bluuebunny/crossref_metadata_2025 huggingface dataset:
Augmentation and facilitation of transmitter release. A quantitative description at the frog neuromuscular junction.
Authors: Zengel J E , Magleby K L
Published: May 2004

Endplate potentials were recorded from frog and toad sartorius neuromuscular junctions under conditions of greatly reduced quantal contents. The magnitudes of augmentation increased with the duration and frequency of stimulation, often increasing at an accelerating rate during 10-20-s conditioning trains. The magnitudes of the first and second components of facilitation also increased, but reached apparent steady state values within the first few seconds of stimulation. These observations could be accounted for by assuming (a) that augmentation and the first and second components of facilitation arise from underlying factors in the nerve terminal that act to increase transmitter release; (b) that each nerve impulse adds an increment to each of the underlying factors; (c) that the magnitude of the increment typically increases during the train for augmentation but remains constant for the components of facilitation; and (d) that the underlying factors decay with first-order kinetics with time constants of approximately 7 s for augmentation and 60 and 500 ms for the first and second components of facilitation, respectively. The increments of facilitation added by each impulse were about twice as large in the toad as in the frog. Facilitation was described better by assuming a power relationship between the underlying factor and the observed facilitation than by assuming a linear relationship. Augmentation was described by assuming either a linear or power relationship.
A quantitative description of stimulation-induced changes in transmitter release at the frog neuromuscular junction.
Authors: Magleby K L , Zengel J E
Published: May 2004

Endplate potentials were recorded from frog sartorius neuromuscular junctions under conditions of greatly reduced quantal contents to develop a quantitative description of stimulation-induced changes in transmitter release. Four general models relating potentiation, augmentation, and the first and second components of facilitation to transmitter release were developed. These models were then tested by incorporating equations for the kinetic properties of the four components of increased transmitter release and examining the ability of the resulting sets of equations to predict stimulation-induced changes in transmitter release. Three of the models were essentially consistent with the observation that augmentation had a multiplicative type relationship to facilitation. These models could also predict the effect of frequency and duration of stimulation on endplate potential (EPP) amplitude during and after prolonged (40 s) trains including the response to step changes in stimulation rate. These models extend by about two orders of magnitude the duration of stimulation-induced changes in transmitter release that can be accounted for, and show that the combined kinetic properties of potentiation, augmentation, and the two components of facilitation are generally sufficient to account for these changes.
Sodium transport effects on the basolateral membrane in toad urinary bladder.
Authors: Davis C W , Finn A L
Published: May 2004

In toad urinary bladder epithelium, inhibition of Na transport with amiloride causes a decrease in the apical (Vmc) and basolateral (Vcs) membrane potentials. In addition to increasing apical membrane resistance (Ra), amiloride also causes an increase in basolateral membrane resistance (Rb), with a time course such that Ra/Rb does not change for 1-2 min. At longer times after amiloride (3-4 min), Ra/Rb rises from its control values to its amiloride steady state values through a secondary decrease in Rb. Analysis of an equivalent electrical circuit of the epithelium shows that the depolarization of Vcs is due to a decrease in basolateral electromotive force (Vb). To see of the changes in Vcs and Rb are correlated with a decrease in Na transport, external current (Ie) was used to clamp Vmc to zero, and the effects of amiloride on the portion of Ie that takes the transcellular pathway were determined. In these studies, Vcs also depolarized, which suggests that the decrease in Vb was due to a decrease in the current output of a rheogenic Na pump. Thus, the basolateral membrane does not behave like an ohmic resistor. In contrast, when transport is inhibited during basolateral membrane voltage clamping, the apical membrane voltage changes are those predicted for a simple, passive (i.e., ohmic) element.
Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site.
Authors: Fröhlich O , Leibson C , Gunn R B
Published: May 2004

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min-1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.
Ionic currents in single isolated bullfrog atrial cells.
Authors: Hume J R , Giles W
Published: May 2004

