bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane properties of isolated mudpuppy taste cells.
Authors: Kinnamon S C , Roper S D
Published: May 2004

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.
The sodium current underlying action potentials in guinea pig hippocampal CA1 neurons.
Authors: Sah P , Gibb A J , Gage P W
Published: May 2004

Neurons were acutely dissociated from the CA1 region of hippocampal slices from guinea pigs. Whole-cell recording techniques were used to record and control membrane potential. When the electrode contained KF, the average resting potential was about -40 mV and action potentials in cells at -80 mV (current-clamped) had an amplitude greater than 100 mV. Cells were voltage-clamped at 22-24 degrees C with electrodes containing CsF. Inward currents generated with depolarizing voltage pulses reversed close to the sodium equilibrium potential and could be completely blocked with tetrodotoxin (1 microM). The amplitude of these sodium currents was maximal at about -20 mV and the amplitude of the tail currents was linear with potential, which indicates that the channels were ohmic. The sodium conductance increased with depolarization in a range from -60 to 0 mV with an average half-maximum at about -40 mV. The decay of the currents was not exponential at potentials more positive than -20 mV. The time to peak and half-decay time of the currents varied with potential and temperature. Half of the channels were inactivated at a potential of -75 mV and inactivation was essentially complete at -40 to -30 mV. Recovery from inactivation was not exponential and the rate varied with potential. At lower temperatures, the amplitude of sodium currents decreased, their time course became longer, and half-maximal inactivation shifted to more negative potentials. In a small fraction of cells studied, sodium currents were much more rapid but the voltage dependence of activation and inactivation was very similar.
Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers.
Authors: Sejersted O M , Wasserstrom J A , Fozzard H A
Published: May 2004

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.
Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues.
Authors: Breitwieser G E , Szabo G
Published: May 2004

The role of a guanine nucleotide-binding protein (Gk) in the coupling between muscarinic receptor activation and opening of an inwardly rectifying K+ channel [IK(M)] was examined in cardiac atrial myocytes, using hydrolysis-resistant GTP analogues. In the absence of muscarinic agonist, GTP analogues produced a membrane current characteristic of IK(M). The initial rate of appearance of this receptor-independent IK(M) was measured for the various analogues in order to explore the kinetic properties of IK(M) activation. We found that IK(M) activation is controlled solely by the intracellular analogue/GTP ratio and not by the absolute concentrations of the nucleotides. Analogues competed with GTP for binding to Gk with the following relative affinities: GTP gamma S greater than GTP greater than GppNHp greater than GppCH2p. At sufficiently high intracellular concentrations, however, all GTP analogues produced the same rate of IK(M) activation. This analogue-independent limiting rate is likely to correspond to the rate of GDP release from inactive, GDP-bound Gk. Muscarinic receptor stimulation by nanomolar concentrations of acetylcholine (ACh), which do not elicit IK(M) under control conditions, catalyzed IK(M) activation in the presence of GTP analogues. The rate of Gk activation by ACh (kACh) was found to be described by the simple relationship kACh = 8.4 X 10(8) min-1 M-1.[ACh] + 0.44 min-1, the first term of which presumably reflects the agonist-catalyzed rate of GDP release from the Gk.GDP complex, while the second term corresponds to the basal rate of receptor-independent GDP release. Combined with the estimated K0.5 of the IK(M)-[ACh] dose-effect relationship, 160 nM, this result also allowed us to estimate the rate of Gk.GTP hydrolysis, kcat, to be near 135 min-1. These results provide, for the first time, a quantitative description of the salient features of G-protein function in vivo.
Selective block of calcium current by lanthanum in single bullfrog atrial cells.
Authors: Nathan R D , Kanai K , Clark R B , Giles W
Published: May 2004

