bluuebunny/crossref_metadata_2025 huggingface dataset:
Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH.
Authors: Pappone P A , Barchfeld G L
Published: May 2004

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.
Gating of single non-Shaker A-type potassium channels in larval Drosophila neurons.
Authors: Solc C K , Aldrich R W
Published: May 2004

The voltage-dependent gating of transient A2-type potassium channels from primary cultures of larval Drosophila central nervous system neurons was studied using whole-cell and single-channel voltage clamp. A2 channels are genetically distinct from the Shaker A1 channels observed in Drosophila muscle, and differ in single-channel conductance, voltage dependence, and gating kinetics. Single A2 channels were recorded and analyzed at -30, -10, +10, and +30 mV. The channels opened in bursts in response to depolarizing steps, with three to four openings per burst and two to three bursts per 480-ms pulse (2.8-ms burst criterion). Mean open durations were in a range of 2-4 ms and mean burst durations in a range of 9-17 ms. With the exception of the first latency distributions, none of the means of the distributions measured showed a consistent trend with voltage. Macroscopic inactivation of both whole-cell A currents and ensemble average currents of single A2 channels was well fitted by a sum of two exponentials. The fast time constants in different cells were in a range of 9-25 ms, and the slow time constants in a range of 60-140 ms. A six-state kinetic model (three closed, one open, two inactivated states) was tested at four command voltages by fitting frequency histograms of open durations, burst durations, burst closed durations, number of openings per burst, and number of bursts per trace. The model provided good fits to these data, as well as to the ensemble averages. With the exception of the rates leading to initial opening, the transitions in the model were largely independent of voltage.
Expression of epithelial Na channels in Xenopus oocytes.
Authors: Palmer L G , Corthesy-Theulaz I , Gaeggeler H P , Kraehenbuhl J P , Rossier B
Published: May 2004

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride-sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.
Volume-activated chloride permeability can mediate cell volume regulation in a mathematical model of a tight epithelium.
Authors: Strieter J , Stephenson J L , Palmer L G , Weinstein A M
Published: May 2004

Cell volume regulation during anisotonic challenge is investigated in a mathematical model of a tight epithelium. The epithelium is represented as compliant cellular and paracellular compartments bounded by mucosal and serosal bathing media. Model variables include the concentrations of Na, K, and Cl, hydrostatic pressure, and electrical potential, and the mass conservation equations have been formulated for both steady-state and time-dependent problems. Ionic conductance is represented by the Goldman constant field equation (Civan, M.M., and R.J. Bookman. 1982. Journal of Membrane Biology. 65:63-80). A basolateral cotransporter of Na, K, and Cl with 1:1:2 stoichiometry (Geck, P., and E. Heinz. 1980. Annals of the New York Academy of Sciences. 341:57-62.) and volume-activated basolateral ion permeabilities are incorporated in the model. MacRobbie and Ussing (1961. Acta Physiologica Scandinavica. 53:348-365.) reported that the cells of frog skin exhibit osmotic swelling followed by a volume regulatory decrease (VRD) when the serosal bath is diluted to half the initial osmolality. Similar regulation is achieved in the model epithelium when both a basolateral cotransporter and a volume-activated Cl permeation path are included. The observed transepithelial potential changes could only be simulated by allowing volume activation of the basolateral K permeation path. The fractional VRD, or shrinkage as percent of initial swelling, is examined as a function of the hypotonic challenge. The fractional VRD increases with increasing osmotic challenge, but eventually declines under the most severe circumstances. This analysis demonstrates that the VRD response depends on the presence of adequate intracellular chloride stores and the volume sensitivity of the chloride channel.
Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers.
Authors: Pape P C , Konishi M , Hollingworth S , Baylor S M
Published: May 2004

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.
Voltage-dependent block of charge movement components by nifedipine in frog skeletal muscle.
Authors: Huang C L
Published: May 2004