Enzymatic dispersion has been used to yield single cells from segments of bullfrog atrium. Previous data (Hume and Giles, 1981) have shown that these individual cells are quiescent and have normal resting potentials and action potentials. The minimum DC space constant is approximately 920 microns. The major goals of the present study were: (a) to develop and refine techniques for making quantitative measurements of the transmembrane ionic currents, and (b) to identify the individual components of ionic current which generate different phases of the action potential. Initial voltage-clamp experiments made using a conventional two-microelectrode technique revealed a small tetrodotoxin (TTX)-insensitive inward current. The small size of this current (2.5-3.0 X 10(-10)A) and the technical difficulty of the two-microelectrode experiments prompted the development of a one-microelectrode voltage-clamp technique which requires impalements using a low-resistance (0.5-2 M omega) micropipette. Voltage-clamp experiments using this new technique in isolated single atrial cells reveal five distinct ionic currents: (a) a conventional transient Na+ current, (b) a TTX-resistant transient inward current, carried mainly by Ca++, (c) a component of persistent inward current, (d) a slowly developing outward K+ current, and (e) an inwardly rectifying time-independent background current. The single suction micropipette technique appears well-suited for use in the quantitative study of ionic currents in these cardiac cells, and in other small cells having similar electrophysiological properties.
Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method.
Authors: Brahm J
Published: May 2004

Human red cell permeability to the homologous series of methanol, ethanol, n-propanol, n-butanol, and n-hexanol was determined in tracer efflux experiments by the continuous flow tube method, whose time resolution is 2-3 ms. Control experiments showed that unstirred layers in the cell suspension were less than 2 X 10(-4) cm, and that permeabilities less than or equal to 10(-2) cm s-1 can be determined with the method. Alcohol permeability varied with the chain length (25 degrees C): Pmeth 3.7 X 10(-3) cm s-1, Peth 2.1 X 10(-3) cm s-1, Pprop 6.5 X 10(-3) cm s-1, Pbut less than or equal to 61 X 10(-3) cm s-1, Phex 8.7 X 10(-3) cm s-1. The permeability for methanol, ethanol, and n-propanol was concentration independent (1-500 mM). The permeability to n-butanol and n-hexanol, however, increased above the upper limit of determination at alcohol concentrations of 100 and 25 mM, respectively. The activation energies for the permeability to methanol, n-propanol, and n-hexanol were similar, 50-63 kJ mol-1. Methanol permeability was not reduced by p-chloromercuribenzene sulfonate (PCMBS), thiourea, or phloretin, which inhibit transport of water or hydrophilic nonelectrolytes. It is concluded (a) that all the alcohols predominantly permeate the membrane lipid bilayer structure; (b) that both the distribution coefficient and the diffusion coefficient of the alcohols within the membrane determine the permeability, and (c) that the relative importance of the two factors varies with changes in the chain length.
Comparison of excitatory currents activated by different transmitters on crustacean muscle. I. Acetylcholine-activated channels.
Authors: Lingle C , Auerbach A
Published: May 2004

The properties of acetylcholine-activated excitatory currents on the gm1 muscle of three marine decapod crustaceans, the spiny lobsters Panulirus argus and interruptus, and the crab Cancer borealis, were examined using either noise analysis, analysis of synaptic current decays, or analysis of the voltage dependence of ionophoretically activated cholinergic conductance increases. The apparent mean channel open time (tau n) obtained from noise analysis at -80 mV and 12 degrees C was approximately 13 ms; tau n was prolonged e-fold for about every 100-mV hyperpolarization in membrane potential; tau n was prolonged e-fold for every 10 degrees C decrease in temperature. Gamma, the single-channel conductance, at 12 degrees C was approximately 18 pS and was not affected by voltage; gamma was increased approximately 2.5-fold for every 10 degrees C increase in temperature. Synaptic currents decayed with a single exponential time course, and at -80 mV and 12 degrees C, the time constant of decay of synaptic currents, tau ejc, was approximately 14-15 ms and was prolonged e-fold about every 140-mV hyperpolarization; tau ejc was prolonged about e-fold for every 10 degrees C decrease in temperature. The voltage dependence of the amplitude of steady-state cholinergic currents suggests that the total conductance increase produced by cholinergic agonists is increased with hyperpolarization. Compared with glutamate channels found on similar decapod muscles (see the following article), the acetylcholine channels stay open longer, conduct ions more slowly, and are more sensitive to changes in the membrane potential.
Single chloride channels from Torpedo electroplax. Activation by protons.
Authors: Hanke W , Miller C
Published: May 2004

