bluuebunny/crossref_metadata_2025 huggingface dataset:
Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland.
Authors: Richards N W , Lowy R J , Ernst S A , Dawson D C
Published: May 2004

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.
Kinetics and stoichiometry of coupled Na efflux and Ca influx (Na/Ca exchange) in barnacle muscle cells.
Authors: Rasgado-Flores H , Santiago E M , Blaustein M P
Published: May 2004

Coupled Na+ exit/Ca2+ entry (Na/Ca exchange operating in the Ca2+ influx mode) was studied in giant barnacle muscle cells by measuring 22Na+ efflux and 45Ca2+ influx in internally perfused, ATP-fueled cells in which the Na+ pump was poisoned by 0.1 mM ouabain. Internal free Ca2+, [Ca2+]i, was controlled with a Ca-EGTA buffering system containing 8 mM EGTA and varying amounts of Ca2+. Ca2+ sequestration in internal stores was inhibited with caffeine and a mitochondrial uncoupler (FCCP). To maximize conditions for Ca2+ influx mode Na/Ca exchange, and to eliminate tracer Na/Na exchange, all of the external Na+ in the standard Na+ sea water (NaSW) was replaced by Tris or Li+ (Tris-SW or LiSW, respectively). In both Na-free solutions an external Ca2+ (Cao)-dependent Na+ efflux was observed when [Ca2+]i was increased above 10(-8) M; this efflux was half-maximally activated by [Ca2+]i = 0.3 microM (LiSW) to 0.7 microM (Tris-SW). The Cao-dependent Na+ efflux was half-maximally activated by [Ca2+]o = 2.0 mM in LiSW and 7.2 mM in Tris-SW; at saturating [Ca2+]o, [Ca2+]i, and [Na+]i the maximal (calculated) Cao-dependent Na+ efflux was approximately 75 pmol#cm2.s. This efflux was inhibited by external Na+ and La3+ with IC50's of approximately 125 and 0.4 mM, respectively. A Nai-dependent Ca2+ influx was also observed in Tris-SW. This Ca2+ influx also required [Ca2+]i greater than 10(-8) M. Internal Ca2+ activated a Nai-independent Ca2+ influx from LiSW (tracer Ca/Ca exchange), but in Tris-SW virtually all of the Cai-activated Ca2+ influx was Nai-dependent (Na/Ca exchange). Half-maximal activation was observed with [Na+]i = 30 mM. The fact that internal Ca2+ activates both a Cao-dependent Na+ efflux and a Nai-dependent Ca2+ influx in Tris-SW implies that these two fluxes are coupled; the activating (intracellular) Ca2+ does not appear to be transported by the exchanger. The maximal (calculated) Nai-dependent Ca2+ influx was -25 pmol/cm2.s. At various [Na+]i between 6 and 106 mM, the ratio of the Cao-dependent Na+ efflux to the Nai-dependent Ca2+ influx was 2.8-3.2:1 (mean = 3.1:1); this directly demonstrates that the stoichiometry (coupling ratio) of the Na/Ca exchange is 3:1. These observations on the coupling ratio and kinetics of the Na/Ca exchanger imply that in resting cells the exchanger turns over at a low rate because of the low [Ca2+]i; much of the Ca2+ extrusion at rest (approximately 1 pmol/cm2.s) is thus mediated by an ATP-driven Ca2+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)
Nonmodal gating of cardiac calcium channels as revealed by dihydropyridines.
Authors: Lacerda A E , Brown A M
Published: May 2004

The hypothesis that dihydropyridine (DHP)-sensitive calcium channels have three distinct modes of gating has been examined. The major prediction is that the relative frequencies among modes depend on DHP concentration while the kinetics within a mode do not. We tested this by studying whole-cell and single-channel calcium currents in neonatal rat and adult guinea pig cardiac myocytes in different concentrations of several DHPs. In the absence of DHPs calcium currents declined with time but the kinetics, which are the focus of this study, were unchanged. Open-time frequency distributions had insignificant numbers of prolonged openings and were well fit by single tau's. Agonist DHP stereoisomers produced concentration-dependent changes in whole-cell tail current tau's. The frequency distribution of single calcium channel current open times became biexponential and the tau's were concentration dependent. The average number of openings per trace of channels with customary open times increased with increases in DHP concentration. Latencies to first opening for the customary openings and for prolonged openings were shorter in the presence of DHPs. A second larger conductance is another important feature of DHP-bound single calcium channels. Thus DHPs not only caused prolonged openings; they produced numerous changes in the kinetics of customary openings and increased channel conductance. It follows that these effects of DHPs do not support the hypothesis of modal gating of calcium channels. The mode model is not the only model excluded by the results; models in which DHPs are allowed to act only or mainly on open states are excluded, as are models in which the effects are restricted to inactivated states. We suggest a different type of model in which cooperative binding of DHPs at two sites produces the essential changes in kinetics and conductance.
Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium.
Authors: Hughes B A , Adorante J S , Miller S S , Lin H
Published: May 2004

