bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes.
Authors: Ebihara L , Steiner E
Published: May 2004

Connexin46 (cxn46) is a gap junctional protein that was cloned from a rat lens cDNA library. Expression of cxn46 in solitary Xenopus oocytes resulted in the development of a large time- and voltage-dependent current that was not observed in noninjected control oocytes or in oocytes injected with mRNA for cxn43 or cxn32. The cxn46-induced current activated at potentials positive to -20 mV. On repolarization to -40 mV, the current deactivated over a period of several seconds. Removal of external calcium caused a marked increase in the amplitude of the cxn46-induced current, shifted the steady-state activation curve to more negative potentials, and altered the kinetics of activation and deactivation. Increasing external calcium had the opposite effect. The ability of cxn46 to induce the formation of cell-to-cell channels was tested in the oocyte pair system. Oocyte pairs injected with cxn46 mRNA + antisense oligonucleotides for Xenopus cxn38 were strongly coupled. In contrast, oocyte pairs injected with antisense alone showed no coupling. The inactivation kinetics of the gap junctional channels resembled the deactivation kinetics of the cxn46-induced current in solitary oocytes.
Proton (or hydroxide) fluxes and the biphasic osmotic response of human red blood cells.
Authors: Bisognano J D , Dix J A , Pratap P R , Novak T S , Freedman J C
Published: May 2004

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).
An allosteric model of the molecular interactions of excitation-contraction coupling in skeletal muscle.
Authors: Ríos E , Karhanek M , Ma J , González A
Published: May 2004

A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88-118), and analogous to one proposed by Marks and Jones for voltage-dependent Ca channels (Marks, T. N., and S. W. Jones. 1992. Journal of General Physiology. 99:367-390). The allosteric protein is the calcium release channel, a homotetramer, with two accessible states, closed and open. The kinetics and equilibrium of this transition are modulated by voltage sensors (dihydropyridine receptors) pictured as four units per release channel, each undergoing independent voltage-driven transitions between two states (resting and activating). For each voltage sensor that moves to the activating state, the tendency of the channel to open increases by an equal (large) factor. The equilibrium and kinetic equations of the model are solved and shown to reproduce well a number of experimentally measured relationships including: charge movement (Q) vs. voltage, open probability of the release channel (Po) vs. voltage, the transfer function relationship Po vs. Q, and the kinetics of charge movement, release activation, and deactivation. The main consequence of the assumption of allosteric coupling is that primary effects on the release channel are transmitted backward to the voltage sensor and give secondary effects. Thus, the model reproduces well the effects of perchlorate, described in the two previous articles, under the assumption that the primary effect is to increase the intrinsic tendency of the release channel to open, with no direct effects on the voltage sensor. This modification of the open-closed equilibrium of the release channel causes a shift in the equilibrium dependency of charge movement with voltage. The paradoxical slowing of charge movement by perchlorate also results from reciprocal effects of the channel on the allosterically coupled voltage sensors. The observations of the previous articles plus the simulations in this article constitute functional evidence of allosteric transmission.
Visual pigment bleaching in isolated salamander retinal cones. Microspectrophotometry and light adaptation.
Authors: Jones G J , Fein A , MacNichol E F , Cornwall M C
Published: May 2004