Single-channel fluctuations of a chloride-specific channel from Torpedo californica electroplax were studied with high current and time resolution. Channels were incorporated into virtually solvent-free planar bilayer membranes formed from phospholipid monolayers, and the substructure of the open channel was analyzed. The single channel displays three well-defined substates of conductances 0, 10, and 20 pS in 200 mM Cl-. These three substates are interpreted in terms of a dimeric channel complex composed of two identical "protochannels" gating independently in parallel on a time scale of milliseconds, but coupled together by a bursting process on a time scale of seconds. The probability of forming an open protochannel is voltage dependent and is increased strongly as aqueous pH is lowered. Variations of pH are effective only on the same side of the bilayer as the addition of electroplax vesicles. The dependence of single-channel kinetics on pH and voltage lead to a minimal four-state model in which both open and closed states can be protonated on a residue that changes its pK from 6 to 9 upon opening of the protochannel.
Intracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation.
Authors: Russell J M , Boron W F , Brodwick M S
Published: May 2004

The ion transport mechanism that regulates intracellular pH (pHi) in giant barnacle muscle fibers was studied by measuring pHi and unidirectional Na+ fluxes in internally dialyzed fibers. The overall process normally results in a net acid extrusion from the cell, presumably by a membrane transport mechanism that exchanges external Na+ and HCO-3 for internal Cl- and possibly H+. However, we found that net transport can be reversed either by lowering [HCO-3]o and pHo or by reducing [Na+]o. This reversal (acid uptake) required external Cl-, was stimulated by raising [Na+]i, and was blocked by SITS. When the transporter was operating in the net forward direction (acid extrusion), we found a unidirectional Na+ influx of approximately 60 pmol . cm-2 . s-1, which required external HCO-3 and internal Cl- and was stimulated by cyclic AMP and blocked by SITS or DIDS. These properties of the Na+ influx are all shared with the net acid extrusion process. We also found that under conditions of net forward transport, the pHi-regulating system mediated a unidirectional Na+ efflux, which was significantly smaller than the simultaneous Na+ influx. These data are consistent with a reversible transport mechanism which, even when operating in the net forward direction, mediates a small amount of reversed transport. We also found that the ouabain-sensitive Na+ efflux was sharply inhibited by acidic pHi, being totally absent at pHi values below approximately 6.8.
Basolateral potassium channel in turtle colon. Evidence for single-file ion flow.
Authors: Kirk K L , Dawson D C
Published: May 2004

Treatment of the apical surface of the isolated, ouabain-inhibited turtle colon with the polyene antibiotic amphotericin B permitted the properties of a barium-sensitive potassium conductance in the basolateral membrane to be discerned from the measurements of transepithelial fluxes and electrical currents. Simultaneous measurements of potassium currents and 42K fluxes showed that the movement of potassium was not in accord with simple diffusion. Two other cations, thallium and rubidium, were also permeable and, in addition, exhibited strong interactions with the potassium tracer fluxes. The results indicate that permeant cations exhibit positive coupling, which is consistent with a single-file mechanism of ion translocation through a membrane channel.
Calcium currents in a fast-twitch skeletal muscle of the rat.
Authors: Donaldson P L , Beam K G
Published: May 2004

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.
Mechanism of epithelial lithium transport. Evidence for basolateral Na:Na and Na:Li exchange.
Authors: Kirk K L , Dawson D C
Published: May 2004

Measurement of transmural sodium fluxes across isolated, ouabain-inhibited turtle colon in the presence of a serosal-to-mucosal sodium gradient shows that in the absence of active transport the amiloride-sensitive cellular path contains at least two routes for the transmural movement of sodium and lithium, one a conductive path and the other a nonconductive, cation-exchange mechanism. The latter transport element can exchange lithium for sodium, and the countertransport of these two cations provides a mechanistic basis for the ability of tight epithelia to actively absorb lithium despite the low affinity of the basolateral Na/K-ATPase for this cation.
Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification.
Authors: Grinstein S , Clarke C A , Rothstein A
Published: May 2004

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.
Chemical excitation of Limulus photoreceptors. II. Vanadate, GTP-gamma-S, and fluoride prolong excitation evoked by dim flashes of light.
Authors: Corson D W , Fein A , Walthall W W
Published: May 2004