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).
Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating.
Authors: Bean B P , Rios E
Published: May 2004

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.
Sodium channel gating in clonal pituitary cells. The inactivation step is not voltage dependent.
Authors: Cota G , Armstrong C M
Published: May 2004

We have determined the time course of Na channel inactivation in clonal pituitary (GH3) cells by comparing records before and after the enzymatic removal of inactivation. The cells were subjected to whole-cell patch clamp, with papain included in the internal medium. Inactivation was slowly removed over the course of 10 min, making it possible to obtain control records before the enzyme acted. Papain caused a large (4-100x) increase in current magnitude for small depolarizations (near -40 mV), and a much smaller increase for large ones (approximately 1.5x at +40 mV). For technical reasons it was sometimes convenient to study outward INa recorded with no Na+ outside. The instantaneous I-V (IIV) curve in this condition was nonlinear before papain, and more nearly linear afterwards. The gNa-V curve after papain, obtained by dividing the INa-V curve by the IIV curve, was left-shifted by at least 20 mV and steepened. A spontaneous 5-10 mV left shift occurred in the absence of papain. The rate of the inactivation step was found to vary only slightly from -100 mV to +60 mV, based on the following evidence. (a) Before papain, inactivation rate saturated with voltage and was constant from +20 to +60 mV. (b) We activated the channels with a brief pulse, and studied the time course of the current on changing the voltage to a second, usually more negative level (Na+ present internally and externally). The time course of inactivation at each voltage was obtained by comparing control traces with those after inactivation was removed. When the 5-10-mV spontaneous shift was taken into account, inactivation rate changed by less than 10% from -100 to +60 mV. The data are considered in terms of existing models of the Na channel.
Kinetic diversity of Na+ channel bursts in frog skeletal muscle.
Authors: Patlak J B , Ortiz M
Published: May 2004

Individual Na+ channels of dissociated frog skeletal muscle cells at 10 degrees C fail to inactivate in 0.02% of depolarizing pulses, thus producing bursts of openings lasting hundreds of milliseconds. We present here a kinetic analysis of 87 such bursts that were recorded in multi-channel patches at four pulse potentials. We used standard dwell-time histograms as well as fluctuation analysis to analyze the gating kinetics of the bursting channels. Since each burst contained only 75-150 openings, detailed characterization of the kinetics from single bursts was not possible. Nevertheless, at this low kinetic resolution, the open and closed times could be well fitted by single exponentials (or Lorentzians for the power spectra). The best estimates of both the open and closed time constants produced by either technique were much more broadly dispersed then expected from experimental or analytical variability, with values varying by as much as an order of magnitude. Furthermore, the values of the open and closed time constants were not significantly correlated with one another from burst to burst. The bursts thus expressed diverse kinetic behaviors, all of which appear to be manifestations of a single type of Na+ channel. Although the opening and closing rates were dispersed, their average values were close to those of alpha m and 2 beta m derived from fits to the early transient Na+ currents over the same voltage range. We propose a model in which the channel has both primary states (e.g., open, closed, and inactivated), as well as "modes" that are associated with independent alterations in the rate constants for transition between each of these primary states.
On the role of sodium ions in the regulation of the inward-rectifying potassium conductance in cat ventricular myocytes.
Authors: Harvey R D , Ten Eick R E
Published: May 2004

