bluuebunny/crossref_metadata_2025 huggingface dataset:
Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane.
Authors: Zimmerberg J , Cohen F S , Finkelstein A
Published: May 2004

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.
The permeability of the endplate channel to organic cations in frog muscle.
Authors: Dwyer T M , Adams D J , Hille B
Published: May 2004

The relative permeability of endplate channels to many organic cations was determined by reversal-potential criteria. Endplate currents induced by iontophoretic "puffs" of acetylcholine were studied by a Vaseline gap, voltage clamp method in cut muscle fibers. Reversal potential changes were measured as the NaCl of the bathing medium was replaced by salts of organic cations, and permeability ratios relative to Na+ ions were calculated from the Goldman-Hodgkin-Katz equation. 40 small monovalent organic cations had permeability ratios larger than 0.1. The most permeant including NH4+, hydroxylamine, hydrazine, methylamine, guanidine, and several relatives of guanidine had permeability ratios in the range 1.3--2.0. However, even cations such as imidazole, choline, tris(hydroxymethyl)aminomethane, triethylamine, and glycine methylester were appreciably permeant with permeability ratios of 0.13--0.95. Four compounds with two charged nitrogen groups were also permeant. Molecular models of the permeant ions suggest that the smallest cross-section of the open pore must be at least as large as a square, 6.5 A x 6.5 A. Specific chemical factors seem to be less important than access or friction in determining the ionic selectivity of the endplate channel.
The permeability of endplate channels to monovalent and divalent metal cations.
Authors: Adams D J , Dwyer T M , Hille B
Published: May 2004

The relative permeability of endplate channels to monovalent and divalent metal ions was determined from reversal potentials. Thallium is the most permeant ion with a permeability ratio relative to Na+ of 2.5. The selectivity among alkali metals is weak with a sequence, Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+, and permeability ratios of 1.4, 1.3, 1.1, 1.0, and 0.9. The selectivity among divalent ions is also weak, with a sequence for alkaline earths of Mg++ greater than Ca++ greater than Ba++ greater than Sr++. The transition metal ions Mn++, Co++, Ni++, Zn++, and Cd++ are also permeant. Permeability ratios for divalent ions decreased as the concentration of divalent ion was increased in a manner consistent with the negative surface potential theory of Lewis (1979 J. Physiol. (Lond.). 286: 417--445). With 20 mM XCl2 and 85.5 mM glucosamine.HCl in the external solution, the apparent permeability ratios for the alkaline earth cations (X++) are in the range 0.18--0.25. Alkali metal ions see the endplate channel as a water-filled, neutral pore without high-field-strength sites inside. Their permeability sequence is the same as their aqueous mobility sequence. Divalent ions, however, have a permeability sequence almost opposite from their mobility sequence and must experience some interaction with groups in the channel. In addition, the concentrations of monovalent and divalent ions are increased near the channel mouth by a weak negative surface potential.
Action potentials in fast- and slow-twitch mammalian muscles during reinnervation and development.
Authors: McArdle J J , Michelson L , D'Alonzo A J
Published: May 2004

Action potentials (APs) were recorded from the extrajunctional membrane of surface fibers of the fast-twitch extensor digitorum longus (extensor) and the slow-twitch soleus muscles of adult rats. APs of the extensor muscle had a significantly faster rate of rise and fall, as well as a shorter duration, than those of the soleus. In addition, the overshoot of APs and the resting membrane potential was greater for the extensor. Whereas the soleus produced only one AP regardless of the stimulus duration, the number of extensor responses was directly proportional to the stimulus duration. This repetitive activity was greatly reduced by a concentration of tetrodotoxin (TTX) as low as 5 X 10(11) g/ml. Within 8 d after crush of the nerves to these two muscles, all differences in AP properties disappeared and both muscles became partially resistant to TTX. Reinnervation brought about a redifferentiation so that differences in AP were again significant at 22 d after nerve crush. However, the rate of rise of extensor APs did not attain normal values even as late as 60 d after nerve crush. APs were found to be the same for extensor and soleus muscles from 12-d-old rats. At 18 d after birth, rate of rise was equivalent to that of adult muscle for the soleus although 50--60 d were required before this parameter was fully mature for the extensor. Nevertheless, APs of the extensor and soleus were clearly differentiated within 25 d after birth. Differences in fast and slow muscle APs are discussed with regard to differences in ion gradients and sarcolemmal conductance.
The effects of external Ca++ and Mg++ on the voltage sensitivity of desensitization in Electrophorus electroplaques.
Authors: Pallotta B S , Webb G D
Published: May 2004