A single suction microelectrode voltage-clamp technique was used to study the actions of lanthanum ions (La3+) on ionic currents in single cells isolated from bullfrog right atrium. La3+, added as LaCl3, blocked the "slow" inward Ca2+ current (ICa) in a dose-dependent fashion; 10(-5) M produced complete inhibition. This effect was best fitted by a dose-response curve that was calculated assuming 1:1 binding of La3+ to a site having a dissociation constant of 7.5 x 10(-7) M. La3+ block was reversed (to 90% of control ICa) following washout and, in the presence of 10(-5) M La3+, was antagonized by raising the Ca2+ concentration from 2.5 to 7.5 mM (ICa recovered to 56% of the control). However, the latter effect took approximately 1 h to develop. Concentrations of La3+ that reduced ICa by 12-67%, 0.1-1.5 x 10(-6) M, had no measurable effect upon the voltage dependence of steady state ICa inactivation, which suggest that at these concentrations there are no significant surface-charge effects of La3+ on this gating mechanism. Three additional findings indicate that doses of La3+ that blocked ICa failed to produce nonspecific effects: (a) 10(-5) M La3+ had no measurable effect on the time-independent inwardly rectifying current, IK1; (b) the same concentration had no effect on the kinetics, amplitude, or voltage dependence of a time- and voltage-dependent K+ current, IK; and (c) 10(-4) M La3+ did not alter the size of the tetrodotoxin-sensitive inward Na+ current, INa, or the voltage dependence of its steady state inactivation. Higher concentrations (0.5-1.0 mM) reduced both IK1 and IK, and shifted the steady state activation curve for IK toward more positive potentials, presumably by reducing the external surface potential. Our results suggest that at a concentration of less than or equal to 10(-5) M, La3+ inhibits ICa selectively by direct blockade of Ca channels rather than by altering the external surface potential. At higher concentrations, La3+ exhibits nonspecific effects, including neutralization of negative external surface charge and inhibition of other time- and voltage-dependent ionic currents.
Na channels and two types of Ca channels in rat pancreatic B cells identified with the reverse hemolytic plaque assay.
Authors: Hiriart M , Matteson D R
Published: May 2004

The reverse hemolytic plaque assay (RHPA) was used to study the secretory properties of single rat pancreatic B cells, and to identify insulin-secreting cells for patch-clamp experiments. In secretion studies using the RHPA, we find that the percentage of secreting B cells and the amount of insulin secreted per B cell increase as the glucose concentration is raised from 0 to 20 mM. Using the whole-cell variation of the patch-clamp technique, we find that identified B cells have three types of channels capable of carrying inward current: (a) tetrodotoxin-sensitive, voltage-dependent Na channels, which are nearly completely inactivated at -40 mV, (b) fast deactivating (FD) Ca channels, and (c) slowly deactivating (SD) Ca channels. We have shown that Na channels are functionally significant to the B cell, because tetrodotoxin partially inhibits glucose-induced insulin secretion. The properties of FD and SD Ca channels differ in several respects. FD channels deactivate at -80 mV, with a time constant of 129 microseconds, they are half-maximally activated near +15 mV, they do not inactivate during 100 ms, they conduct Ba2+ better than Ca2+, and they are very sensitive to washout during intracellular dialysis. SD channels, on the other hand, deactivate with a time constant of 2.8 ms, they are half-maximally activated near -5 mV, they inactivate rapidly, they conduct Ba2+ and Ca2+ equally well, and they are insensitive to washout.
Distributing and delivering vessels of the human heart.
Authors: Zamir M
Published: May 2004

The branching characteristics of the right coronary artery, acute marginal, posterior descending, left anterior descending, circumflex, and obtuse marginal arteries are compared with those of diagonal branches, left and right ventricular branches, septal, and higher-order branches, to test a newly proposed functional classification of the coronary arteries in which the first group rank as distributing vessels and the second as delivering vessels. According to this classification, the function of the first type is merely to convey blood to the borders of myocardial zones, while the function of the second is to implement the actual delivery of blood into these zones. This functional difference is important in the hemodynamic analysis of coronary heart disease, as it provides an assessment of the role of a vessel within the coronary network and hence an assessment of the functional importance of that vessel in a particular heart. Measurements from casts of human coronary arteries are used to examine the relevant characteristics of these vessels and hence to test the basis of this classification.
Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells.
Authors: Warshaw D M , Rees D D , Fay F S
Published: May 2004

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.
Calcium currents in embryonic and neonatal mammalian skeletal muscle.
Authors: Beam K G , Knudson C M
Published: May 2004