Visual pigment bleaching desensitizes rod photoreceptors greatly in excess of that due to loss of quantum catch. Whether this phenomenon also occurs in cone photoreceptors was investigated for isolated salamander red-sensitive cones. In parallel experiments, (a) visual pigment depletion by steps of bleaching light was measured by microspectrophotometry, and (b) flash sensitivity was measured by recording light-sensitive membrane current. In isolated cones, visual pigment bleaching permanently reduced flash sensitivity significantly below that due to the reduction in quantum catch, and there was little spontaneous recovery of visual pigment. The "extra" desensitization due to bleaching was most prominent up to bleaches of approximately 80% visual pigment and reached a level approximately 1 log unit beyond that due to loss of quantum catch. At higher bleaches, the effect of loss of quantum catch became more important. Bleaching did not greatly reduce the maximum light-suppressible membrane current. A 99% reduction of the visual pigment permanently reduced the circulating current by only 30%. Visual pigment bleaching speeded up the kinetics of dim flash responses. All electrical effects of bleaching were reversed on exposure to 11-cis retinal, which probably caused visual pigment regeneration. Light adaptation in photopic vision is known to involve significant visual pigment depletion. The present results indicate that cones operate with a maintained circulating current even after a large pigment depletion. It is shown how Weber/Fechner behavior may still be observed in photopic vision when the contributions of bleaching to adaptation are included.
Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.
Authors: Hamada E , Nakajima T , Ota S , Terano A , Omata M , Nakade S , Mikoshiba K , Kurachi Y
Published: May 2004

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)
Block of cardiac ATP-sensitive K+ channels by external divalent cations is modulated by intracellular ATP. Evidence for allosteric regulation of the channel protein.
Authors: Kwok W M , Kass R S
Published: May 2004

We have investigated the interactions between extracellular divalent cations and the ATP-sensitive potassium channel in single guinea pig ventricular cells and found that, under whole-cell patch clamp recording conditions, extracellularly applied Co2+, Cd2+, and Zn2+ block current through the ATP-sensitive K channel (IKATP). The respective Kd's for block of IKATP by Cd2+ and Zn2+ are 28 and 0.46 microM. The Kd for Co2+ is > 200 microM. Extracellular Ca2+ and Mg2+ appear to have no effect at concentrations up to 1 and 2 mM, respectively. Block of IKATP by extracellular cations is not voltage dependent, and both onset and recovery from block occur within seconds. Single-channel experiments using the inside-out patch configuration show that internally applied Cd2+ and Zn2+ are not effective blockers of IKATP. Experiments in the outside-out patch configuration confirm that the divalent cations interact directly with IKATP channel activity. Our study also shows that this block of IKATP is dependent on intracellular ATP concentrations. Under whole-cell conditions, when cells are dialyzed with [ATP]pipette = 0, the degree of cation block is reduced. This dependence on intracellular ATP was confirmed at the single-channel level by experiments in excised, inside-out patch configurations. Our results show that some, but not all, divalent cations inhibit current through IKATP channels by binding to sites that are not within the transmembrane electric field, but are on the extracellular membrane surface. The interdependence of internal ATP and external divalent cation binding is consistent with an allosteric interaction between two binding sites and is highly suggestive of a modulatory mechanism involving conformational change of the channel protein.
A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages.
Authors: Kapus A , Romanek R , Qu A Y , Rotstein O D , Grinstein S
Published: May 2004

Phagocytes generate large amounts of metabolic acid during activation. Therefore, the presence of a conductive pathway capable of H+ extrusion has been suggested (Henderson, L. M., J. B. Chappell, and O. T. G. Jones. 1987. Biochemical Journal. 246:325-329). In this report, electrophysiological and fluorimetric methods were used to probe the existence of a H+ conductance in murine peritoneal macrophages. In suspended cells, recovery of the cytosolic pH (pHi) from an acid-load in Na+ and HCO3(-)-free medium was detectable in depolarizing but not in hyperpolarizing media. The rate of alkalinization was potentiated by the rheogenic ionophore valinomycin. These findings are consistent with the existence of a conductive H+ (equivalent) pathway. This notion was confirmed by patch-clamping and fluorescence ratio measurements of single adherent cells. When voltage was clamped in the whole-cell configuration, depolarizing pulses induced a sizable outward current which was accompanied by cytosolic alkalinization. Several lines of evidence indicate that H+ (equivalents) carry this current: (a) the conductance was unaffected by substitution of the major ionic constituents of the intra-and/or extracellular media, (b) the reversal potential of the tail currents approached the H+ equilibrium potential; and (c) the voltage-induced currents and pHi changes were both Zn2+ sensitive and had similar time course and potential dependence. The peak whole-cell current displayed marked outward rectification and was exquisitely H+ selective. At constant voltage, the H+ permeability was increased by lowering pHi but was inhibited by extracellular acidification. Together with the voltage dependence of the conductance, these features ensure that H+ extrusion can occur during activation, while potentially deleterious acid uptake is precluded. The properties of the conductance appear ideally suited for pHi regulation during phagocyte activation, because these cells undergo a sustained depolarization and an incipient acidification when stimulated. Comparison of the magnitude of the current with the amount of metabolic acid generated during macrophage activation indicates that the conductance is sufficiently large to contribute to the H+ extrusion required for maintenance of pHi.
Whole-cell currents in single and confluent M-1 mouse cortical collecting duct cells.
Authors: Korbmacher C , Segal A S , Fejes-Tóth G , Giebisch G , Boulpaep E L
Published: May 2004