We treated Limulus ventral photoreceptors with the phosphatase inhibitors fluoride, vanadate, and GTP-gamma-S [guanosine-5'0-(3-thiotriphosphate)] under various conditions of illumination and external calcium concentrations. In the dark in low-calcium (1 mM) artificial seawater (ASW), fluoride-induced discrete waves cluster together in time. Under these conditions, the intervals between waves were found to be correlated, and there were excess short intervals beyond the number expected from an exponential interval distribution. To assess the effects of the inhibitors on the light response, we stimulated ventral receptors with a series of dim flashes and averaged the current response under voltage clamp. In ASW, vanadate and GTP-gamma-S prolong the decay of the averaged response to dim test flashes, but prolongation does not always accompany the induction of discrete waves in the dark. Prolongation induced by vanadate in normal-calcium (10 mM) ASW was enhanced in low-calcium (1 mM Ca2+) ASW. Many individual response records suggest that prolongation results from extra discrete waves late in the light response, whereas others reveal long-lasting complex waveforms that cannot easily be resolved into discrete waves. The apparent effect of the inhibitors on the light response is to allow a single photoactivated rhodopsin molecule to produce multiple discrete waves and complex long-lasting events. We suggest that both prolongation of the light response and clustering of waves in the dark result from inhibition of a step in the pathway of visual transduction, in which GTP hydrolysis normally helps to turn off the production of both light-evoked and spontaneous waves.
Sodium channel gating currents in frog skeletal muscle.
Authors: Campbell D T
Published: May 2004

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.
Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.
Authors: Nørby J G , Klodos I , Christiansen N O
Published: May 2004

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.
The water permeability of toad urinary bladder. I. Permeability of barriers in series with the luminal membrane.
Authors: Levine S D , Jacoby M , Finkelstein A
Published: May 2004

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.
Metaretinochrome in membranes as an effective donor of 11-cis retinal for the synthesis of squid rhodopsin.
Authors: Seki T
Published: May 2004

Aporetinochrome, which is a protein moiety of retinochrome without chromophore retinal, is found in the membrane containing retinochrome. All of the prosthetic retinal of retinochrome in membranes, which is all-trans retinal, is bound to the chromophoric site on the protein moiety, with protonated Schiff bases showing an absorption band with the maximum at 495 nm. On exposure to light, retinochrome is converted to metaretinochrome at room temperature. The prosthetic retinals of metaretinochrome in membranes, which are 11-cis retinals, are in two states: retinals bound to the chromophoric site with protonated Schiff bases, and the free retinals, which are separated from the protein moiety. These states are suggested from the following observations. (a) The ratio of the absorbance at 470 nm of metaretinochrome to that at 495 nm of the parental retinochrome differs because of differences in samples and is higher in the purer preparations. (b) The difference spectrum of absorption of metaretinochrome caused by alkalinization shows two minimum peaks at approximately 420 and 470 nm. (c) The rate of bleaching of metaretinochrome in membranes with dilute NH2OH is much faster than that of retinochrome, and the absorption band in the near-UV region is more susceptible to NH2OH than the visible absorption band. The state of the prosthetic retinals in metaretinochrome was confirmed directly by the reaction of metaretinochrome in membranes with NaBH4. After treatment with NaBH4, the sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern shows two fluorescent bands: one at the position that corresponds to the retinochrome protein (mol wt 27,000 +/- 2,000), and another at the front of migration, where no band of protein is observed. Retinoids extracted from the NaBH4-treated metaretinochrome in membranes and analyzed with high-pressure liquid chromatography show a main peak of 11-cis retinol. The results of this and earlier (Seki et al., 1982) papers are summarized, and it is strongly suggested that metaretinochrome in the squid retina may play the role of 11-cis retinal donor for opsin and contribute to the synthesis of the squid rhodopsin.
Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells.
Authors: Yellen G
Published: May 2004

Single channel currents through Ca2+-activated K+ channels of bovine chromaffin cells were measured to determine the effects of small ions on permeation through the channel. The channel selects strongly for K+ over Na+ and Cs+, and Rb+ carries a smaller current through the channel than K+. Tetraethylammonium ion (TEA+) blocks channel currents when applied to either side of the membrane; it is effective at lower concentrations when applied externally. Millimolar concentrations of internal Na+ reduce the average current through the channel and produce large fluctuations (flicker) in the open channel currents. This flickery block is analyzed by a new method, amplitude distribution analysis, which can measure block and unblock rates in the microsecond time range even though individual blocking events are not time-resolved by the recording system. The analysis shows that the rate of block by Na+ is very voltage dependent, but the unblock rate is voltage independent. These results can be explained easily by supposing that current flow through the channel is diffusion limited, a hypothesis consistent with the large magnitude of the single channel current.
Volume-responsive sodium and proton movements in dog red blood cells.
Authors: Parker J C , Castranova V
Published: May 2004