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.
The organization of taste sensibilities in hamster chorda tympani nerve fibers.
Authors: Frank M E , Bieber S L , Smith D V
Published: May 2004

Electrophysiological measurements of nerve impulse frequencies were used to explore the organization of taste sensibilities in single fibers of the hamster chorda tympani nerve. Moderately intense taste solutions that are either very similar or easily discriminated were applied to the anterior lingual surface. 40 response profiles or 13 stimulus activation patterns were considered variables and examined with multivariate statistical techniques. Three kinds of response profiles were seen in fibers that varied in their overall sensitivity to taste solutions. One profile (S) showed selectivity for sweeteners, a second (N) showed selectivity for sodium salts, and a third (H) showed sensitivity to salts, acids, and other compounds. Hierarchical cluster analysis indicated that profiles fell into discrete classes. Responses to many pairs of effective stimuli were covariant across profiles within a class, but some acidic stimuli had more idiosyncratic effects. Factor analysis of profiles identified two common factors, accounting for 77% of the variance. A unipolar factor was identified with the N profile, and a bipolar factor was identified with the S profile and its opposite, the H profile. Three stimulus activation patterns were elicited by taste solutions that varied in intensity of effect. Hierarchical cluster analysis indicated that the patterns fell into discrete classes. Factor analysis of patterns identified three common unipolar factors accounting for 82% of the variance. Eight stimuli (MgSO4, NH4Cl, KCl, citric acid, acetic acid, urea, quinine HCl, HCl) selectively activated fibers with H profiles, three stimuli (fructose, Na saccharin, sucrose) selectively activated fibers with S profiles, and two stimuli (NaNO3, NaCl) activated fibers with N profiles more strongly than fibers with H profiles. Stimuli that evoke different patterns taste distinct to hamsters. Stimuli that evoke the same pattern taste more similar. It was concluded that the hundreds of peripheral taste neurons that innervate the anterior tongue play one of three functional roles, providing information about one of three features that are shared by different chemical solutions.
Adaptation of a deep-sea cephalopod to the photic environment. Evidence for three visual pigments.
Authors: Matsui S , Seidou M , Horiuchi S , Uchiyama I , Kito Y
Published: May 2004

Watasenia scintillans, a bioluminescent deep-sea squid, has a specially developed eye with a large open pupil and three visual pigments. Photoreceptor cells (outer segment: 476 micron; inner segment: 99 micron) were long in the small area of the ventral retina receiving downwelling light, whereas they were short (outer segment: 207 micron; inner segment: 44 micron) in the other regions of the retina. The short photoreceptor cells contained the visual pigment with retinal (lambda max approximately 484 nm), probably for the purpose of adapting to their environmental light. The outer segment of the long photoreceptor cells consisted of two strata, a pinkish proximal area and a yellow distal area. The visual pigment with 3-dehydroretinal (lambda max approximately 500 nm) was located in the pinkish proximal area, giving high sensitivity at longer wavelengths. A newly found pigment (lambda max approximately 471 nm) was in the yellow distal area. The small area of the ventral retina containing two visual pigments is thought to have a high and broad spectral sensitivity, which is useful for distinguishing the bioluminescence of squids of the same species in their environmental downwelling light. These findings were obtained by partial bleaching of the extracted pigment from various areas of the retina and by high-performance liquid chromatographic analysis of the chromophore, complemented by microscopic observations.
Potassium conductance of the squid giant axon. Single-channel studies.
Authors: Llano I , Webb C K , Bezanilla F
Published: May 2004