Potential-dependent inhibition of charge movement components by nifedipine was studied in intact, voltage-clamped, frog skeletal muscle fibers. Available charge was reduced by small shifts in holding potential (from -100 mV to -70 mV) in 2 microM nifedipine, without changes in the capacitance deduced from control (-120 mV to -100 mV) voltage steps made at a fully polarized (-100 mV) holding potential. These voltage-dependent effects did not occur in lower (0-0.5 microM) nifedipine concentrations. The voltage dependence of membrane capacitance at higher (10 microM) nifedipine concentrations was reduced even in fully polarized fibers, but shifting the holding voltage produced no further block. Voltage-dependent inhibition by nifedipine was associated with a fall in available charge, and a reduction in the charge and capacitance-voltage relationships and of late (q gamma) charging transients. It thus separated a membrane-capacitance with a distinct and steep steady-state voltage dependence. Tetracaine (2 mM) reduced voltage-dependent membrane capacitance and nonlinear charge more than did nifedipine. However, nifedipine did not exert voltage-dependent effects on charging currents, membrane capacitance, or inactivation of tetracaine-resistant (q beta) charge. This excludes participation of q beta, or the membrane charge as a whole, from the voltage-dependent effects of nifedipine. Rather, the findings suggest that the charge susceptible to potential-dependent block by nifedipine falls within the tetracaine-sensitive (q gamma) category of intramembrane charge.
Slowing of velocity during isotonic shortening in single isolated smooth muscle cells. Evidence for an internal load.
Authors: Harris D E , Warshaw D M
Published: May 2004

In single smooth muscle cells, shortening velocity slows continuously during the course of an isotonic (fixed force) contraction (Warshaw, D.M. 1987. J. Gen. Physiol. 89:771-789). To distinguish among several possible explanations for this slowing, single smooth muscle cells were isolated from the gastric muscularis of the toad (Bufo marinus) and attached to an ultrasensitive force transducer and a length displacement device. Cells were stimulated electrically and produced maximum stress of 144 mN/mm2. Cell force was then reduced to and maintained at preset fractions of maximum, and cell shortening was allowed to occur. Cell stiffness, a measure of relative numbers of attached crossbridges, was measured during isotonic shortening by imposing 50-Hz sinusoidal force oscillations. Continuous slowing of shortening velocity was observed during isotonic shortening at all force levels. This slowing was not related to the time after the onset of stimulation or due to reduced isometric force generating capacity. Stiffness did not change significantly over the course of an isotonic shortening response, suggesting that the observed slowing was not the result of reduced numbers of cycling crossbridges. Furthermore, isotonic shortening velocity was better described as a function of the extent of shortening than as a function of the time after the onset of the release. Therefore, we propose that slowing during isotonic shortening in single isolated smooth muscle cells is the result of an internal load that opposes shortening and increases as cell length decreases.
Circadian rhythms in Limulus photoreceptors. II. Quantum bumps.
Authors: Kaplan E , Barlow R B , Renninger G , Purpura K
Published: May 2004

The light response of the lateral eye of the horseshoe crab, Limulus polyphemus, increases at night, while the frequency of spontaneous discrete fluctuations of its photoreceptor membrane potential (quantum bumps) decreases. These changes are controlled by a circadian clock in the brain, which transmits activity to the eye via efferent optic nerve fibers (Barlow, R. B., S. J. Bolanski, and M. L Brachman. 1977. Science. 197:86-89). Here we report the results of experiments in which we recorded from single Limulus photoreceptors in vivo for several days and studied in detail changes in their physiological and membrane properties. We found that: (a) The shape of (voltage) quantum bumps changes with the time of day. At night, spontaneous bumps and bumps evoked by dim light are prolonged. The return of the membrane potential to its resting level is delayed, but the rise time of the bump is unaffected. On average, the area under a bump is 2.4 times greater at night than during the day. (b) The rate of spontaneous bumps decreases at night by roughly a factor of 3, but their amplitude distribution remains unchanged. (c) The resting potential and resistance of the photoreceptor membrane do not change with the time of day. (d) the relationship between injected current and impulse rate of the second order neuron, the eccentric cell, also remains unchanged with the time of day. Thus the efferent input from the brain to the retina modulates some of the membrane properties of photoreceptor cells. Our findings suggest that the efferent input acts on ionic channels in the membrane to increase the sensitivity of the photoreceptor to light.
A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers.
Authors: Lukács G L , Moczydlowski E
Published: May 2004