M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl-Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M-1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.
Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor.
Authors: Shirokov R , Levis R , Shirokova N , Ríos E
Published: May 2004

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately -30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)-dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.
Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides.
Authors: Murrell-Lagnado R D , Aldrich R W
Published: May 2004

Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates.
Measurement of calcium transients and slow calcium current in myotubes.
Authors: García J , Beam K G
Published: May 2004

The purpose of this study was to characterize excitation-contraction (e-c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the calcium indicator dye Fluo-3 to study the calcium transients and slow calcium currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow calcium current. The slow calcium current had a threshold for activation of about 0 mV; the peak amplitude of the current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong (> or = 50 mV) that the amplitude of the slow calcium current became very small. Thus, the slow calcium current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the calcium transient by mechanisms that appeared to be unrelated to the block of current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large calcium releases and becomes more efficient when the size of the release is reduced.
Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier.
Authors: Lopatin A N , Nichols C G
Published: May 2004

Delayed rectifier potassium channels were expressed in the membrane of Xenopus oocytes by injection of rat brain DRK1 (Kv2.1) cRNA, and currents were measured in cell-attached and inside-out patch configurations. In intact cells the current-voltage relationship displayed inward going rectification at potentials > +100 mV. Rectification was abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions, but was restored by introducing Mg2+ or Na+ ions into the bath solution. At +50 mV, half-maximum blocking concentrations for Mg2+ and Na+ were 4.8 +/- 2.5 mM (n = 6) and 26 +/- 4 mM (n = 3) respectively. Increasing extracellular potassium concentration reduced the degree of rectification of intact cells. It is concluded that inward going rectification resulting from voltage-dependent block by internal cations can be observed with normally outwardly rectifying DRK1 channels.
Ionic permeability characteristics of the N-methyl-D-aspartate receptor channel.
Authors: Zarei M M , Dani J A
Published: May 2004

N-methyl-D-aspartate (NMDA) receptor channels in cultured CA1 hippocampal neurons were studied using patch-clamp techniques. The purpose of the research was to determine the occupancy of the channel by permeant cations and to determine the influence of charged residues in or near the pore. The concentration dependence of permeability ratios, the mole-fraction dependence of permeability ratios, the concentration dependence of the single-channel conductance, and a single-channel analysis of Mg2+ block all independently indicated that the NMDA receptor behaves as a singly-occupied channel. More precisely, there is one permeant cation at a time occupying the site or sites that are in the narrow region of the pore directly in the permeation pathway. Permeability-ratio measurements in mixtures of monovalent and divalent cations indicated that local charges in or near the pore do not produce a large local surface potential in physiologic solutions. In low ionic strength solutions, a local negative surface potential does influence the ionic environment near the pore, but in normal physiologic solutions the surface potential appears too small to significantly influence ion permeation. The results indicate that the mechanism for the high Ca2+ conductance of the NMDA receptor channel is not the same as for the voltage-dependent Ca2+ channel (VDCC). The VDCC has two high affinity, interacting binding sites that provide high Ca2+ selectivity and conductance. The binding site of the NMDA receptor is of lower affinity. Therefore, the selectivity for Ca2+ is not as high, but the lower affinity of binding provides a faster off rate so that interacting sites are not required for high conductance.
Shaker potassium channel gating. II: Transitions in the activation pathway.
Authors: Zagotta W N , Hoshi T , Dittman J , Aldrich R W
Published: May 2004