Shrinkage of dog red blood cells (RBC) activates a Na transport pathway that is Cl dependent, amiloride sensitive, and capable of conducting Na-proton counterflow. It is possible to establish transmembrane gradients for either Na or protons and to demonstrate that each cation species can drive reciprocal movements of the other. The nature of the coupling between Na and proton movements was investigated using the fluorescent probe diS-C3(5) and also by an indirect method in which K movements through valinomycin channels were used to draw inferences about the membrane potential. No evidence was found to suggest that the Na-proton pathway activated by shrinkage of dog RBC is a conductive one. By exclusion, it is presumed that the coupling between the counterflow of Na and protons is electroneutral. The volume-activated Na-proton fluxes in dog RBC have certain properties that distinguish them from similar transport pathways in other cell types.
Inactivation viewed through single sodium channels.
Authors: Vandenberg C A , Horn R
Published: May 2004

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.
Active ion transport in the renal proximal tubule. II. Ionic dependence of the Na pump.
Authors: Soltoff S P , Mandel L J
Published: May 2004

The dependence of Na pump activity on intracellular and extracellular Na+ and K+ was investigated using a suspension of rabbit cortical tubules that contained mostly (86%) proximal tubules. The ouabain-sensitive rate of respiration (QO2) was used to measure the Na pump activity of intact tubules, and the Na,K-ATPase hydrolytic activity was measured using lysed proximal tubule membranes. The dependence (K0.5) of the Na pump on intracellular Na+ was affected by the relative intracellular concentration of K+, ranging from approximately 10 to 15 mM at low K+ and increasing to approximately 30 mM as the intracellular K+ was increased. The Na pump had a K0.5 for extracellular K+ of 1.3 mM in the presence of saturating concentrations of intracellular Na+. Measurements of the Na,K-ATPase activity under comparable conditions rendered similar values for the K0.5 of Na+ and K+. The Na pump activity in the intact tubules saturated as a function of extracellular Na at approximately 80 mM Na, with a K0.5 of 30 mM. Since Na pump activity under these conditions could be further stimulated by increasing Na+ entry with the cationophore nystatin, these values pertain to the Na+ entry step and not to the Na+ dependence of the intracellular Na+ site. When tubules were exposed to different extracellular K+ concentrations and the intracellular Na+ concentration was subsaturating, the Na pump had an apparent K0.5 of 0.4 mM for extracellular K. Under normal physiological conditions, the Na pump is unsaturated with respect to intracellular Na+, and indirect analysis suggests that the proximal cell may have an intracellular Na+ concentration of approximately 35 mM.
Effect of membrane polarization on contractile threshold and time course of prolonged contractile responses in skeletal muscle fibers.
Authors: Caputo C , Bolaños P , Gonzalez G F
Published: May 2004

Short muscle fibers (less than 1.5 mm) from the m. lumbricalis IV digiti of Rana pipiens were voltage-clamped at -100 mV with a two-microelectrode technique, in normal Ringer's solution containing 10(-6) g/ml tetrodotoxin. The activation curve relating peak tension to membrane potential could be shifted toward more negative or less negative potential values by hyperpolarizing or depolarizing the fiber membrane to -130, -120, or -70 mV, respectively, which indicates that contractile threshold depends on the fiber membrane potential. Long (greater than 5 s) depolarizing (90 mV) pulses induce prolonged contractile responses showing a plateau and a rapid relaxation phase similar to K contractures. Conditioning hyperpolarizations prolong the time course of these responses, while conditioning depolarizations shorten it. The shortening of the response time course, which results in a decrease of the area under the response, is dependent on the amplitude and duration of the conditioning depolarization. Depending on the magnitude and duration, a conditioning depolarization may also reduce peak tension. When the area under the response is reduced by 50%, the level of membrane potential also affects the repriming rate. During repriming, peak tension is restored before the contracture area. Thus, when peak tension is reprimed to 80%, the area is reprimed by 50% of its normal value. Repriming has a marked temperature dependency with a Q10 higher than 4. These results are compatible with the idea that an inactivation process, voltage and time dependent, regulates the release of calcium from the sarcoplasmic reticulum during these responses.
Sodium channel activation in the squid giant axon. Steady state properties.
Authors: Stimers J R , Bezanilla F , Taylor R E
Published: May 2004