A novel, small conductance of Cl- channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaCl, 10 mM Tris-HCl, pH 7.4, single Cl- channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 +/- 0.8 pS at positive voltages and 15.1 +/- 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na+ compared with Cl- and displays the following sequence of anion permeability relative to Cl- as measured under near bi-ionic conditions: I- (2.7) greater than NO3- (1.8) greater than Br- (1.5) greater than Cl- (1.0) greater than CH3CO2- (0.18) greater than HCO3- (0.10) greater than gluconate (0.06) greater than F- (0.05). The unitary conductance saturates with increasing Cl- concentration in a Michaelis-Menten fashion with a Km of 100 mM and gamma max = 33 pS at positive voltage. The I-V curve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaCl with 100 mM tetraethylammonium chloride on the cis side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at +75 mV. Channel gating is sensitive to cis pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster Cl- channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance Cl- channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate Cl- channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.
Arachidonic acid metabolites alter G protein-mediated signal transduction in heart. Effects on muscarinic K+ channels.
Authors: Scherer R W , Breitwieser G E
Published: May 2004

The muscarinic acetylcholine receptor (mAChR)-stimulated, inwardly rectifying K+ current (IK [ACh]) was examined in single bullfrog atrial cells using the whole-cell patch clamp technique. IK[ACh] was activated either by bath addition of 1 microM ACh or via activation of the G protein, Gk, with guanosine-gamma-thiotriphosphate (GTP gamma S). Arachidonic acid (AA) modulated IK[ACh] under both conditions. AA decreased mAChR-stimulated IK[ACh] and increased the rate of decay from the peak current (desensitization). In addition, AA affected GTP gamma S-activated IK[ACh] by modulation of Gk. The effects of AA and its metabolites on Gk were assessed by examining their effects on both the basal rate of Gk activation by GTP gamma S, and the mAChR-mediated increase in activation rate produced by nanomolar ACh. AA increased the basal rate of GTP gamma S-mediated IK[ACh] activation, but reduced the ACh-induced augmentation of this rate. All of the effects of AA on GTP gamma S-mediated IK[ACh] activation were produced by metabolites. A lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), decreased the basal and ACh-enhanced rate of IK[ACh] activation in both the presence and absence of exogenous AA. In contrast, indomethacin (INDO), a cyclooxygenase inhibitor, increased the basal rate of IK[ACh] activation by GTP gamma S in both the presence and absence of exogenous AA, and reversed the effects of AA on the ACh-augmented basal rate. AA metabolites produced via lipoxygenase and cyclooxygenase pathways thus have opposing effects on the signal transduction pathway from mAChR to IK[ACh]. We directly tested a lipoxygenase pathway metabolite, LTC4, on GTP gamma S-mediated IK[ACh] activation and found that it not only overcame the inhibitory effects of NDGA, but also increased both the basal and ACh-augmented rate of IK[ACh] activation. From these data, we propose that AA metabolites modulate the function of Gk by altering its kinetic properties.
Translocation mechanism of Na-Ca exchange in single cardiac cells of guinea pig.
Authors: Li J M , Kimura J
Published: May 2004

We have studied in single cardiac ventricular cells of guinea pig the ionic translocation mechanism of the electrogenic Na-Ca exchange, i.e., whether Na and Ca ions countercross the membrane simultaneously or consecutively with "ping pong" kinetics. The dose-response relation between the external Ca concentrations [( Ca]o) and the current density of the outward Na-Ca exchange current were measured at three different intracellular Na concentrations [( Na]i) in the absence of external Na. Nonlinear regression curves of the dose-response relation obtained by computer revealed Michaelis-Menten type hyperbola from which the [Ca]o giving a half-maximal response (apparent KmCao or K'mCao) and the apparent maximum current magnitude (I'max) were estimated at each [Na]i. As [Na]i increased, the K'mCao increased progressively and the value of K'mCao/I'max tended to decrease. These results are consistent with the simultaneous mechanism. The K'mCao/I'max values, however, were small and close to each other, so it was not possible to completely preclude a consecutive mechanism.
Regulation of the formation and water permeability of endosomes from toad bladder granular cells.
Authors: Shi L B , Wang Y X , Verkman A S
Published: May 2004

Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers.
N-ethylmaleimide uncouples muscarinic receptors from acetylcholine-sensitive potassium channels in bullfrog atrium.
Authors: Nakajima T , Irisawa H , Giles W
Published: May 2004