The patch-clamp technique was implemented in the cut-open squid giant axon and used to record single K channels. We present evidence for the existence of three distinct types of channel activities. In patches that contained three to eight channels, ensemble fluctuation analysis was performed to obtain an estimate of 17.4 pS for the single-channel conductance. Averaged currents obtained from these multichannel patches had a time course of activation similar to that of macroscopic K currents recorded from perfused squid giant axons. In patches where single events could be recorded, it was possible to find channels with conductances of 10, 20, and 40 pS. The channel most frequently encountered was the 20-pS channel; for a pulse to 50 mV, this channel had a probability of being open of 0.9. In other single-channel patches, a channel with a conductance of 40 pS was present. The activity of this channel varied from patch to patch. In some patches, it showed a very low probability of being open (0.16 for a pulse to 50 mV) and had a pronounced lag in its activation time course. In other patches, the 40-pS channel had a much higher probability of being open (0.75 at a holding potential of 50 mV). The 40-pS channel was found to be quite selective for K over Na. In some experiments, the cut-open axon was exposed to a solution containing no K for several minutes. A channel with a conductance of 10 pS was more frequently observed after this treatment. Our study shows that the macroscopic K conductance is a composite of several K channel types, but the relative contribution of each type is not yet clear. The time course of activation of the 20-pS channel and the ability to render it refractory to activation only by holding the membrane potential at a positive potential for several seconds makes it likely that it is the predominant channel contributing to the delayed rectifier conductance.
Fast-deactivating calcium channels in chick sensory neurons.
Authors: Swandulla D , Armstrong C M
Published: May 2004

Whole-cell Ca and Ba currents were studied in chick dorsal root ganglion (DRG) cells kept 6-10 in culture. Voltage steps with a 15-microseconds rise time were imposed on the membrane using an improved patch-clamp circuit. Changes in membrane current could be measured 30 microseconds after the initiation of the test pulse. Currents through Ca channels were recorded under conditions that eliminate Na and K currents. Tail currents, associated with Ca channel closing, decayed in two distinct phases that were very well fitted by the sum of two exponentials. The time constants tau f and tau s were near 160 microseconds and 1.5 ms at -80 mV, 20 degrees C. The tail current components, called FD and SD (fast-deactivating and slowly deactivating), are Ca channel currents. They were greatly reduced when Mg2+ replaced all other divalent cations in the bath. The SD component inactivated almost completely as the test pulse duration was increased to 100 ms. It was suppressed when the cell was held at membrane potentials positive to -50 mV and was blocked by 100-200 microM Ni2+. This behavior indicates that the SD component was due to the closing of the low-voltage-activated (LVA) Ca channels previously described in this preparation. The FD component was fully activated with 10-ms test pulses to +20 mV at 20 degrees C, and inactivated to approximately 30% during 500-ms test pulses. It was reduced in amplitude by holding at -40 mV, but was only slightly reduced by micromolar concentrations of Ni2+. Replacement of Ca2+ with Ba2+ increased the FD tail current amplitudes by a factor of approximately 1.5. The deactivation kinetics did not change (a) as channels inactivated during progressively longer pulses or (b) when the degree of activation was varied. Further, tau f was affected neither by changing the holding potential nor by varying the test pulse amplitude. Lowering the temperature from 20 to 10 degrees C decreased tau f by a factor of 2.5. In all cases, the FD component was very well fitted by a single exponential. There was no indication of an additional tail component of significant size. Our findings indicate that the FD component is due to closing of a single class of Ca channels that coexist with the LVA Ca channel type in chick DRG neurons.
Removal of inactivation causes time-invariant sodium current decays.
Authors: Hahin R
Published: May 2004

The kinetic properties of the closing of Na channels were studied in frog skeletal muscle to obtain information about the dependence of channel closing on the past history of the channel. Channel closing was studied in normal and modified channels. Chloramine-T was used to modify the channels so that inactivation was virtually removed. A series of depolarizing prepulse potentials was used to activate Na channels, and a -140-mV postpulse was used to monitor the closing of the channels. Unmodified channels decay via a biexponential process with time constants of 72 and 534 microseconds at 12 degrees C. The observed time constants do not depend upon the potential used to activate the channels. The contribution of the slow component to the total decay increases as the activating prepulse is lengthened. After inactivation is removed, the biexponential character of the decay is retained, with no change in the magnitude of the time constants. However, increases in the duration of the activating prepulse over the range where the current is maximal 1-75 ms) produce identical biexponential decays. The presence of biexponential decays suggests that either two subtypes of Na channels are found in muscle, or Na channels can exist in one of two equally conductive states. The time-invariant decays observed suggest that channel closure does not depend upon their past history.
Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles.
Authors: Smith G L , Valdeolmillos M , Eisner D A , Allen D G
Published: May 2004