Desensitization onset was studied in voltage-clamped Electrophorus electroplaques during prolonged exposure to bath-applied carbamylcholine. The time-course of desensitization was described by a first-order rate constant kappa obs, which increased exponentially with membrane hyperpolarization from -20 to -90 mV. When Ca++ was increased from 2 to 10 mM, the voltage sensitivity of kappa obs decreased; kappa obs decreased for voltages more negative than -40 mV, and increased slightly at voltages more positive than -40 mV. 10 mM Mg++ had a less pronounced effect and the voltage sensitivity of kappa obs was unchanged. The equilibrium level of desensitization, estimated from the carbamylcholine-dependent conductance which remained after desensitization was apparently complete, also increased with hyperpolarization.
LK sheep reticulocytosis: effect of anti-L on K influx and in vitro maturation.
Authors: Kim H D , Theg B E , Lauf P K
Published: May 2004

After massive hemorrhage, adult sheep with genotypically low potassium (LK) red cells temporarily produce high potassium (HK) cells with ouabain-sensitive K+ pump fluxes equivalent to mature HK red cells. In light of recent reports of different red cell volume populations accompanying the HK-LK transition also occurring in newborn LK sheep and the unresolved controversy over the effect of anti-L on K+ transport in these immature red cells, we have reexamined the K+ transport changes and the effect of anti-L in the newly formed HK cells at various times after anemic stress and under in vitro conditions. We found that approximately 7 d after bleeding, maximum reticulocytosis occurred in the peripheral blood. After separation by density centrifugation, the top 10% cell fraction contained 100% reticulocytes, with a mean cell volume 2.5 times larger than that of mature erythrocytes. These immature red cells were of HK type, and their K+ pump and leak fluxes were 30 and 10 times higher, respectively, than those found in mature LK cells. The new cells may possess HK- and LK-type pumps because K+ pump influx was significantly stimulated by anti-L. When separated by density centrifugation on days 9, 17, and 23 after bleeding, some of the cells apparently maintained their large size while gaining higher density. Large cells from day 9, kept in vitro for 22 h, showed anti-L-sensitive K+ pump and leak fluxes that declined within hours, paralleling the behavior of these cells in vivo, whereas cellular K+ levels changed much less. It is concluded that the newly formed red cells may belong to a stress-induced macrocytic cell population that does not acquire all of the characteristics of adult LK cells.
Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder.
Authors: Narvarte J , Finn A L
Published: May 2004

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.
Sensitivities of single nerve fibers in the hamster chorda tympani to mixtures of taste stimuli.
Authors: Hyman A M , Frank M E
Published: May 2004

Responses of three groups of neural fibers from the chorda tympani of the hamster to binary mixtures of taste stimuli applied to the tongue were analyzed. The groups displayed different sensitivities to six chemicals at concentrations that had approximately equal effects on the whole nerve. Sucrose-best fibers responded strongly only to sucrose and D-phenylalanine. NaCl-best and HCl-best fibers, responded to four electrolytes: equally to CaCl2 and nearly equally to HCl, but the former responded more to NaCl, and the latter responded more to NH4Cl. The groups of fibers dealt differently with binary mixtures. Sucrose-best fibers responded to a mixture of sucrose and D-phenylalanine as if one of the chemicals had been appropriately increased in concentration, but they responded to a mixture of either one and an electrolyte as if the concentration of sucrose or D-phenylalanine had been reduced. NaCl-best fibers responded to a mixture as if it were a "mixture" of two appropriate concentrations of one chemical, or somewhat less. But, responses of HCl-best fibers to mixtures were greater than that, approaching a sum of responses to components. These results explain effects on the whole nerve, suggest that the sensitivity of a mammalian taste receptor to one chemical can be affected by a second, which may or may not be a stimulus for that receptor, and suggest that some effects of taste mixtures in humans may be the result of peripheral processes.
Calcium flux across disk membranes. Studies with intact rod photoreceptors and purified disks.
Authors: Szuts E Z
Published: May 2004