The conductance of the inward-rectifying K+ current (IK1) in isolated cat ventricular myocytes is decreased by reducing the extracellular Na+ concentration. Using a whole-cell patch-clamp technique, possible mechanisms underlying this Na+ dependence were investigated. These included (a) block of inward K+ current by the Na+ substitute, (b) changes in membrane surface charge associated with removal of extracellular Na+, (c) increases of intracellular Ca2+ due to suppression of Na-Ca exchange, (d) reduction of a Na+-dependent K+ conductance due to a subsequent decrease of intracellular Na+, (e) reduction of IK1 conductance (gK1) associated with reduction of intracellular pH due to suppression of Na-proton exchange. The findings support the hypothesis that the effect of removing Na+ is mediated through a decrease in intracellular pH. These include observations that: (a) reducing internal pH by reducing external pH caused a decrease in gK1, and the conductance changes caused by reducing extracellular pH and removing extracellular Na+ were not additive: (b) the effect of reducing pHo was attenuated by dialyzing with a low pH internal solution; (c) gK1 was reduced by exposure to the Na-proton exchange inhibitor dimethylamiloride, and this effect was absent in the absence of Na+. These findings imply that physiological or pathological processes such as ischemia and metabolic or respiratory acidosis which can produce intracellular acidosis should be expected to affect K+ permeation through the IK1 channel.
Voltage-dependent block of cardiac inward-rectifying potassium current by monovalent cations.
Authors: Harvey R D , Ten Eick R E
Published: May 2004

The inward-rectifying K+ current (IK1) in cat ventricular myocytes, like inward-rectifying K+ currents in many other preparations, exhibited a negative slope conductance region at hyperpolarized membrane potentials that was time-dependent. This was evident as an inactivation of inward current elicited by hyperpolarizing voltage-clamp pulses resulting in a negative slope region of the steady-state current-voltage relationship at potentials negative to -140 mV. Removing extracellular Na+ prevented the development of the negative slope in this voltage region, suggesting that Na+ can block IK1 channels in a time- and voltage-dependent manner. The time and voltage dependence of Cs+-induced block of IK1 was also examined. Cs+ blocked inward current in a manner similar to that of Na+, but the former was much more potent. The fraction of current blocked by Cs+ in the presence of Na+ was reduced in a time- and voltage-dependent manner, which suggested that these blocking ions compete for a common or at least similar site of action. In the absence of Na+, inactivation of IK1 could also be induced by both Cs+ and Li+. However, Li+ was less potent than Na+ in this respect. Calculation of the voltage sensitivity of current block by each of these ions suggests that the mechanism of block by each is similar.
Calcium-induced calcium release mechanism in guinea pig taenia caeci.
Authors: Iino M
Published: May 2004

Fura-2 was used to measure the amount of Ca released from the intracellular Ca store of a saponin-skinned smooth muscle fiber bundle of the guinea pig taenia caeci (width, 150-250 microns) placed in a capillary cuvette at 20-22 degrees C. The amount of Ca actively loaded into the store was assayed when released by the application of 50 mM caffeine and/or 10 microM inositol 1,4,5-trisphosphate (IP3) in the absence of ATP, and was found to have a biphasic dependence on the loading [Ca2+] with a peak near pCa 6. After Ca loading at pCa 6, IP3 released almost all the releasable Ca, whereas caffeine discharged Ca from only approximately 40% of the store. The maximum amount of Ca in the store was some 220 mumol/liter cell water. Ca in the caffeine-releasable store was released approximately exponentially to zero with time when Ca2+ was applied in the absence of ATP, and the rate constant of the Ca-induced Ca release (CICR) increased steeply with the concentration of Ca2+ applied. Increase in [Mg2+] (0.5-5.0 mM) or decrease in pH (7.3-6.7) shifted the relation between pCa and the rate of CICR roughly in parallel toward the lower pCa. An adenine nucleotide increased the rate of the CICR, but it did not change the range of effective [Ca2+]. 5 mM caffeine greatly enhanced the CICR mechanism, making it approximately 30 times more sensitive to [Ca2+]. However the drug had no Ca-releasing action in the absence of Ca2+. Procaine in millimolar concentrations inhibited the rate of the CICR. These properties are similar to those of the skeletal muscle CICR and ryanodine receptor channels. Rates of the CICR under a physiological ionic milieu were estimated from the results, and a [Ca2+] greater than 1 microM was expected to be necessary for the activation of the Ca release. This Ca sensitivity seems too low for the CICR mechanism to play a primary physiological role in Ca mobilization, unless assisted by other mechanisms.
Glutaraldehyde fixation of the cAMP-dependent Na+/H+ exchanger in trout red cells.
Authors: Motais R , Borgese F , Scheuring U , Garcia-Romeu F
Published: May 2004