In this paper we investigate the effects of caffeine (5-20 mM) on ferret papillary muscle. The intracellular Ca2+ concentration ( [Ca2+]i) was measured from the light emitted by the photoprotein aequorin, which had previously been microinjected into superficial cells. Isometric tension was measured simultaneously. The rapid application of caffeine produced a transient increase of [Ca2+]i, which decayed spontaneously within 2-3 s and was accompanied by a transient contracture. The removal of extracellular Na+ or an increase in the concentration of intracellular Na+ (produced by strophanthidin) increased the magnitude of the caffeine response. Cessation of stimulation for several minutes or stimulation at low rates decreased the magnitude of the stimulated twitch and Ca2+ transient. These maneuvers also decreased the size of the caffeine response. These results are consistent with the hypothesis that the caffeine-releasable pool of Ca2+ (sarcoplasmic reticulum) is modulated by maneuvers that affect contraction. Ryanodine (10 microM) decreased the magnitude of the caffeine response as well as that of the stimulated twitch. In contrast, the rapid removal of external Ca2+ abolished the systolic Ca2+ transient within 5 s, but had no effect on the caffeine response. From this we conclude that the abolition of twitch by Ca2+-free solutions is not due to depletion of the sarcoplasmic reticulum of Ca2+, but may be due to a requirement of Ca2+ entry into the cell to trigger Ca2+ release from the sarcoplasmic reticulum.
Dissociation of changes in apparent myofibrillar Ca2+ sensitivity and twitch relaxation induced by adrenergic and cholinergic stimulation in isolated ferret cardiac muscle.
Authors: McIvor M E , Orchard C H , Lakatta E G
Published: May 2004

In isolated, aequorin-injected ferret cardiac muscle we measured the apparent myofilament Ca2+ sensitivity and its relationship to twitch relaxation time in the presence of autonomic perturbations. The Ca2+-tension relation was determined from the peak aequorin luminescence and peak twitch tension measured in muscles across a broad range of bathing [Ca2+] in the presence and absence of acetylcholine (ACh) (1 microM) or isoproterenol (ISN) (1 microM), or both drugs. ACh shifted the relationship of peak tension to (peak) aequorin light leftward, which suggests an increase in myofilament Ca2+ sensitivity, but it did not alter relaxation, which was measured as the time for peak tension to decay by 50% (t 1/2 R). ISN produced its previously documented effects, i.e., a rightward shift of the relationship of peak tension to peak aequorin light and a decrease in t1/2R. ACh abolished the ISN effect on the peak tension-aequorin light relationship but did not reverse the effect of ISN to decrease t1/2R. The effects of ACh and ISN of modulating the apparent myofilament Ca2+ sensitivity in intact muscles, corroborate findings of previous studies in isolated myofibrillar preparations. However, these perturbations of myofilament Ca2+ sensitivity in the intact muscle do not relate to twitch relaxation, measured as t1/2R, since (a) ACh affects the former but not the later and (b) the effect of ISN on the Ca2+-tension relationship is abolished by ACh, while the relaxant effect persists.
Potassium blocks barium permeation through a calcium-activated potassium channel.
Authors: Neyton J , Miller C
Published: May 2004

Single high-conductance Ca2+-activated K+ channels from rat skeletal muscle were inserted into planar lipid bilayers, and discrete blocking by the Ba2+ ion was studied. Specifically, the ability of external K+ to reduce the Ba2+ dissociation rate was investigated. In the presence of 150 mM internal K+, 1-5 microM internal Ba2+, and 150 mM external Na+, Ba2+ dissociation is rapid (5 s-1) in external solutions that are kept rigorously K+ free. The addition of external K+ in the low millimolar range reduces the Ba2+ off-rate 20-fold. Other permeant ions, such as Tl+, Rb+, and NH4+ show a similar effect. The half-inhibition constants rise in the order: Tl+ (0.08 mM) less than Rb+ (0.1 mM) less than K+ (0.3 mM) less than Cs+ (0.5 mM) less than NH4+ (3 mM). When external Na+ is replaced by 150 mM N-methyl glucamine, the Ba2+ off-rate is even higher, 20 s-1. External K+ and other permeant ions reduce this rate by approximately 100-fold in the micromolar range of concentrations. Na+ also reduces the Ba2+ off-rate, but at much higher concentrations. The half-inhibition concentrations rise in the order: Rb+ (4 microM) less than K+ (19 microM) much less than Na+ (27 mM) less than Li+ (greater than 50 mM). The results require that the conduction pore of this channel contains at least three sites that may all be occupied simultaneously by conducting ions.
Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+ -activated K+ channel.
Authors: Neyton J , Miller C
Published: May 2004