Calcium accumulation by rod disks was studied in excised bullfrog retinas with 45Ca tracer-exchange methods. Ca uptake by disks is a necessary requirement if light-induced Ca releases from disks mediate photoreceptor excitation. In an hour-long incubation, disks exchanged less than or equal to 0.01 mole of Ca per mole of rhodopsin, or less than or equal to 10% of their total Ca. This corresponds to a unidirectional flux of less than or equal to 0.01 fmol/cm2 S, or less than or equal to 5 ions/disk-second across the disk membrane. Neither incubation in 10 mM Ca (which increases cytoplasmic activity 10--100-fold) nor photostimulation (which photoactivated up to 50% rhodopsin/h) had measurable effect on exchange rate, though an increase of several orders of magnitude would have been expected according to the hypothesis. The observed exchange could not be explained by: (a) 45Ca losses from disks before measurement (neither the net efflux nor the Ca-Ca exchange property of disks adequately explains such losses), (b) a limited pool of exchangeables Ca from strongly binding intradiskal sites, or (c) rate-limiting flux across the plasma membrane during incubation. For the study of the Ca efflux properties of disks, separate experiments were performed with 45Ca-loaded disks. Intradiskal activity could be estimated from the disks' hyperosmotically sensitive 45Ca pool and from their intradiskal volume (indirectly assayed by density). Ca-Ca exchange was undetectable (less than or equal to 0.1 fmol/cm2 S) in disks whose intradiskal activity was at least 0.3 mM. Net efflux was 0.2 fmol/cm2 S for an intradiskal activity of approximately 1 mM and is comparable to published fluxes for phospholipid vesicles. These results seem to exclude the internal space of disks as the source of Ca for photoreceptor excitation.
Thermodynamic and kinetic studies of the gating behavior of a K+-selective channel from the sarcoplasmic reticulum membrane.
Authors: Labarca P , Coronado R , Miller C
Published: May 2004

A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose [corrected] of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K+. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple two-state conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.
Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum.
Authors: Coronado R , Rosenberg R L , Miller C
Published: May 2004

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.
Stereospecificity of multiple receptor sites in a labellar sugar receptor of the fleshfly for amino acids and small peptides.
Authors: Shimada I , Tanimura T
Published: May 2004

N-Formylation and N-methylation of the alpha-amino group of L-phenylalanine result in extremely decreased responses of the labellar sugar receptor of the fleshfly, whereas the same structural alteration of L-valine hardly affects the response. Methyl esterification of the alpha-carboxyl group of phenylalanine, on the other hand, maintains the response to some extent, but similar treatment of valine completely diminishes the response. The aromatic structure in phenylalanine is not essential for stimulation. These results suggest a substantial difference in the stereospecificities and functional group specificities of the furnase (F) and aliphatic carboxylate (T) sites in the sugar receptor. The effect of small peptides on the sugar receptor was examined systematically. Their effectiveness depends mainly on the place of the constituent amino acids rather than on their composition, indicating the decisive role that certain aliphatic amino acids in the C-terminal position play in stimulation. Remarkable regularities in the stimulating effectiveness of small peptides exactly correspond to the stereospecificity of each receptor site. We propose two hypothetical models of the F and T sites, which involve three and two subsites, respectively, that are capable of hydrogen bond formation. The F and T sites also have a hydrophobic subsite that discriminates the R groups of the stimulants and a few spatial barriers.
Properties of the pH-sensitive site that controls the lambda max of Limulus metarhodopsin.
Authors: Lisman J E , Schulman S , Sheline Y , Brown P K
Published: May 2004

A pH-sensitive site controls the lambda max of Limulus metarhodopsin. The properties of this site were examined using intracellular recordings of the early receptor potential (ERP) as a pigment assay. ERPs recorded over a range of extracellular pHs indicate that the apparent pK of the site is in the range of 8.3-8.6. Several lines of evidence indicate that the site responds directly to changes in extracellular pH (pHo) rather than to changes in intracellular pH(pHi) that follow as a secondary result of changing pHo : (a) the effect of changing pHo was rapid (less than 60 s); (b) when pHo was raised, the simultaneous rise in pHi, as measured with phenol red, was relatively small; (c) raising pHi by intracellular injection of pH 10 glycine buffer did not affect the site; and (d) the effect of changing pH0 could not be blocked by increasing the intracellular pH buffering capacity. It is concluded that the pH-sensitive site on metarhodopsin is on the extracellular surface of the plasma membrane.
Saxitoxin binding in nerves from walking legs of the lobster Homarus americanus. Two classes of receptors.
Authors: Strichartz G R , Hanson Bay C M
Published: May 2004