It has been shown that the addition of a beta-adrenergic catecholamine to a trout red blood cell suspension induces a 60-100-fold increase of sodium permeability resulting from the activation of a cAMP-dependent Na+/H+ antiport. Subsequent addition of propranolol almost instantaneously reduces the intracellular cAMP concentration, and thus the Na permeability, to their basal values (Mahé et al., 1985). If glutaraldehyde (0.06-0.1%) is added when the Na+/H+ exchanger is activated after hormonal stimulation, addition of propranolol no longer inhibits Na permeability: once activated and fixed by glutaraldehyde, the cAMP dependence disappears. Glutaraldehyde alone causes a rapid decrease in the cellular cAMP concentration. In its fixed state the antiporter is fully amiloride sensitive. The switching on of the Na+/H+ exchange by cAMP is rapidly (2 min) followed by acute but progressive desensitization of the exchanger (Garcia-Romeu et al., 1988). The desensitization depends on the concentration of external sodium, being maximal at a normal Na concentration (145 mM) and nonexistent at a low Na concentration (20 mM). If glutaraldehyde is added after activation in nondesensitizing conditions (20 mM Na), transfer to a Na-rich medium induces only a very slight desensitization: thus the fixative can "freeze" the exchanger in the nondesensitizing conformation. NO3- inhibits the activity of the cAMP-dependent Na+/H+ antiporter of the trout red blood cell (Borgese et al., 1986). If glutaraldehyde is added when the cells are activated by cAMP in a chloride-containing medium, the activity of the exchanger is no longer inhibited when Cl- is replaced by NO3-. Conversely, after fixation in NO3- medium replacement of NO3- by Cl- has very little stimulatory effect. This indicates that the anion dependence is not a specific requirement for the exchange process but that the anion environment is critical for the switching on of the Na+/H+ exchanger and for the maintenance of its activated configuration.
Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process.
Authors: Parent L , Coronado R
Published: May 2004

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.
Chemical excitation and inactivation in photoreceptors of the fly mutants trp and nss.
Authors: Suss E , Barash S , Stavenga D G , Stieve H , Selinger Z , Minke B
Published: May 2004

The Drosophila and Lucilia photoreceptor mutants, trp and nss, respond like wild-type flies to a short pulse of intense light or prolonged dim light; however, upon continuous intense illumination, the trp and nss mutants are unable to maintain persistent excitation. This defect manifests itself by a decline of the receptor potential toward baseline during prolonged intense illumination with little change in the shape or amplitude of the quantal responses to single photons (quantum bumps). Previous work on the trp and nss mutants suggests that a negative feedback loop may control the rate of bump production. Chemical agents affecting different steps of the phototransduction cascade were used in conjunction with light to identify a possible branching point of the feedback loop and molecular stages which are affected by the mutation. Fluoride ions, which in the dark both excite and adapt the photoreceptors of wild-type flies, neither excite nor adapt the photoreceptors of the trp and nss mutants. The hydrolysis-resistant analogue, GTP gamma S, which excites the photoreceptors of wild-type flies, resulting in noisy depolarization, markedly reduces the light response of both mutant flies. Intracellular recordings revealed, however, that the inhibitory effect of GTP gamma S on the nss mutant was accompanied neither by any significant depolarization nor by an increase in the noise, and thus was very different from the effect of a dim background light. The combination of inositol trisphosphate and diphosphoglycerate (InsP3 + DPG), which efficiently excites the photoreceptors of wild-type Lucilia, also excites the photoreceptors of nss Lucilia mutant. The InsP3 + DPG together act synergistically with light to accelerate the decline of the response to light in the mutant flies. These results suggest that the fly phototransduction pathway involves a feedback regulatory loop, which branches subsequent to InsP3 production and regulates guanine nucleotide-binding protein (G protein)-phospholipase C activity. A defect in this regulatory loop, which may cause an unusually low level of intracellular Ca2+, severely reduces the triggering of bumps in the mutants during intense prolonged illumination.
Na-K pump current in the Amphiuma collecting tubule.
Authors: Horisberger J D , Giebisch G
Published: May 2004