Treatment of giant axons from the squid, Loligo pealei, with pronase removes Na channel inactivation. It was found that the peak Na current is increased, but the activation kinetics are not significantly altered, by pronase. Measurements of the fraction of open channels as a function of voltage (F-V) showed an e-folding at 7 mV and a center point near -15 mV. The rate of e-folding implies that a minimum of 4 e-/channel must cross the membrane field to open the channel. The charge vs. voltage (Q-V) curve measured in a pronase-treated axon is not significantly different from that measured when inactivation is intact: approximately 1,850 e-/micron2 were measured over the voltage range -150 to 50 mV, and the center point was near -30 mV. Normalizing these two curves (F-V and Q-V) and plotting them together reveals that they cross when inactivation is intact but saturate together when inactivation is removed. This illustrates the error one makes when measuring peak conductance with intact inactivation and interpreting that to be the fraction of open channels. A model is described that was used to interpret these results. In the model, we propose that inactivation must be slightly voltage dependent and that an interaction occurs between the inactivating particle and the gating charge. A linear sequence of seven states (a single open state with six closed states) is sufficient to describe the data presented here for Na channel activation in pronase-treated axons.
The effect of nucleotides on the rate of spontaneous quantum bumps in Limulus ventral photoreceptors.
Authors: Stern J , Chinn K , Robinson P , Lisman J
Published: May 2004

The effect of intracellular nucleotides on the rate of spontaneous quantum bumps in Limulus ventral photoreceptors has been examined. Internal dialysis of photoreceptors with solutions lacking nucleotide leads to an elevation of the quantum bump rate that can be reversed by introduction of nucleotide. Similarly, elevation occurs after treating intact cells with the metabolic inhibitor 2-deoxyglucose. This effect can be reversed by intracellular injection of ATP. The rate of spontaneous quantum bumps in unpoisoned cells can be reduced to below normal levels by injection of ATP. These results support the hypothesis that high-energy nucleotides suppress the rate of spontaneous quantum bumps.
Calcium influx in contracting and paralyzed frog twitch muscle fibers.
Authors: Curtis B A , Eisenberg R S
Published: May 2004

Calcium uptake produced by a potassium contracture in isolated frog twitch fibers was 6.7 +/- 0.8 pmol in 0.7 cm of fiber (mean +/- SEM, 21 observations) in the presence of 30 microM D600. When potassium was applied to fibers paralyzed by the combination of 30 microM D600, cold, and a prior contracture, the calcium uptake fell to 3.0 +/- 0.7 pmol (11): the fibers were soaked in 45Ca in sodium Ringer for 3 min before 45Ca, in a potassium solution, was added for 2 min; each estimate of uptake was corrected for 5 min of resting influx, measured from the same fiber (average = 2.3 +/- 0.3 pmol). The calcium influx into paralyzed fibers is unrelated to contraction. This voltage-sensitive, slowly inactivating influx, which can be blocked by 4 mM nickel, has properties similar to the calcium current described by several laboratories. The paired difference in calcium uptake between contracting and paralyzed fibers, 2.9 +/- 0.8 pmol (16), is a component of influx related to contraction. Its size varies with contracture size and it occurs after tension production: 45Ca applied immediately after contracture is taken up in essentially the same amounts as 45Ca added before contraction. This delayed uptake is probably a "reflux" refilling a binding site on the cytoplasmic side of the T membrane, which had been emptied during the prior contracture, perhaps to initiate it. We detect no component of calcium uptake related to excitation-contraction coupling occurring before or during a contracture.
Cyclic AMP inhibits Na+/H+ exchange at the apical membrane of Necturus gallbladder epithelium.
Authors: Reuss L , Petersen K U
Published: May 2004