The binding of exchange-labeled saxitoxin (STX) to sodium channels has been investigated in the nonmyelinated fibers of the walking leg nerves of the lobster. The properties of the STX binding site differed systematically among the nerves from different walking legs. The equilibrium dissociation constant for STX binding (KSTX) to the front legs is approximately twice that for the binding to the rear legs; the average ratio of KSTX (front): KSTX (rear) from five separate experiments was 1.80 +/- 0.21 (mean +/- SE). The actual KSTX values ranged from 124.0 to 22.7 nM for the front leg nerves and from 8.6 to 12.7 nM for the rear leg nerves. KSTX values for the middle two walking leg nerves fell between those for the front and rear legs. The inhibitory dissociation constant for tetrodotoxin (KTTX), calculated from tetrodotoxin's inhibition of labeled STX binding, was 3.02 +/- 0.27 nM for the front legs and 2.20 +/- 0.33 nM for the rear legs. The ratio KSTX: KTTX was different in the front and rear leg nerves, being 5.5 and 4.2, respectively. The apparent P pKa of the STX receptor also differed between the two legs, being 4.6 +/- 0.3 for the front legs and 5.1 +/- 0.1 for the rear legs. These results demonstrate that one tissue type in one organism can contain different toxin binding sites. The difference in the receptors can be qualitatively accounted for by the location of an additional negative charge near the receptor site of the rear walking leg.
Inactivation of potassium current in squid axon by a variety of quaternary ammonium ions.
Authors: Swenson R P
Published: May 2004

The characteristics of potassium channel block by a diverse group of quaternary ammonium (QA) ions was examined in squid axons. Altering the size and nature of the head and/or tail groups of the QA ions applied internally produced only quantitative differences in the potassium current block. Although their entry rate is diminished, compounds with head groups as large as 11 X 12 A are capable of occluding the channel, whereas the smallest QA ions, with head groups approximately 5 X 6 A, are not potent blockers. When one or three terminal hydrogens of the head group were replaced by hydroxyl moieties, the compound's blocking ability was diminished, suggesting that QA binding is not improved by hydrogen bonding at these positions. QA ions bound to their site within the potassium channel with 1:1 stoichiometry, and the site is perhaps 20% or more of the distance through the membrane electric field. Raising external potassium concentration did not alter the steady-state or kinetic features of the QA block of outward potassium currents; however, increasing temperature or adding Ba2+ internally increased the rate of decay of the QA-blocked currents. From the structure-function analysis of the QA ions, projections concerning both the architecture of the potassium channel's inner mouth and the significance of various chemical constituents of the ions were made. The potassium channel may now be pictured as having a wider mouth (up to 11 X 12 A) extending to the QA binding site and then narrowing quickly to the region of channel selectivity. Important alterations that improve the blocking ability of the compounds include: (a) lengthening the alkyl hydrocarbon tail group (up to 10 carbon), (b) lengthening a second hydrocarbon chain of the head group (e.g., decyldimethylphenylammonium bromide [C10DM phi]), and (c) adding a carbonyl moiety to the tail (e.g., ambutonium).
Rhodopsin kinetics in the cat retina.
Authors: Ripps H , Mehaffey L , Siegel I M
Published: May 2004

The bleaching and regeneration of rhodopsin in the living cat retina was studied by means of fundus reflectometry. Bleaching was effected by continuous light exposures of 1 min or 20 min, and the changes in retinal absorbance were measured at 29 wavelengths. For all of the conditions studied (fractional bleaches of from 65 to 100%), the regeneration of rhodopsin to its prebleach levels required greater than 60 min in darkness. After the 1-min exposures, the difference spectra recorded during the first 10 min of dark adaptation were dominated by photoproduct absorption, and rhodopsin regeneration kinetics were obscured by these intermediate processes. Extending the bleaching duration to 20 min gave the products of photolysis an opportunity to dissipate, and it was possible to follow the regenerative process over its full time-course. It was not possible, however, to fit these data with the simple exponential function predicted by first-order reaction kinetics. Other possible mechanisms were considered and are presented in the text. Nevertheless, the kinetics of regeneration compared favorably with the temporal changes in log sensitivity determined electrophysiologically by other investigators. Based on the bleaching curve for cat rhodopsin, the photosensitivity was determined and found to approximate closely the value obtained for human rhodopsin; i.e., the energy Ec required to bleach 1-e-1 of the available rhodopsin was 7.09 log scotopic troland-seconds (corrected for the optics of the cat eye), as compared with approximately 7.0 in man.
"Rapid regeneration" in the cat retina: a case for spreading depression.
Authors: Ripps H , Mehaffey L , Siegel I M
Published: May 2004