There is strong evidence supporting the hypothesis of an electrogenic Na-K pump in the basolateral membrane of several epithelia. Thermodynamic considerations and results in nonepithelial cells indicate that the current carried by the pump could be voltage dependent. In order to measure the pump current and to determine its voltage dependence in a tight epithelium, we have used the isolated perfused collecting tubule of Amphiuma and developed a technique for clamping the basolateral membrane potential (Vbl) through transepithelial current injection. The transcellular current was calculated by subtracting the paracellular current (calculated from the transepithelial conductance measured in the presence of luminal amiloride) from the total transepithelial current. Basolateral membrane current-voltage (I-V) curves were obtained in conditions where the ratio of the pump current to the total basolateral membrane current had been maximized by loading the cells with Na+ (exposure to low-K+ bath), and by blocking the basolateral K+ conductance with barium. The pump current was defined as the difference of the current across the basolateral membrane measured before and 10-15 s after the addition of strophanthidin (20 microM) to the bath solution. With a bath solution containing 3 mM K+, the pump current was nearly constant in the Vbl range of -20 to -80 mV (52 +/- 5 microA.cm-2 at -60 mV) but showed a marked voltage dependence at higher negative Vbl (pump current decreased to 5 +/- 9 microA.cm-2 at -180 mV). In a 1.0 mM K bath, the shape of the pump I-V curve was similar but the amplitude of the current was decreased (24 +/- 4 microA.cm-2 at -60 mV). In a 0.1 mM K bath, the pump current was not significantly different from 0. Our results indicate that the basolateral Na-K pump generates a current which depends on the extracellular potassium concentration. With physiological peritubular concentration of K+ and in the physiological range of potential, the pump activity, measured as the pump-generated current, was independent of the membrane potential.
[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes.
Authors: Nakao M , Gadsby D C
Published: May 2004

Na/K pump current was determined between -140 and +60 mV as steady-state, strophanthidin-sensitive, whole-cell current in guinea pig ventricular myocytes, voltage-clamped and internally dialyzed via wide-tipped pipettes. Solutions were designed to minimize all other components of membrane current. A device for exchanging the solution inside the pipette permitted investigation of Na/K pump current-voltage (I-V) relationships at several levels of pipette [Na] [( Na]pip) in a single cell; the effects of changes in external [Na] [( Na]o) or external [K] [( K]o) were also studied. At 50 mM [Na]pip, 5.4 mM [K]o, and approximately 150 mM [Na]o, Na/K pump current was steeply voltage dependent at negative potentials but was approximately constant at positive potentials. Under those conditions, reduction of [Na]o enhanced pump current at negative potentials but had little effect at positive potentials: at zero [Na]o, pump current was only weakly voltage dependent. At 5.4 mM [K]o and approximately 150 mM [Na]o, reduction of [Na]pip from 50 mM scaled down the sigmoid pump I-V relationship and shifted it slightly to the right (toward more positive potentials). Pump current at 0 mV was activated by [Na]pip according to the Hill equation with best-fit K0.5 approximately equal to 11 mM and Hill coefficient nH approximately equal to 1.4. At zero [Na]o, reduction of [Na]pip seemed to simply scale down the relatively flat pump I-V relationship: Hill fit parameters for pump activation by [Na]pip at 0 mV were K0.5 approximately equal to 10 mM, nH approximately equal to 1.4. At 50 mM [Na]pip and high [Na]o, reduction of [K]o from 5.4 mM scaled down the sigmoid I-V relationship and shifted it slightly to the right: at 0 mV, K0.5 approximately equal to 1.5 mM and nH approximately equal to 1.0. At zero [Na]o, lowering [K]o simply scaled down the flat pump I-V relationships yielding, at 0 mV, K0.5 approximately equal to 0.2 mM, nH approximately equal to 1.1. The voltage-independent activation of Na/K pump current by both intracellular Na ions and extracellular K ions, at zero [Na]o, suggests that neither ion binds within the membrane field. Extracellular Na ions, however, seem to have both a voltage-dependent and a voltage-independent influence on the Na/K pump: they inhibit outward Na/K pump current in a strongly voltage-dependent fashion, with higher apparent affinity at more negative potentials (K0.5 approximately equal to 90 mM at -120 mV, and approximately 170 mM at -80 mV), and they compete with extracellular K ions in a seemingly voltage-independent manner.(ABSTRACT TRUNCATED AT 400 WORDS)
ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.
Authors: Ribalet B , Ciani S , Eddlestone G T
Published: May 2004