The effects of elevating intracellular cAMP levels on Na+ transport across the apical membrane of Necturus gallbladder epithelium were studied by intracellular and extracellular microelectrode techniques. Intracellular cAMP was raised by serosal addition of the phosphodiesterase inhibitor theophylline (3 mM) or mucosal addition of either 8-Br-cAMP (1 mM) or the adenylate cyclase activator forskolin (10 microM). During elevation of intracellular cAMP, intracellular Na+ activity (alpha Nai) and intracellular pH (pHi) decreased significantly. In addition, acidification of the mucosal solution, which contained either 100 or 10 mM Na+, was inhibited by approximately 50%. The inhibition was independent of the presence of Cl- in the bathing media. The rates of change of alpha Nai upon rapid alterations of mucosal [Na+] from 100 to 10 mM and from 10 to 100 mM were both decreased, and the rate of pHi recovery upon acid loading was also reduced by elevated cAMP levels. Inhibition was approximately 50% for all of these processes. These results indicate that cAMP inhibits apical membrane Na+/H+ exchange. The results of measurements of pHi recovery at 10 and 100 mM mucosal [Na+] and a kinetic analysis of recovery as a function of pHi suggest that the main or sole mechanism of the inhibitory effect of cAMP is a reduction in the maximal rate of acid extrusion. In conjunction with the increase in apical membrane electrodiffusional Cl- permeability, produced by cAMP, which causes a decrease in net Cl- entry (Petersen, K.-U., and L. Reuss, 1983, J. Gen. Physiol., 81:705), inhibition of Na+/H+ exchange contributes to the reduction of fluid absorption elicited by this agent. Similar mechanisms may account for the effects of cAMP in other epithelia with similar transport properties. It is also possible that inhibition of Na+/H+ exchange by cAMP plays a role in the regulation of pHi in other cell types.
Activation of squid axon K+ channels. Ionic and gating current studies.
Authors: White M M , Bezanilla F
Published: May 2004

We have used data obtained from measurements of ionic and gating currents to study the process of K+ channel activation in squid giant axons. A marked improvement in the recording of K+ channel gating currents (IKg) was obtained by total replacement of Cl- in the external solution by NO-3, which eliminates approximately 50% of the Na+ channel gating current with no effect on IKg. The midpoint of the steady state charge-voltage (Qrel - V) relationship is approximately 40 mV hyperpolarized to that of the steady state activation (fo - V) curve, which is an indication that the channel has many nonconducting states. Ionic and gating currents have similar time constants for both ON and OFF pulses. This eliminates any Hodgkin-Huxley nx scheme for K+ channel activation. An interrupted pulse paradigm shows that the last step in the activation process is not rate limiting. IKg shows a nonartifactual rising phase, which indicates that the first step is either the slowest step in the activation sequence or is voltage independent. These data are consistent with the following general scheme for K+ channel activation: (formula; see text)
Relationship between light sensitivity and intracellular free Ca concentration in Limulus ventral photoreceptors. A quantitative study using Ca-selective microelectrodes.
Authors: Levy S , Fein A
Published: May 2004

The possible role of Ca ions in mediating the drop in sensitivity associated with light adaptation in Limulus ventral photoreceptors was assessed by simultaneously measuring the sensitivity to light and the intracellular free Ca concentration (Cai); the latter was measured by using Ca-selective microelectrodes. In dark-adapted photoreceptors, the mean resting Cai was 3.5 +/- 2.5 microM SD (n = 31). No correlation was found between resting Cai and absolute sensitivity from cell to cell. Typically, photoreceptors are not uniformly sensitive to light; the Cai rise evoked by uniform illumination was 20-40 times larger and faster in the most sensitive region of the cell (the rhabdomeral lobe) than it was away from it. In response to a brief flash, the Cai rise was barely detectable when 10(2) photons were absorbed, and it was saturated when approximately 10(5) photons were absorbed. During maintained illumination, starting near the threshold of light adaptation, steady Cai increases were associated with steady desensitizations over several log units of light intensity: a 100-fold desensitization was associated with a 2.5-fold increase in Cai. After a bright flash, sensitivity and Cai recovered with different time courses: the cell was still desensitized by approximately 0.5 log units when Cai had already recovered to the prestimulus level, which suggests that under those conditions Cai is not the rate-limiting step of dark adaptation. Ionophoretic injection of EGTA markedly decreased the light-induced Cai rise and increased the time to peak of the light response, but did not alter the resting Cai, which suggests that the time to peak is affected by a change in the capacity to bind Ca2+ and not by resting Cai. Lowering the extracellular Ca2+ concentration (Cao) first decreased Cai and increased sensitivity. Longer exposure to low Cao resulted in a further decrease of Cai but decreased rather than increased sensitivity, which suggests that under certain conditions it is possible to uncouple Cai and sensitivity.