Fundus reflectometry of the cat retina showed that under certain circumstances a rapid increase in density may follow intense bleaching exposures. The spectral characteristics of the density changes indicated that neither rhodopsin nor its bleach products could be responsible for this effect. The poor condition of the animals in which the phenomenon was observed and its conspicuous absence in the majority of the experimental runs suggested that the effect was associated with a process other than the resynthesis of rhodopsin. It was shown that an extrareceptoral event, spreading depression (SD) of the retina, is the most likely source of the rapid spectral change. The well-known tissue alterations associated with SD were induced in the retina independently of pigment density change. The resultant difference spectra resembled those produced when the rapid density increase occurred spontaneously. It seems likely that the abnormal physiological condition of those cats in which the phenomenon is more frequently observed primes the retina for the light-induced generation of spreading depression.
Electrical resistance of a capillary endothelium.
Authors: Crone C , Christensen O
Published: May 2004

The electrical resistance of consecutive segments of capillaries has been determined by a method in which the microvessels were treated as a leaky, infinite cable. A two-dimensional analytical model to describe the potential field in response to intracapillary current injection was formulated. The model allowed determination of the electrical resistance from four sets of data: the capillary radius, the capillary length constant, the length constant in the mesentery perpendicular to the capillary, and the relative potential drop across the capillary wall. Of particular importance were the mesothelial membranes covering the mesenteric capillaries with resistances several times higher than that of the capillary endothelium. 27 frog mesenteric capillaries were characterized. The average resistance of the endothelium was 1.85 omega cm2, which compares well with earlier determinations of the ionic permeability of such capillaries. However, heterogeneity with respect to resistance was observed, that of 10 arterial capillaries being 3.0 omega cm2 as compared with 0.95 omega cm2 for 17 mid- and venous capillaries. The average in situ length constant was 99 micrometers for the arterial capillaries and 57 micrometers for the mid- and venous capillaries. It is likely that the ions that carry the current must move paracellularly, through junctions that are leaky to small solutes.
Adsorption of divalent cations to bilayer membranes containing phosphatidylserine.
Authors: McLaughlin S , Mulrine N , Gresalfi T , Vaio G , McLaughlin A
Published: May 2004

The Stern equation, a combination of the Langmuir adsorption isotherm, the Boltzmann relation, and the Grahame equation from the theory of the diffuse double layer, provides a simple theoretical framework for describing the adsorption of charged molecules to surfaces. The ability of this equation to describe the adsorption of divalent cations to membranes containing brain phosphatidylserine (PS) was tested in the following manner. Charge reversal measurements were first made to determine the intrinsic 1:1 association constants of the divalent cations with the anionic PS molecules: when the net charge of a PS vesicle is zero one-half of the available sites are occupied by divalent cations. The intrinsic association constant, therefore, is equal to the reciprocal of the divalent cation concentration at which the mobility of a PS vesicle reverses sign. The Stern equation with this association constant is capable of accurately describing both the zeta potential data obtained with PS vesicles at other concentrations of the divalent cations and the data obtained with with vesicles formed from mixtures of PS and zwitterionic phospholipids. Independent measurements of the number of ions adsorbed to sonicated PS vesicles were made with a calcium-sensitive electrode. The results agreed with the zeta potential results obtained with multilamellar vesicles. When membranes are formed at 20 degrees C in 0.1 M NaCl, the intrinsic 1:1 association constants of Ni, Co, Mn, Ba, Sr, Ca, and Mg with PS are 40, 28, 25, 20, 14, 12, and 8 M-1, respectively.
pH dependence of the acetylcholine receptor channel: a species variation.
Authors: Landau E M , Gavish B , Nachshen D A , Lotan I
Published: May 2004