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.
Effects of magnesium on inactivation of the voltage-gated calcium current in cardiac myocytes.
Authors: Hartzell H C , White R E
Published: May 2004

The effects of changes in intracellular and extracellular free ionized [Mg2+] on inactivation of ICa and IBa in isolated ventricular myocytes of the frog were investigated using the whole-cell configuration of the patch-clamp technique. Intracellular [Mg2+] was varied by internal perfusion with solutions having different calculated free [Mg2+]. Increasing [Mg2+]i from 0.3 mM to 3.0 mM caused a 16% reduction in peak ICa amplitude and a 36% reduction in peak IBa amplitude, shifted the current-voltage relationship and the inactivation curve approximately 10 mV to the left, decreased relief from inactivation, and caused a dramatic increase in the rate of inactivation of IBa. The shifts in the current-voltage and inactivation curves were attributed to screening of internal surface charge by Mg2+. The increased rate of inactivation of IBa was due to an increase in both the steady-state level of inactivation as well as an increase in the rate of inactivation, as measured by two-pulse inactivation protocols. Increasing external [Mg2+] decreased IBa amplitude and shifted the current-voltage and inactivation curves to the right, but, in contrast to the effect of internal Mg2+, had little effect on the inactivation kinetics or the steady-state inactivation of IBa at potentials positive to 0 mV. These observations suggest that the Ca channel can be blocked quite rapidly by external Mg2+, whereas the block by [Mg2+]i is time and voltage dependent. We propose that inactivation of Ca channels can occur by both calcium-dependent and purely voltage-dependent mechanisms, and that a component of voltage-dependent inactivation can be modulated by changes in cytoplasmic Mg2+.
Application of a fiber-matrix model to transport in renal tubules.
Authors: Fraser W D , Baines A D
Published: May 2004

The effects of tight junction structure on water and solute fluxes across proximal tubular epithelium were examined with fiber-matrix equations previously derived by Curry and Michel (1980. Microvascular Research. 20:96-99). Using plausible estimates of tight junction fiber length and width the model predicts solute (Ps) and water permeability (Lp) coefficients that agree with the measured values. When fiber-matrix and pore models were compared for physiologically relevant ranges of matrix void fraction (80-98%) and pore radii (0-20 A), the fiber-matrix model predicted a 10-fold higher Lp/Ps ratio. Lp/Ps was most sensitive to small changes in tight junction structure when void fractions exceeded 90%. Void fractions of 96.5% and 97.1% predicted previously measured values for Lp and solute permeabilities in rat and rabbit proximal tubules. These values are consistent with void fractions and permeabilities of artificial membranes. The fiber-matrix tight junction model was incorporated into a model of reabsorption from the rat proximal tubule developed by Weinstein (1984). American Journal of Physiology. 247:F848-F862.) A void fraction of 98% predicted the experimental results for isosmotic reabsorption driven by active transport. Changing void fraction over the range of 97-99% produced a 50-75% change in predicted volume reabsorption with active transport. According to the fiber-matrix model: (a) solute permeabilities alone cannot be used to predict Lp, (b) previously measured solute permeabilities in the proximal tubule are compatible with significant water reabsorption through a water-permeable tight junction, and (c) hydraulic and solute permeabilities may be sensitive to small changes in tight junction fiber length and diameter or ionic strength within the tight junction.
Identification and regulation of whole-cell chloride currents in airway epithelium.
Authors: McCann J D , Li M , Welsh M J
Published: May 2004