In this study, high-conductance Ca2+-activated K+ channels from rat skeletal muscle were incorporated into planar phospholipid bilayers, and discrete blockade of single channels by Ba2+ was studied. With 150 mM K+ held constant in the internal solution, increasing external K+ over the range 100-1,000 mM raises the rate of Ba2+ dissociation. This "enhancement effect," which operates at K+ concentrations 3-4 orders of magnitude higher than those required for the "lockin" effect described previously, depends on applied voltage, saturates with K+ concentration, and is not observed with Na+. The voltage dependence of the Ba2+ off-rate varies with external K+ in a way suggesting that K+, entering the channel from the external side, forces Ba2+ dissociation to the internal solution. With K+ held fixed in the external solution, the Ba2+ off-rate decreases as internal K+ is raised over the range 0-50 mM. This "lock-in" effect is similar to that seen on the external side (Neyton and Miller, 1988), except that the internal lock-in site is of lower affinity and shows only a fivefold preference for K+ over Na+. All the results taken together argue strongly that this channel's conduction pathway contains four sites of very high affinity for K+, all of which may be simultaneously occupied under normal conducting conditions. According to this view, the mutual destabilization resulting from this high ionic occupancy leads to the unusually high conductance of this K+-specific channel.
Intracellular Na+ and K+ activities and membrane conductances in the collecting tubule of Amphiuma.
Authors: Horisberger J D , Giebisch G
Published: May 2004

Membrane potentials and conductances, and intracellular ionic activities were studied in isolated perfused collecting tubules of K+-adapted Amphiuma. Intracellular Na+ (aNai) and K+ (aKi) activities were measured, using liquid ion-exchanger double-barreled microelectrodes. Apical and basolateral membrane conductances were estimated by cable analysis. The effects of inhibition of the apical conductance by amiloride (10(-5) M) and of inhibition of the basolateral Na-K pump by either a low K+ (0.1 mM) bath or by ouabain (10(-4) M) were studied. Under control conditions, aNai was 8.4 +/- 1.9 mM and aKi 56 +/- 3 mM. With luminal amiloride, aNai decreased to 2.2 +/- 0.4 mM and aKi increased to 66 +/- 3 mM. Ouabain produced an increase of aNai to 44 +/- 4 mM, and a decrease of aKi to 22 +/- 6, and similar changes were observed when the tubule was exposed to a low K+ bath solution. During pump inhibition, there was a progressive decrease of the K+-selective basolateral membrane conductance and of the Na+ permeability of the apical membrane. A similar inhibition of both membrane conductances was observed after pump inhibition by low K+ solution. Upon reintroduction of K+, a basolateral membrane hyperpolarization of -23 +/- 4 mV was observed, indicating an immediate reactivation of the electrogenic Na-K pump. However, the recovery of the membrane conductances occurred over a slower time course. These data imply that both membrane conductances are regulated according to the intracellular ionic composition, but that the basolateral K+ conductance is not directly linked to the pump activity.
Role of Na+/H+ exchange in the control of intracellular pH and cell membrane conductances in frog skin epithelium.
Authors: Harvey B J , Ehrenfeld J
Published: May 2004

