+, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba2+ occupies either site, K+ efflux is further reduced. The reduction of Ba2+-sensitivity by increasing [K+]0 at high [K+]0 is attributable to the displacement of Ba2+ from the control sites by K+. The increased Ba2+ sensitivity produced by going from [K+]0 = 0 to [K+] greater than or equal to 75 mM when Vi greater than or equal to -30 mV is attributable to states in which Ba2+ occupies one site and K+ the other when [K+]0 not equal to 0.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]

Efflux of 42K+ was measured in frog sartorius muscles equilibrated in hyperosmotic depolarizing solutions. At the internal potentials obtained, K+ passes mainly through the inward rectifier potassium channels. Inhibition of K+ efflux by external Zn2+ (0.25 to 15 mM) differs in three significant ways from inhibition by Ba2+. (1) The dose-response relation does not correspond to action at a single site. (2) The Zn2+-sensitivity of K+ efflux does not depend on [K+]0 at constant internal potential. (3) Zn2+ inhibition is reduced by hydrogen ions, while Ba2+ inhibition is unaffected. Further, the Ba2+-sensitivity of K+ efflux is not altered by a half-inhibiting Zn2+ concentration, suggesting that the two ions do not interact at a common site. The histidine-modifying reagent diethylpyrocarbonate (DEPC) reduces Zn2+ inhibition. After DEPC treatment Zn2+ inhibition is further reduced by low pH. DEPC has little effect on Ba2+ inhibition. Zn2+ inhibition is not altered by treatment with the sulfhydryl reagents 5,5'-dithio-bis(2-nitrobenzoic acid) or dithiothreitol. The results can be described by either of two models in which two sites can bind Zn2+ and one or both of the sites may also bind H+. When both sites bind Zn2+, K+ efflux is inhibited, and a third site may then bind H+. The effects of DEPC can be accounted for by a decrease in H+ affinity of the first two sites by a factor of 50, and a decrease in Zn2+ affinity of these sites and of the H+ affinity of the third site by about one order of magnitude. [ABSTRACT FROM AUTHOR]

The effects of external Rb+ on the efflux of 42K+ from whole frog sartorius muscles loaded with 305 mM K+ and 120 mM Cl- were studied. K+ efflux is activated by [Rb+]o less than about 40 mM according to a sigmoid relation similar to that for activation by [K+]o. At [Rb+]o greater than 40 mM, K+ efflux declines, although at [Rb+]o = 300 mM it is still greater than at [Rb+]o = 0 mM. For low concentrations, the increment in K+ efflux over that in K+-and Rb+-free solution, delta K, is described by the relation delta k = a[X+]on, for both K+ and Rb+. The value of a is larger for Rb+ than for K+, while the values of n are similar; the activation produced by a given [Rb+]o is larger than that by an equal [K+]o for concentrations less than about 40 mM. Adding a small amount of Rb+ to a K+-containing solution has effects on K+ efflux which depend on [K+]o. At low [K+]o, adding Rb+ increases K+ efflux, the effect being greatest near [K+]o = 30 mM and declining at higher [K+]o; at [K+]o above 40 mM, addition of Rb+ decreases K+ efflux. At [K+]o above 75 mM, where K+ efflux is largely activated, Rb+ reduces K+ efflux by a factor b, described by the relation b = 1/(1+c[Rb+]o). Activation is discussed in terms of binding to at least two sites in the membrane, and the reduction in K+ efflux by Rb+ at high [K+]o in terms of association with an additional inhibitory site. [ABSTRACT FROM AUTHOR]

The binding of the cardiosteroidH-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release. The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding ( B) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant ( K) of 2.2×10 m. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one. Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (τ≃25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (τ≃15 hr). Increasing [K] from 2.5 to 10 mm slows the time course of binding without any significant change in binding capacity of the muscle fibers. Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979) Biochim. Biophys. Acta 552:247] on the distribution of (Na+K)-ATPase among different cell membrane fractions from frog skeletal muscle. [ABSTRACT FROM AUTHOR]

Efflux of sodium from frog sartorius muscles was measured during and after exposure to Ringer's fluid made hypertonic by addition of 400 mm glycerol. Effects of strophanthidin, removal of external Na, and variation of external K were determined. During exposure to glycerol-containing solutions, Na efflux increased. Upon return to Ringer's fluid, Na efflux at first increased further. After the initial increase, Na efflux gradually declined; for the first two hours the efflux of Na from treated muscles was higher than that from untreated muscles. In the second hour, the strophanthidin-sensitive fractions of Na efflux were slightly increased while the strophanthidin-insensitive fractions were slightly decreased when compared with untreated muscles. The responses of Na efflux to removal of external sodium and to varying external K were comparable in both treated and untreated muscles. This shows that, at first, the membranes which remained after glycerol treatment exhibited the normal characteristics of Na extrusion. For at least eight hours after glycerol withdrawal the Na efflux from treated muscles declined relative to that of untreated muscles. The decline was largely due to reduction in strophanthidinsensitive fractions of efflux. Six to eight hours after glycerol withdrawal the Na efflux in treated muscles was less responsive to alterations in external K and Na than it was in untreated muscles. This indicates that aged glycerol-treated sartorii lost a substantial part of their capacity to actively transport sodium. [ABSTRACT FROM AUTHOR]

Sartorius muscles were exposed to a hypertonic Ringer's fluid containing 400 mm glycerol and subsequently returned to normal Ringer's fluid. Employing lanthanum as an extracellular marker and after suitable preparation, sections of muscle fibers were examined with an electron-microscope. Extensive alteration in 'transverse' tubular morphology occurs after glycerol treatment. The number of sites at the Z line level usually containing complete triads decreases by about two-thirds. In about half of the sites with altered morphology no 'transverse' tubules were present, while in the other half the sites were completely empty. Fibers at all depths in glycerol-treated muscles were equally affected. The remnants of the 'transverse' tubules which remain were not very accessible to extracellular lanthanum. Estimates based on measurements of the presence or absence of lanthanum indicate that glycerol treatment disconnects 90% of the 'transverse' tubules from the external solution. The remaining tubules connected to the external solution are largely but not entirely located in a surface layer of about one-tenth the fiber radius in depth. [ABSTRACT FROM AUTHOR]

The efflux of(36)Cl and(42)K from frog's sartorius muscles equilibrated in Ringer's fluid with added KCl were measured in the absence and presence of salicylate, benzoate, and acetylsalicylate. The transmembrane potential and resistance were also measured in sartorii under similar conditions. Although the rate coefficient for loss of(42)K remained reasonably constant over extended experimental periods for untreated muscles, the rate coefficient for loss of(36)Cl fluctuated in many muscles giving rise to minima and maxima. The aromatic anions mentioned increased the efflux of chloride while having no detectable effect on the potassium efflux. The aromatic anion-stimulated chloride efflux was insensitive to alterations of external pH and was markedly reduced when nitrate replaced external chloride. No detectable changes in transmembrane potential or resistance were produced by salicylate, the most extensively studied aromatic anion. The results suggest that salicylate and the other aromatic anions stimulate an exchange diffusion mechanism for chloride. [ABSTRACT FROM AUTHOR]