The effect of N-ethylmaleimide (NEM), a sulphydryl alkylating agent, on the acetylcholine-activated K+ current, IK(ACh), has been studied in single cells from bullfrog atrium using a tight-seal, whole-cell voltage clamp technique. Addition of NEM (5 x 10(-5) M) produced a time-dependent complete block of IK(ACh). Dialysis of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S, 5-10 x 10(-4) M), a nonhydrolyzable GTP analogue, into the myoplasm from the recording pipette gradually activated IK(ACh) even in the absence of acetylcholine. This effect is thought to be due to a GTP gamma S-induced dissociation of GTP-binding proteins (Gi and/or Go) into subunits that can directly activate these K+ channels. When NEM (5 x 10(-5) M) was applied after the GTP gamma S effect had fully developed, it failed to inhibit the GTP gamma S-induced K+ current, indicating that the NEM effect is unlikely to be on the dissociated subunits of the GTP-binding protein(s) or on the K+ channels. In contrast, pretreatment with NEM before GTP gamma S application markedly reduced the muscarinic K+ current, suggesting that NEM can block this K+ current by inhibition of the dissociation of the GTP-binding proteins into functional subunits. In NEM-treated cells the stimulatory effect of isoproterenol on ICa was present, but the inhibitory action of ACh on ICa was completely abolished. These results demonstrated that NEM can preferentially inhibit muscarinic receptor-effector interactions, probably by alkylating the GTP-binding proteins that are essential for these responses.
Stretch of active muscle during the declining phase of the calcium transient produces biphasic changes in calcium binding to the activating sites.
Authors: Gordon A M , Ridgway E B
Published: May 2004

In voltage-clamped barnacle single muscle fibers, muscle shortening during the declining phase of the calcium transient increases myoplasmic calcium. This extra calcium is probably released from the activating sites by a change in affinity when cross-bridges break (Gordon, A. M., and E. B. Ridgway, 1987. J. Gen. Physiol. 90:321-340). Stretching the muscle at similar times causes a more complex response, a rapid increase in intracellular calcium followed by a transient decrease. The amplitudes of both phases increase with the rate and amplitude of stretch. The rapid increase, however, appears only when the muscle is stretched more than approximately 0.4%. This is above the length change that produces the breakpoint in the force record during a ramp stretch. This positive phase in response to large stretches is similar to that seen on equivalent shortening at the same point in the contraction. For stretches at different times during the calcium transient, the peak amplitude of the positive phase has a time course that is delayed relative to the calcium transient, while the peak decrease during the negative phase has an earlier time course that is more similar to the calcium transient. The amplitudes of both phases increase with increasing strength of stimulation and consequent force. When the initial muscle the active force. A large decrease in length (which drops the active force to zero) decreases the extra calcium seen on a subsequent restretch. After such a shortening step, the extra calcium on stretch recovers (50 ms half time) toward the control level with the same time course as the redeveloped force. Conversely, stretching an active fiber decreases the extra calcium on a subsequent shortening step that is imposed shortly afterward. Enhanced calcium binding due to increased length alone cannot explain our data. We hypothesize that the calcium affinity of the activating sites increases with cross-bridge attachment and further with cross-bridge strain. This accounts for the biphasic response to stretch as follows: cross-bridges detached by stretch first decrease calcium affinity, then upon reattachment increase calcium affinity due to the strained configuration brought on by the stretch. The experiments suggest that cross-bridge attachment and strain can modify calcium binding to the activating sites in intact muscle.
Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils.
Authors: Simchowitz L , Davis A O
Published: May 2004

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl-/HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization.
Sustained subthreshold-for-twitch depolarization in rat single ventricular myocytes causes sustained calcium channel activation and sarcoplasmic reticulum calcium release.
Authors: Talo A , Stern M D , Spurgeon H A , Isenberg G , Lakatta E G
Published: May 2004