We used the whole-cell patch-clamp technique to study membrane currents in human airway epithelial cells. The conductive properties, as described by the instantaneous current-voltage relationship, rectified in the outward direction when bathed in symmetrical CsCl solutions. In the presence of Cl concentration gradients, currents reversed near ECl and were not altered significantly by cations. Agents that inhibit the apical membrane Cl conductance inhibited Cl currents. These conductive properties are similar to the conductive properties of the apical membrane Cl channel studied with the single-channel patch-clamp technique. The results suggest that the outwardly rectifying Cl channel is the predominant Cl-conductive pathway in the cell membrane. The steady-state and non-steady-state kinetics indicate that current flows through ion channels that are open at hyperpolarizing voltages and close with depolarization. These Cl currents were regulated by the cAMP-dependent protein kinase: when the catalytic subunit of cAMP-dependent protein kinase was included in the pipette solution, Cl channel current more than doubled. We also found that reducing extracellular osmolarity by 30% increased Cl current, suggesting that cell-swelling stimulated Cl current. Studies of transepithelial Cl transport in cell monolayers suggest that a reduction in solution osmolarity activates the apical Cl channel: reducing extracellular osmolarity stimulated a short-circuit current that was inhibited by Cl-free solution, by mucosal addition of a Cl channel antagonist, and by submucosal addition of a loop diuretic. These results suggest that apical membrane Cl channels may be regulated by cell volume and by the cAMP-dependent protein kinase.
Kinetic time constants independent of previous single-channel activity suggest Markov gating for a large conductance Ca-activated K channel.
Authors: McManus O B , Magleby K L
Published: May 2004

Models for the gating of ion channels usually assume that the rate constants for leaving any given kinetic state are independent of previous channel activity. Although such discrete Markov models have been successful in describing channel gating, there is little direct evidence for the Markov assumption of time-invariant rate constants for constant conditions. This paper tests the Markov assumption by determining whether the single-channel kinetics of the large conductance Ca-activated K channel in cultured rat skeletal muscle are independent of previous single-channel activity. The experimental approach is to examine dwell-time distributions conditional on adjacent interval durations. The time constants of the exponential components describing the distributions are found to be independent of adjacent interval duration, and hence, previous channel activity. In contrast, the areas of the different components can change. Since the observed time constants are a function of the underlying rate constants for transitions among the kinetic states, the observation of time constants independent of previous channel activity suggests that the rate constants are also independent of previous channel activity. Thus, the channel kinetics are consistent with Markov gating. An observed dependent (inverse) relationship between durations of adjacent open and shut intervals together with Markov gating indicates that there are two or more independent transition pathways connecting open and shut states. Finally, no evidence is found to suggest that gating is not at thermodynamic equilibrium: the inverse relationship was independent of the time direction of analysis.
Dependence of center radius on temporal frequency for the receptive fields of X retinal ganglion cells of cat.
Authors: Troy J B , Enroth-Cugell C
Published: May 2004

We examined the dependence of the center radius of X cells on temporal frequency and found that at temporal frequencies above 40 Hz the radius increases in a monotonic fashion, reaching a size approximately 30% larger at 70 Hz. This kind of spatial expansion has been predicted with cable models of receptive fields where inductive elements are included in modeling the neuronal membranes. Hence, the expansion of the center radius is clearly important for modeling X cell receptive fields. On the other hand, we feel that it might be of only minor functional significance, since the responsivity of X cells is attenuated at these high temporal frequencies and the signal-to-noise ratio is considerably worse than at low and midrange temporal frequencies.
Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons.
Authors: Müller T H , Swandulla D , Lux H D
Published: May 2004

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.
Calcium-independent cell volume regulation in human lymphocytes. Inhibition by charybdotoxin.
Authors: Grinstein S , Smith J D
Published: May 2004

The properties of the K+ pathway underlying regulatory volume decrease (RVD) in human blood lymphocytes were investigated. Evidence is presented for the existence of three types of K+ conductance in these cells. Ionomycin, a Ca2+ ionophore, induced a K(+)-dependent hyperpolarization, indicating the presence of Ca2(+)-activated K+ channels, which were blocked by charybdotoxin (CTX). CTX also induced a depolarization of the resting membrane potential, even at subphysiological cytosolic [Ca2+]([Ca2+]i), which suggests the existence of a second CTX-sensitive, but Ca2(+)-independent conductance. A CTX-resistant K+ conductance was also detected. RVD in blood lymphocytes was partially (approximately 75%) blocked by CTX. However, volume regulation was not accompanied by detectable changes in [Ca2+]i, nor was it prevented by removal of extracellular Ca2+ and depletion or buffering of intracellular Ca2+. These observations suggest that K+ loss during RVD is mediated by Ca2(+)-independent, CTX-sensitive channels or that Ca2(+)-dependent channels can be activated by cell swelling at normal or subnormal [Ca2+]i. The former interpretation is supported by findings in rat thymic lymphocytes. These cells also displayed a CTX-sensitive Ca2(+)-dependent hyperpolarization. However, CTX did not significantly alter the resting potential, suggesting the absence of functional Ca2(+)-independent, toxin-sensitive channels. Volume regulation in thymic lymphocytes was less efficient than in human blood cells. In contrast to blood lymphocytes, RVD in thymocytes was not affected by CTX. These observations indicate that, though present in lymphocytes, Ca2(+)-activated K+ channels do not play an important role in volume regulation. Instead, RVD seems to be mediated by Ca2(+)-independent K+ channels. We propose that two types of channels, one CTX sensitive and the other CTX insensitive, mediate RVD in human blood lymphocytes, whereas only the latter type is involved in rat thymocytes.
G protein control of potassium channel activity in a mast cell line.
Authors: McCloskey M A , Cahalan M D
Published: May 2004