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.
Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes.
Authors: Dolmetsch R E , Lewis R S
Published: May 2004

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert-butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)-ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.
Binding of benzocaine in batrachotoxin-modified Na+ channels. State-dependent interactions.
Authors: Wang G K , Wang S Y
Published: May 2004

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.
A scheme to account for the effects of Rb+ and K+ on inward rectifier K channels of bovine artery endothelial cells.
Authors: Pennefather P S , DeCoursey T E
Published: May 2004

An electrochemical gating model is presented to account for the effects described in the companion paper by M. R. Silver, M. S. Shapiro, and T. E. DeCoursey (1994. Journal of General Physiology, 103:519-548) of Rb+ and Rb+/K+ mixtures on the kinetics and voltage dependence of an inwardly rectifying (IR) K+ channel. The model proposes that both Rb+ and K+ act as allosteric modulators of an intrinsically voltage dependent isomerization between open and closed states. Occupancy of binding sites on the outside of the channel promotes channel opening and stabilizes the open state. Rb+ binds to separate sites within the pore and plugs IR channels. Occupancy of the pore by Rb+ can modify the rates of isomerization and the affinity of the allosteric sites for activator ions. The model also incorporates the proposed triple-barreled nature of the IR channel (Matsuda, H., 1988. Journal of Physiology. 397:237-258.) by proposing that plugging of the channel is a cooperative process involving a single site in each of the three bores, 80% of the way through the membrane field. Interaction between bores during plugging and permeation is consistent with correlated flux models of the properties of the IR channel. Parallel bores multiply the number allosteric sites associated with the macromolecular channel and allow for steep voltage dependence without compromising the parallel shift of the half-activation potential with reversal potential. Our model proposes at least six and possibly 12 such allosteric binding sites for activator ions. We derive algebraic relations that permit derivation of parameters that define simple versions of our model from the data of Silver et al. (1994). Numerical simulations based on those parameters closely reproduce that data. The model reproduces the RS+ induced slowing of IR kinetics and the negative shift of the relation between the half-activation voltage (V1/2) and reversal potential when channel plugging is associated with (a) a slowing of the isomerization rates; (b) an increase in the affinity of allosteric sites on closed channels that promote opening; and (c) a decrease in the affinity of sites on open channels that slow closing. Rb+ also slows closing at positive potentials where open channel blockade is unlikely. Allowing Rb+ to be 1.5 times more potent than K+ as an activator in the model can account for this effect and improves the match between the predicted and observed relation between the Rb+ to K+ mole fraction and the opening rate at V1/2.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium channel current of vascular smooth muscle cells: extracellular protons modulate gating and single channel conductance.
Authors: Klöckner U , Isenberg G
Published: May 2004

Modulation of L-type Ca2+ channel current by extracellular pH (pHo) was studied in vascular smooth muscle cells from bovine pial and porcine coronary arteries. Relative to pH 7.4, alkaline pH reversibly increased and acidic pH reduced ICa. The efficacy of pHo in modulating ICa was reduced when the concentration of the charge carrier was elevated ([Ca2+]o or [Ba2+]o varied between 2 and 110 mM). Analysis of whole cell and single Ca2+ channel currents suggested that more acidic pHo values shift the voltage-dependent gating (approximately 15 mV per pH-unit) and reduce the single Ca2+ channel conductance gCa due to screening of negative surface charges. pHo effects on gCa depended on the pipette [Ba2+] ([Ba2+]p), pK*, the pH providing 50% of saturating conductance, increased with [Ba2+]p according to pK* = 2.7-2.log ([Ba2+]p) suggesting that protons and Ba2+ ions complete for a binding site that modulates gCa. The above mechanisms are discussed in respect to their importance for Ca2+ influx and vasotonus.
Actions of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) in retinal ON bipolar cells indicate that it is an agonist at L-AP4 receptors.
Authors: Thoreson W B , Miller R F
Published: May 2004