The effects of pH changes on the miniature endplate current (mepc) and on endplate current fluctuations (acetylcholine [ACh] noise) were examined at the neuromuscular junction in vitro in two species of frogs. In Rana pipiens the relationship between the decay time constant of the mepc (tau') and pH had a symmetrical bell shape; the value of tau' being largest at pH 7 and decreasing at more acid or more alkaline pH. In acid pH the mepc amplitude (A) decreased relative to its value at pH 7, and in alkaline pH A increased. In Rana ridibunda a narrower and asymmetric bell-shaped dependence of tau' on pH, having a maximum of pH 5.5, was found. The mepc amplitude was again reduced in acid pH but had a peak at pH 5.5. Also, its value at pH 9 was larger than at pH 7. These results were obtained with a number of different buffers and were not found to be sensitive to the nature of the buffer chosen. By performing ACh-noise analysis we found that in Rana pipiens at acid pH (5.5-5.0), the single channel conductance (gamma) and the single channel open time (tau) were significantly reduced relative to their value at pH 7. However, in Rana ridibunda at acid pH (5.4) gamma was unchanged and tau was markedly increased relative to their values at pH 7. The results can be explained quantitatively by electrostatic interaction between two fixed and titratable ionic groups and a mobile charge in the receptor molecule. The model fits the data for groups having pKs approximately 4.8 and approximately 9.8 for Rana pipiens and approximately 4.6 and approximately 6.3 for Rana ridibunda. The groups can be tentatively identified as amino acid residues; glutamic or aspartic and lysine or tyrosine for Rana pipiens; glutamic or aspartic and histidine for Rana ridibunda. The difference in the fitted values of the other model parameters for these two species can be attributed to differences in the spatial configuration of the charged groups.
Glycolytic and oxidative metabolism in relation to retinal function.
Authors: Winkler B S
Published: May 2004

Measurements of lactate production and ATP concentration in superfused rat retinas were compared with extracellular photoreceptor potentials (Fast PIII). The effect of glucose concentration, oxygen tension, metabolic inhibition, and light were studied. Optimal conditions were achieved with 5-20 mM glucose and oxygen. The isolated retina had a high rate of lactate production and maintained the ATP content of a freshly excised retina, and Fast PIII potentials were similar to in vivo recordings. Small (less than 10%) decreases in aerobic and anaerobic lactate production were observed after illumination of dark-adapted retinas. There were no significant differences in ATP content in dark- and light-adapted retinas. In glucose-free medium, lactate production ceased, and the amplitude of Fast PIII and the level of ATP declined, but the rates of decline were slower in oxygen than in nitrogen. ATP levels were reduced and the amplitude of Fast PIII decreased when respiration was inhibited, and these changes were dependent on glucose concentration. Neither glycolysis alone nor Krebs cycle activity alone maintained the superfused rat retina at an optimal level. Retinal lactate production and utilization of ATP were inhibited by ouabain. Mannose but not galactose or fructose produced lactate and maintained ATP content and Fast PIII. Iodoacetate blocked lactate production and Fast PIII and depleted the retina of ATP. Pyruvate, lactate, and glutamine maintained ATP content and Fast PIII reasonably well (greater than 50%) in the absence of glucose, even in the presence of iodoacetate. addition of glucose, mannose, or 2-deoxyglucose to medium containing pyruvate and iodoacetate abolished Fast PIII and depleted the retina of its ATP. It is suggested that the deleterious effects of these three sugars depend upon their cellular uptake and phosphorylation during the blockade of glycolysis by iodoacetate.
Local movement in stimulated frog sartorius muscle.
Authors: Stienen G J , Blangé T
Published: May 2004