Using the patch-clamp technique, we studied regulation of potassium channels by G protein activators in the histamine-secreting rat basophilic leukemia (RBL-2H3) cell line. These cells normally express inward rectifier K+ channels, with a macroscopic whole-cell conductance in normal Ringer ranging from 1 to 16 nS/cell. This conductance is stabilized by including ATP or GTP in the pipette solution. Intracellular dialysis with any of three different activators of G proteins (GTP gamma S, GppNHp, or AlF-4) completely inhibited the inward rectifier K+ conductance with a half-time for decline averaging approximately 300 s after "break-in" to achieve whole-cell recording. In addition, with a half-time averaging approximately 200 s, G protein activators induced the appearance of a novel time-independent outwardly rectifying K+ conductance, which reached a maximum of 1-14 nS. The induced K+ channels are distinct from inward rectifier channels, having a smaller single-channel conductance of approximately 8 pS in symmetrical 160 mM K+, and being more sensitive to block by quinidine, but less sensitive to block by Ba2+. The induced K+ channels were also highly permeable to Rb+ but not to Na+ or Cs+. The current was not activated by the second messengers Ca2+, inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, or by cyclic AMP-dependent phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 12-13 h) prevented this current's induction both by guanine nucleotides and aluminum fluoride, but had no effect on the decrease in inward rectifier conductance. Since GTP gamma S is known to stimulate secretion from patch-clamped rat peritoneal mast cells, it is conceivable that K+ channels become inserted into the plasma membrane from secretory granules. However, total membrane capacitance remained nearly constant during appearance of the K+ channels, suggesting that secretion induced by GTP gamma S was minimal. Furthermore, pertussis toxin had no effect on secretion triggered by antigen, and triggering of secretion before electrical recording failed to induce the outward K+ current. Finally, GTP gamma S activated the K+ channel in excised inside-out patches of membrane. We conclude that two different GTP-binding proteins differentially regulate two subsets of K+ channels, causing the inward rectifier to close and a novel K+ channel to open when activated.
Phosphorylation modulates potassium conductance and gating current of perfused giant axons of squid.
Authors: Augustine C K , Bezanilla F
Published: May 2004

The presence of internal Mg-ATP produced a number of changes in the K conductance of perfused giant axons of squid. For holding potentials between -40 and -50 mV, steady-state K conductance increased for depolarizations to potentials more positive than approximately -15 mV and decreased for smaller depolarizations. The voltage dependencies of both steady-state activation and inactivation also appears shifted toward more positive potentials. Gating kinetics were affected by internal ATP, with the activation time constant slowed and the characteristic delay in K conductance markedly enhanced. The rate of deactivation also was hastened during perfusion with ATP. Internal ATP affected potassium channel gating currents in similar ways. The voltage dependence of gating charge movement was shifted toward more positive potentials and the time constants of ON and OFF gating current also were slowed and hastened, respectively, in the presence of ATP. These effects of ATP on the K conductance occurred when no exogenous protein kinases were added to the internal solution and persisted even after removing ATP from the internal perfusate. Perfusion with a solution containing exogenous alkaline phosphatase reversed the effects of ATP. These results provide further evidence that the effects of ATP on the K conductance are a consequence of a phosphorylation reaction mediated by a kinase present and active in perfused axons. Phosphorylation appears to alter the K conductance of squid giant axons via a minimum of two mechanisms. First, the voltage dependence of gating parameters are shifted toward positive potentials. Second, there is an increase in the number of functional closed states and/or a decrease in the rates of transition between these states of the K channels.