Metabotropic glutamate receptors (mGluRs) include receptors sensitive to L-2-amino-4-phosphonobutyrate (L-AP4) and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). To determine whether 1S,3R-ACPD is an agonist at retinal L-AP4 receptors, whole cell voltage clamp recordings were obtained from mudpuppy ON bipolar cells in a superfused retinal slice and L-AP4 and 1S,3R-ACPD were bath applied. Both compounds evoked similar outward currents which reversed near 0 mV and were accompanied by an increased input resistance. Responses to both agonists washed out in parallel suggesting they act through the same second messenger pathway(s). Inhibitors of cGMP-PDE activity suppressed responses to both L-AP4 and 1SR,3RS-ACPD, suggesting that both compounds activate cGMP-PDE. Responses to 1S,3R-ACPD were occluded by prior activation of L-AP4 receptors, but not blocked by the non-AP4, mGluR antagonists, L-aminophosphonopropionic acid (L-AP3) or 4-carboxy-3-hydroxyphenylglycine (4C3H-PG). These results indicate that 1S,3R-ACPD is an agonist at L-AP4 receptors. 1S,3S-ACPD and 4C3H-PG evoked outward currents similar to L-AP4 suggesting they may also be L-AP4 receptor agonists. Using the b-wave of the ERG as an assay for ON bipolar cell responses, concentration/response curves were obtained for ACPD enantiomers. The rank-order potency of ACPD enantiomers at L-AP4 receptors in ON bipolar cells is similar to their rank-order potency at non-AP4, mGluRs in brain which suggests that the receptors possess similar binding sites and may be members of a common receptor family.
Basic properties and potential regulators of the apical K+ channel in macula densa cells.
Authors: Hurst A M , Lapointe J Y , Laamarti A , Bell P D
Published: May 2004

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside-out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside-out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.
White noise analysis of a chromatic type horizontal cell in the Xenopus retina.
Authors: Stone S L
Published: May 2004

The dynamics of color-coded signal transmission in the light-adapted Xenopus retina were studied by a combination of white noise (Wiener) analysis and simultaneous recordings from two types of horizontal cells: chromatic-type horizontal cells (C-HCs) are hyperpolarized by blue light and depolarized by red light, whereas luminosity-type horizontal cells (L-HCs) are hyperpolarized by all wave-lengths. The retina was stimulated by two superimposed fields of red and blue light modulated by two independent white noise signals, and the resulting intracellular responses were decomposed into red and blue components (first-order kernels). The first-order kernels predict the intracellular responses with a small degree of error (3.5-9.5% in terms of mean square error) under conditions where modulated responses exceeded 30 mV in amplitude peak-to-peak, thus demonstrating that both red and blue modulation responses are linear. Moreover, there is little or no interaction between the red- and blue-evoked responses; i.e., nearly identical first-order kernels were obtained for one color whether the other color was modulated or not. In C-HCs (but not L-HCs), there were consistent differences in the dynamics of the red and blue responses. In the C-HC, the cutoff frequency of the red response was higher than for the blue (approximately 12 vs 5 Hz), and the red kernel was more bandpass than the blue. In the L-HC, kernel waveform and cutoff frequencies were similar for both colors (approximately 12 Hz or greater), and the time-to-peak of the L-HC kernel was always shorter than either the red or blue C-HC kernel. These results have implications for the mechanisms underlying color coding in the distal retina, and they further suggest that nonlinear phenomena, such as voltage-dependent conductances in HCs, do not contribute to the generation of modulation responses under the experimental conditions used here.
Partial reactions of the Na,K-ATPase: determination of rate constants.
Authors: Heyse S , Wuddel I , Apell H J , Stürmer W
Published: May 2004

Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)