Local movement was recorded in tetanically contracting frog sartorius muscle to estimate the nonuniformity in the distribution of compliance in the muscle preparation and the compliance that resides in the attachments of the preparation to the measuring apparatus. The stimulated muscle was also subjected to rapid length changes, and the local movements and tension responses were recorded. The results indicate that during tension development at resting length the central region of the muscle shortens at the expense of the ends. After stimulation the "shoulder" in the tension, which divided the relaxation into a slow decline and a subsequent, rather exponential decay toward zero, was accompanied by an abrupt increase in local movement. We also examined the temperature sensitivity of the two phases of relaxation. The results are consistent with the view that the decrease in tension during relaxation depends on mechanical conditions. The local movement brought about by the imposed length changes indicates that the peak value of the relative length change of the uniformly acting part was approximately 20% less than the relative length change of the whole preparation. From these observations, corrections were obtained for the compliance data derived from the tension responses. These corrections allow a comparison with data in the literature obtained from single fiber preparations. The implications for the stiffness measured during the tension responses are discussed.
The kinetics of monazomycin-induced voltage-dependent conductance. II. Theory and a demonstration of a form of memory.
Authors: Muller R U , Peskin C S
Published: May 2004

The empirical differential equation that describes the kinetics of monazomycin-induced voltage-dependent conductance is derived using a standard chemical kinetic formulation and the assumption that monazomycin entry into and exit from the membrane is autocatalytic. The predicted form of gating currents is shown and numerical calculations for this process are made using a range of values for two unmeasured variables. A form of "memory" is then demonstrated, along with the ability of the theoretical equation to explain the nature of the memory.
Isotonic contraction of skinned muscle fibers on a slow time base: effects of ionic strength and calcium.
Authors: Gulati J , Podolsky R J
Published: May 2004

The force development by calcium-activated skinned frog skeletal muscle fibers and the motion on a slow time base after a quick decrease in load were studied at 0-1 degrees C as a function of the ionic strength and the degree of activation. The ionic strength was varied between 50 and 190 mM by adding appropriate concentrations of KCl to the bathing solution. Under these conditions, the fibers could be maximally activated for several cycles at low ionic strength without developing residual tension. We found that the steady isometric force in fully activated fibers linearly decreased when the KCl concentration was increased from 0 to 140 mM. The steady isotonic motion at a given relative load in fully activated fibers was almost the same at KCl concentration greater than or equal to 50 mM. In 0 and 20 mM KCl, the isotonic velocity decreased continuously for more than 300 ms. At a given relative load, the initial velocity of the motion in 0 and 20 mM KCl was about 0.6 and 0.9 times, respectively, that in 140 mM KCl. The initial velocity decreased further when residual tension developed; this observation provides additional evidence that residual tension may reflect the presence of an internal load. The effect of calcium on the motion was examined at 70 mM KCl. In this solution, the motion during the velocity transient at a given relative load appeared to be the same at different levels of activation. The speed of the subsequent motion was almost steady at high calcium levels but decreased continuously in low calcium levels. These results support the idea that at low ionic strength the response of the fiber to calcium is switch-like, but that other factors also affect the contraction mechanism under these conditions.
Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating.
Authors: Cohen C J , Bean B P , Colatsky T J , Tsien R W
Published: May 2004

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.
Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes.
Authors: Mercer R W , Dunham P B
Published: May 2004

ATP stimulates Na transport into inside-out vesicles (IOVs) made from human red cell membranes; strophanthidin inhibits the ATP-stimulated transport. The substrates for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) (glycolytic enzymes bound to the cytoplasmic surface of the red cell membrane) also stimulate Na transport into IOVs without added ATP. The elution of GAPDH from the membranes prevents the stimulation by the substrates, but not by exogenous ATP. Hexokinase plus glucose (agents that promote breakdown of ATP) prevent stimulation of Na transport by exogenous ATP but not by the substrates for GAPDH and PGK. [32P]orthophosphate is incorporated into a membrane-bound organic phosphate compound shown chromatographically to be ATP. The level of membrane-bound ATP is decreased when Na is added, and this decrease is inhibited by strophanthidin. When further synthesis of [32P]ATP is blocked by the addition of unlabeled orthophosphate, all of the membrane-bound [32P]ATP is dissipated by the addition of Na. From these observations it was concluded that membrane-bound glycolytic enzymes synthesize ATP and deposit it in a membrane-associated compartment from which it is used by the Na/K pump.
Distribution and kinetics of membrane dielectric polarization. 1. Long-term inactivation of gating currents.
Authors: Bezanilla F , Taylor R E , Fernández J M
Published: May 2004

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.
Effects of cyclic adenosine 3',5'-monophosphate on photoreceptor disc shedding and retinomotor movement. Inhibition of rod shedding and stimulation of cone elongation.
Authors: Besharse J C , Dunis D A , Burnside B
Published: May 2004