Single rat ventricular myocytes, voltage-clamped at -50 to -40 mV, were depolarized in small steps in order to define the mechanisms that govern the increase in cytosolic [Ca2+] (Cai) and contraction, measured as a reduction in myocyte length. Small (3-5 mV), sustained (seconds) depolarizations that caused a small inward or no detectable change in current were followed after a delay by small (less than 2% of the resting length), steady reductions in cell length measured via a photodiode array, and small, steady increases in Cai measured by changes in Indo-1 fluorescence. Larger (greater than -30 and less than -20 mV), sustained depolarizations produced phasic Ca2+ currents, Cai transients, and twitch contractions, followed by a steady current and a steady increase in Cai and contraction. Nitrendipine (or Cd, verapamil, or Ni) abolished the steady contraction and always produced an outward shift in steady current. The steady, nitrendipine-sensitive current and sustained increase in Cai and contraction exhibited a similar voltage dependence over the voltage range between -40 and -20 mV. 2 microM ryanodine in the presence of intact Ca2+ channel activity also abolished the steady increase in Cai and contraction over this voltage range. We conclude that when a sustained depolarization does not exceed about -20 mV, the resultant steady, graded contraction is due to SR Ca2+ release graded by a steady ("window") Ca2+ current. The existence of appreciable, sustained, graded Ca2+ release in response to Ca2+ current generated by arbitrarily small depolarizations is not compatible with any model of Ca2(+)-induced Ca2+ release in which the releasing effect of the Ca2+ channel current is mediated solely by Ca2+ entry into a common cytosolic pool. Our results therefore imply a distinction between the triggering and released Ca2+ pools.
Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance.
Authors: Blaustein R O , Finkelstein A
Published: May 2004

In a recent paper (Blaustein, R. O., T. M. Koehler, R. J. Collier, and A. Finkelstein, 1989. Proc. Natl. Acad. Sci. USA. 86:2209-2213) we described the general channel-forming properties of the PA65 fragment of anthrax toxin in planar phospholipid bilayer membranes. In the present paper we extend our previous studies of the permeability properties of this channel, using a series of symmetric tetraalkylammonium (TAA) ions. Our main finding is that at micromolar concentrations on either the cis (toxin-containing) or trans side of a membrane containing many (greater than 1,000) channels, these ions, ranging in size from tetramethylammonium to tetrahexylammonium, induce a voltage-dependent reduction of membrane conductance. (We attribute a similar voltage-dependent reduction of membrane conductance by millimolar concentrations of HEPES to a cationic form of this buffer present at micromolar concentrations.) In going from large negative to large positive voltages (on the TAA side) one sees that the conductance first decreases from its value in the absence of TAA, reaches a minimum, and then rises back at larger positive voltages toward the level in the absence of TAA. Our interpretation of this behavior is that these symmetric TAA ions block the cation-selective PA65 channel in a voltage-dependent manner. We postulate that there is a single site within the channel to which TAA ions can bind and thereby block the passage of the major current-carrying ion (potassium). A blocking ion is driven into the site by modest positive voltages, but is driven off the site and through the channel by larger positive voltages, thus explaining the relief of block. (In the accompanying paper [Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942] we confirm this interpretation of the data by analysis at the single-channel level.) This means that these blocking ions can pass through the channel; the permeability to tetrahexylammonium, the largest ion studied, implies that the narrowest part of the channel has a diameter of at least 11 A.
Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport.
Authors: Adorante J S , Miller S S
Published: May 2004

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.
The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation.
Authors: Sweitzer N K , Moss R L
Published: May 2004

The effect of changes in temperature on the calcium sensitivity of tension development was examined in permeabilized cellular preparations of rat ventricle and rabbit psoas muscle. Maximum force and Ca2+ sensitivity of force development increased with temperature in both muscle types. Cardiac muscle was more sensitive to changes in temperature than skeletal muscle in the range 10-15 degrees C. It was postulated that the level of thin filament activation may be decreased by cooling. To investigate this possibility, troponin C (TnC) was partially extracted from both muscle types, thus decreasing the level of thin filament activation independent of temperature and, at least in skeletal muscle fibers, decreasing cooperative activation of the thin filament as well. TnC extraction from cardiac muscle reduced the calcium sensitivity of tension less than did extraction of TnC from skeletal muscle. In skeletal muscle the midpoint shift of the tension-pCa curve with altered temperature was greater after TnC extraction than in control fibers. Calcium sensitivity of tension development was proportional to the maximum tension generated in cardiac or skeletal muscle under all conditions studied. Based on these results, we conclude that (a) maximum tension-generating capability and calcium sensitivity of tension development are related, perhaps causally, in fast skeletal and cardiac muscles, and (b) thin filament activation is less cooperative in cardiac muscle than in skeletal muscle, which explains the differential sensitivity of the two fiber types to temperature and TnC extraction. Reducing thin filament cooperativity in skeletal muscle by TnC extraction results in a response to temperature similar to that of control cardiac cells. This study provides evidence that force levels in striated muscle influence the calcium binding affinity of TnC.
Zn2(+)-induced subconductance events in cardiac Na+ channels prolonged by batrachotoxin. Current-voltage behavior and single-channel kinetics.
Authors: Schild L , Ravindran A , Moczydlowski E
Published: May 2004