Ion-sensitive microelectrodes and current-voltage analysis were used to study intracellular pH (pHi) regulation and its effects on ionic conductances in the isolated epithelium of frog skin. We show that pHi recovery after an acid load is dependent on the operation of an amiloride-sensitive Na+/H+ exchanger localized at the basolateral cell membranes. The antiporter is not quiescent at physiological pHi (7.1-7.4) and, thus, contributes to the maintenance of steady state pHi. Moreover, intracellular sodium ion activity is also controlled in part by Na+ uptake via the exchanger. Intracellular acidification decreased transepithelial Na+ transport rate, apical Na+ permeability (PNa) and Na+ and K+ conductances. The recovery of these transport parameters after the removal of the acid load was found to be dependent on pHi regulation via Na+/H+ exchange. Conversely, variations in Na+ transport were accompanied by changes in pHi. Inhibition of Na+/K+ ATPase by ouabain produced covariant decreases in pHi and PNa, whereas increases in Na+ transport, occurring spontaneously or after aldosterone treatment, were highly correlated with intracellular alkalinization. We conclude that cytoplasmic H+ activity is regulated by a basolateral Na+/H+ exchanger and that transcellular coupling of ion flows at opposing cell membranes can be modulated by the pHi-regulating mechanism.
Intracellular pH-regulating mechanism of the squid axon. Interaction between DNDS and extracellular Na+ and HCO3-.
Authors: Boron W F , Knakal R C
Published: May 2004

Intracellular pH (pHi) of the squid axon is regulated by a stilbenesensitive transporter that couples the influx of Na+ and HCO3- (or the equivalent) to the efflux of Cl-. According to one model, the extracellular ion pair NaCO3- exchanges for intracellular Cl-. In the present study, the ion-pair model was tested by examining the interaction of the reversible stilbene derivative 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) with extracellular Na+ and HCO3-. Axons (initial pHi approximately 7.4) were internally dialyzed with a pH 6.5 solution containing 400 mM Cl- but no Na+. After pHi, as measured with a glass microelectrode, had fallen to approximately 6.6, dialysis was halted. In the presence of both external Na+ and HCO3- (pHo = 8.0, 22 degrees C), pHi increased due to the pHi-regulating mechanism. At a fixed [Na+]o of 425 mM and [HCO3-]o of 12 mM, DNDS reversibly reduced the equivalent acid-extrusion rate (JH) calculated from the rate of pHi recovery. The best-fit value for maximal inhibition was 104%, and for the [DNDS]o at half-maximal inhibition, 0.3 mM. At a [Na+]o of 425 mM, the [HCO3-]o dependence of JH was examined at 0, 0.1, and 0.25 mM DNDS. Although Jmax was always approximately 20 pmol cm-2 s-1, Km(HCO3-) was 2.6, 5.7, and 12.7 mM, respectively. Thus, DNDS is competitive with HCO3-. At a [HCO3-]o of 12 mM, the [Na+]o dependence of JH was examined at 0 and 0.1 mM DNDS. Although Jmax was approximately 20 pmol cm-2 s-1 in both cases, Km(Na+) was 71 and 179 mM, respectively. At a [HCO3-]o of 48 mM, Jmax was approximately 20 pmol cm-2 s-1 at [DNDS]o levels of 0, 0.1, and 0.25 mM. However, Km(Na+) was 22, 45, and 90 mM, respectively. Thus, DNDS (an anion) is also competitive with Na+. The results are consistent with simple competition between DNDS and NaCO3-, and place severe restrictions on other kinetic models.
Excitation of skinned muscle fibers by imposed ion gradients. III. Distribution of permeant ions in unstimulated and stimulated fibers.
Authors: Stephenson E W
Published: May 2004