As a test of the hypothesis that cyclic nucleotides play a role in the regulation of retinomotor movements and disc shedding in the photoreceptor-pigment epithelial complex, we have used an in vitro eyecup preparation that sustains both disc shedding and cone retinomotor movements, Eyecups were prepared in white light from animals in which both shedding and cone movement had been blocked by 4 d of constant-light treatment. In eyecups incubated for 3 h in light, disc shedding was negligible and cones remained in the light-adapted (contracted) position. In eyecups incubated in darkness, however, a massive shedding response (dominated by rod photoreceptors) was induced, and at the same time cone photoreceptors elongated to their dark-adapted position. In eyecups incubated in light dbcAMP promoted cone elongation and thus mimicked darkness; the dbcAMP effect was potentiated by the phosphodiesterase inhibitors papaverine and 3-isobutylmethylxanthine. In eyecups incubated in darkness, on the other hand, both phosphodiesterase inhibitors and dbcAMP reduced the phagosome content of the pigment epithelium. The effects of dbcAMP on the cone elongation and rod shedding appear to be specific in that dbcGMP, adenosine, and adenosine 5'-monophosphate had no significant effect. Our results suggest that cAMP plays a role in the regulation of both retinomotor movements and disc shedding.
Weak acid permeability through lipid bilayer membranes. Role of chemical reactions in the unstirred layer.
Authors: Walter A , Hastings D , Gutknecht J
Published: May 2004

The premeabilities of planar lipid bilayer (egg phosphatidylcholine-decane) membranes to butyric and formic acids were measured by tracer and pH electrode techniques. The purposes of the study were (a) to establish criteria for the applicability of each method and (b) to resolve a discrepancy between previously published permeabilities determined using the different techniques. Tracer fluxes of butyric acid were measured at several concentrations and pH's. Under symmetrical conditions the one-way flux of butyric acid(J) is described by 1/J = 1/Pul ([HA] + [A-]) + 1/Pm([HA]), where Pul and Pm are the unstirred layer and membrane permeability coefficients. Pm determined in this manner is 950 x 10(4) cm s-1. Published values for the butyric acid permeability for egg phosphatidylcholine-decane bilayers are 11.5 x 10(-4) (Wolosin and Ginsburg, 1975) and 640 x 10(-4) cm s-1 (Orbach and Finkelstein, 1980). Wolosin and Ginsburg measured net fluxes from a solution of pH = Pka into an unbuffered solution containing a pH electrode. Orbach and Finkelstein measured tracers fluxes under symmetrical conditions at pH 7.4. We reproduced the results of Wolosin and Ginsburg and showed that their apparently low Pm was caused by unstirred layer effects in their poorly buffered solutions. The permeability to formic acid (pKa = 3.75) measured by both tracer and pH electrode techniques was approximately 10(-2) cm s-1. However, if pm greater than Pul, the pH electrode technique cannot be used for measuring the permeabilities of weak acids with pKa's greater than approximately 4.
Divalent cations and the activation kinetics of potassium channels in squid giant axons.
Authors: Gilly W F , Armstrong C M
Published: May 2004

The effects of external Zn+2 and other divalent cations on K channels in squid giant axons were studied. At low concentration (2 mM) Zn+2 slows opening kinetics without affecting closing kinetics. Higher concentrations (5-40 mM) progressively slow opening and speed channel closing to a lesser degree. In terms of "shifts," opening kinetics are strongly shifted to the right on the voltage axis, and off kinetics much less so. The shift of the conductance-voltage relation along the axis is intermediate. Zinc's kinetic effects show little sign of saturation at the highest concentration attainable. Zn does not alter the shape of the instantaneous current-voltage relation of open channels. Some other divalent cations have effects similar to Zn+2, Hg2+ being the most potent and Ca+2 the least. After treatment with Hg+2, which is irreversible, Zn+2 still slows opening kinetics, which suggests that each channel has at least two sites for divalent cation action. The results are not compatible with a simple theory of fixed, uniform surface charges. They suggest that external cations interact directly with a negatively charged element of the gating apparatus that moves inward from the membrane's outer surface during activation. Examination of normal kinetics shows that there is a slow step somewhere in the chain leading to channel opening. But the slowest step must not be the last one.