The mechanism of voltage-dependent substate production by external Zn2+ in batrachotoxin-modified Na+ channels from canine heart was investigated by analysis of the current-voltage behavior and single-channel kinetics of substate events. At the single-channel level the addition of external Zn2+ results in an increasing frequency of substate events with a mean duration of approximately 15-25 ms for the substate dwell time observed in the range of -70 to +70 mV. Under conditions of symmetrical 0.2 M NaCl, the open state of cardiac Na+ channels displays ohmic current-voltage behavior in the range of -90 to +100 mV, with a slope conductance of 21 pS. In contrast, the Zn2(+)-induced substate exhibits significant outward rectification with a slope conductance of 3.1 pS in the range of -100 to -50 mV and 5.1 pS in the range of +50 to +100 mV. Analysis of dwell-time histograms of substate events as a function of Zn2+ concentration and voltage led to the consideration of two types of models that may explain this behavior. Using a simple one-site blocking model, the apparent association rate for Zn2+ binding is more strongly voltage dependent (decreasing e-fold per +60 mV) than the Zn2+ dissociation rate (increasing e-fold per +420 mV). However, this simple blocking model cannot account for the dependence of the apparent dissociation rate on Zn2+ concentration. To explain this result, a four-state kinetic scheme involving a Zn2(+)-induced conformational change from a high conductance conformation to a substate conformation is proposed. This model, similar to one introduced by Pietrobon et al. (1989. J. Gen. Physiol. 94:1-24) for H(+)-induced substate behavior in L-type Ca2+ channels, is able to simulate the kinetic and equilibrium behavior of the primary Zn2(+)-induced substate process in heart Na+ channels. This model implies that binding of Zn2+ greatly enhances conversion of the open, ohmic channel to a low conductance conformation with an asymmetric energy profile for Na+ permeation.
Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra.
Authors: Nasi E
Published: May 2004

Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could also be identified. The relevance of such conductance mechanisms in the generation of the light response in Lima photoreceptors is discussed.
Effect of osmotic compression on the force-velocity properties of glycerinated rabbit skeletal muscle cells.
Authors: Ford L E , Nakagawa K , Desper J , Seow C Y
Published: May 2004

The force-velocity relations of single glycerinated rabbit psoas muscle fibers at 5 degrees C were studied at maximum and half-maximum activation in the presence of 0 (control) and 39-145 g/liter dextran T-70. Resting fiber diameter decreased progressively to approximately 70% of the nondextran control as the dextran concentration was increased. Isometric force at full activation increased to a maximum of 136% of control at 111 g/liter dextran and then fell to 80% of control in 145 g/liter dextran. Maximum velocity, which fell to 49% of the control value in the highest concentration of dextran, was nearly constant at approximately 65% control over the range of 58-111 g/liter dextran. Relative maximum power, which gives an estimate of changes in intermediate velocity, was not significantly reduced by dextran concentrations up to 76 g/liter, but then fell progressively to 62% of control in the highest concentration of dextran. At half-maximum activation, maximum velocity and relative maximum power were not significantly different from the values at full activation. The results obtained at partial activation indicate that the decline of velocity seen in the presence of dextran is not due to a passive internal load and that the dextran does not cause a viscous resistance to shortening. The increased velocity in the absence of dextran can be explained by the reduced ability of cross-bridges to resist shortening, as proposed by Goldman (1987. Biophys. J. 51:57).
Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels.
Authors: Ravindran A , Schild L , Moczydlowski E
Published: May 2004

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers.
Authors: Mejía-Alvarez R , Fill M , Stefani E
Published: May 2004

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.