Ion gradients imposed across an internal membrane system stimulate skinned muscle fibers; to evaluate the sarcoplasmic reticulum (SR) as the primary target site, SR polarization under resting and stimulatory conditions was assessed from fiber uptake of permeant probe ions. Solvent spaces were estimated from simultaneous [14C]urea (U) or [3H]deoxyglucose (DOG) uptake in segments of fibers from bullfrog semitendinosus muscle, skinned by microdissection. The distribution spaces, i.e., virtual solvent volumes at bath concentrations (Vu and VDOG), of these uncharged probes correlated well with the protein content of the same segments, which validated the tracer methodology for volume normalization. The membrane-bounded volume fraction (Vm), derived from the difference between total solvent volume (Vs) and the non-membrane-bounded solvent volume (Vc), was sufficient to detect appreciable SR ion accumulation. The Vm estimated from the difference between VU and VDOG assayed simultaneously with 2 or 5-6 min exposures was 10-11%, which is consistent with the morphometric volume fraction (mostly SR) in frog fibers; however, the change in this difference after membrane permeabilization corresponded to Vm only 5%. The change in permeant ion distribution space caused by member permeabilization was used to assess SR membrane polarization, assuming the free ions distribute across the intact membrane according to the Nernst ratio. Resting polarization (SR lumen positive) was assessed from [14C]SCN- or [14C]propionate- distribution spaces in unstimulated fibers, expressed relative to VDOG (assayed simultaneously). The ratios for (a) [14C]SCN- space (carrier 2 mM) and (b) [14C]propionate- space (carrier 120 mM) were not decreased by membrane permeabilization. This indicated that anion distribution was independent of membrane integrity and did not reflect an SR transmembrane potential, although a was more and b was less than 1. Polarization under stimulatory conditions (lumen negative) was assessed from 86Rb+ distribution, before and after an imposed ion gradient (choline Cl replacement of K methanesulfonate (KMes) at constant [K+] [Cl-]) that theoretically could generate a 48-fold transmembrane cation ratio; Ca release was minimized by EGTA. The ratio of 86Rb+ space to VU, greater than 1 in KMes (120 mM K, the effective carrier), was higher in choline Cl (2.5 mM K) but not decreased by membrane permeabilization; this indicated that 86Rb+ distribution did not reflect an SR transmembrane potential. Similar results in the presence of valinomycin ruled out the possibility of inadequate 86Rb+ equilibration.(ABSTRACT TRUNCATED AT 400 WORDS)
Excitation of skinned muscle fibers by imposed ion gradients. IV. Effects of stretch and perchlorate ion.
Authors: Stephenson E W
Published: May 2004

Depolarizing ion gradients stimulate 45Ca release in skeletal muscle fibers skinned by microdissection. Several lines of indirect evidence suggest that sealed transverse (T) tubules rather than sarcoplasmic reticulum (SR) are the locus of such stimulatory depolarization. Two implications of this hypothesis were tested. (a) A requirement for signal transmission was evaluated from the stimulation of 45Ca efflux in fibers that had been highly stretched, an intervention that can impair the electrical stimulation of intact fibers. Length was increased over approximately 95-115 s, after loading with 45Ca and rinsing at normal length; prestimulus 45Ca loss due to stretch itself was very small. In the first study, stimulation of 45Ca release by KCl replacement of K propionate was inhibited completely in fibers stretched to twice slack length, compared with fibers at 1.05-1.1 times slack length. Identical protocols did not alter 45Ca release stimulated by caffeine or Mg2+ reduction, implying that SR Ca release per se was fully functional and inhibition was selective for a preceding step in ionic stimulation. In a second study, stimulation by choline Cl replacement of K methanesulfonate, at constant [K+] [Cl-] product, was inhibited strongly; total 45Ca release decreased 69%, and stimulation above control loss decreased 78%, in segments stretched to twice the length at which sarcomere spacing had been 2.2 micron, compared with paired controls from the same fibers kept at 2.3 micron. (b) Perchlorate potentiation of T tubule activation was evaluated in fibers stimulated at constant [K+] [Cl-] at normal length (2.3 micron); this anion shifts the voltage dependence of intramembrane charge movement and contractile activation in intact fibers. Perchlorate (8 mM) potentiated both submaximal stimulation of Ca2+-dependent 45Ca release by partial choline Cl replacement of K methanesulfonate and the small Ca2+-insensitive 45Ca efflux component stimulated by nearly full replacement in the presence of 5 mM EGTA. These results provide independent support for the hypothesis that the T tubules are the locus of stimulation by depolarizing ion gradients, with junctional transmission of this signal causing SR 45Ca release.
Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties.
Authors: Behrens M I , Oberhauser A , Bezanilla F , Latorre R
Published: May 2004

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.
Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure.
Authors: Cohen F S , Niles W D , Akabas M H
Published: May 2004

Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.