bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.
Authors: MacKinnon R , Miller C
Published: May 2004

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.
The interaction of intracellular Mg2+ and pH on Cl- fluxes associated with intracellular pH regulation in barnacle muscle fibers.
Authors: Russell J M , Brodwick M S
Published: May 2004

The intracellular dialysis technique was used to measure unidirectional Cl- fluxes and net acid extrusion by single muscle fibers from the giant barnacle. Decreasing pHi below normal levels of 7.35 stimulated both Cl- efflux and influx. These increases of Cl- fluxes were blocked by disulfonic acid stilbene derivatives such as SITS and DIDS. The SITS-sensitive Cl- efflux was sharply dependent upon pHi, increasing approximately 20-fold as pHi was decreased from 7.35 to 6.7. Under conditions of normal intracellular Mg2+ concentration, the apparent pKa for the activation of Cl- efflux was 7.0. We found that raising [Mg2+]i, but not [Mg2+]o, had a pronounced inhibitory effect on both SITS-sensitive unidirectional Cl- fluxes as well as on SITS-sensitive net acid extrusion. Increasing [Mg2+]i shifted the apparent pKa of Cl- efflux to a more acid value without affecting the maximal flux that could be attained. This relation between pHi and [Mg2+]i on SITS-sensitive Cl- efflux is consistent with a competition between H ions and Mg ions. We conclude that the SITS-inhibitable Cl- fluxes are mediated by the pHi-regulatory transport mechanism and that changes of intracellular Mg2+ levels can modify the activity of the pHi regulator/anion transporter.
Functional pacemaking area in the early embryonic chick heart assessed by simultaneous multiple-site optical recording of spontaneous action potentials.
Authors: Kamino K , Komuro H , Sakai T , Hirota A
Published: May 2004

Pacemaking areas in the early embryonic chick hearts were quantitatively assessed using simultaneous multiple-site optical recordings of spontaneous action potentials. The measuring system with a 10- X 10- or a 12 X 12-element photodiode array had a spatial resolution of 15-30 microns. Spontaneous action potential-related optical signals were recorded simultaneously from multiple contiguous regions in the area in which the pacemaker site was located in seven- to nine-somite embryonic hearts stained with a voltage-sensitive merocyanine-rhodanine dye (NK 2761). In the seven- to early eight-somite embryonic hearts, the location of the pacemaking area is not uniquely determined, and as development proceeds to the nine-somite stage, the pacemaking area becomes confined to the left pre-atrial tissue. Analysis of the simultaneous multiple-site optical recordings showed that the pacemaking area was basically circular in shape in the later eight- to nine-somite embryonic hearts. An elliptical shape also was observed at the seven- to early eight-somite stages of development. The size of the pacemaking area was estimated to be approximately 1,200-3,000 micron2. We suggest that the pacemaking area is composed of approximately 60-150 cells, and that the pacemaking area remains at a relatively constant size throughout the seven- to nine-somite stages. It is thus proposed that a population of pacemaking cells, rather than a single cell, serves as a rhythm generator in the embryonic chick heart.
Characterization of the inward-rectifying potassium current in cat ventricular myocytes.
Authors: Harvey R D , Ten Eick R E
Published: May 2004

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.
Hydrogen ion modulation of Ca channel current in cardiac ventricular cells. Evidence for multiple mechanisms.
Authors: Krafte D S , Kass R S
Published: May 2004

We have investigated the effects of H ions on (L-type) Ca channel current in isolated ventricular cells. We find that the current amplitude is enhanced in solutions that are alkaline relative to pH 7.4 and reduced in solutions acidic to this pH. We measured pH0-induced shifts in channel gating and analyzed our results in terms of surface potential theory. The shifts are well described by changes in surface potential caused by the binding of H ions to negative charges on the cell surface. The theory predicts a pK of 5.8 for this binding. Gating shifts alone cannot explain all of our observations on modulation of current amplitude. Our results suggest that an additional mechanism contributes to modification of the current amplitude.
Interaction among different sensory units within a single fungiform papilla in the frog tongue.
Authors: Murayama N
Published: May 2004

The possible interaction among different sensory units in the frog tongue was studied using several single papillae dually innervated by the medial and lateral branches of the glossopharyngeal (IXth) nerve. The afferent activity in one branch exposed to NaCl stimulation of the papilla revealed marked inhibition after antidromic electrical stimulation (100 Hz, 30 s, and 3 V) of the other branch. The degree of inhibition depended on the number of sensory responses observed in the electrically stimulated branch as well as the nature of the stimulated sensory units. Statistical analysis suggested that antidromic activation of gustatory units conducting the responses to NaCl and quinine and slowly adapting mechanosensitive units produced a large antidromic inhibition amounting to 19-25%, but that of gustatory units conducting the responses to acetic acid and rapidly adapting mechanosensitive units gave rise to only a slight inhibition. To examine the differential effects of these sensory units in antidromic inhibition, antidromic impulses were evoked by chemical stimulation of the adjacent papilla neuronally connected with the dually innervated papilla under study. Antidromic volleys of impulses elicited by NaCl or quinine stimulation produced a large inhibition of the afferent activity in the other branch, as induced by NaCl stimulation of the dually innervated papilla. Plausible mechanisms of synaptic interaction in peripheral gustatory systems are considered.
Inactivation of excitation-contraction coupling in rat extensor digitorum longus and soleus muscles.
Authors: Chua M , Dulhunty A F
Published: May 2004

K contractures and two-microelectrode voltage-clamp techniques were used to measure inactivation of excitation-contraction coupling in small bundles of fibers from rat extensor digitorum longus (e.d.l.) and soleus muscles at 21 degrees C. The rate of spontaneous relaxation was faster in e.d.l. fibers: the time for 120 mM K contractures to decay to 50% of maximum tension was 9.8 +/- 0.5 s (mean +/- SEM) in e.d.l. and 16.8 +/- 1.7 s in soleus. The rate of decay depended on membrane potential: in e.d.l., the 50% decay time was 14.3 +/- 0.7 s for contractures in 80 mM K (Vm = 25 mV) and 4.9 +/- 0.4 s in 160 mM K (Vm = -3 mV). In contrast to activation, which occurred with less depolarization in soleus fibers, steady state inactivation required more depolarization: after 3 min at -40 mV in 40 mM K, the 200 mM K contracture amplitude in e.d.l. fell to 28 +/- 10% (n = 5) of control, but remained at 85 +/- 2% (n = 6) of control in soleus. These different inactivation properties in e.d.l. and soleus fibers were not influenced by the fact that the 200 mM K solution used to test for steady state inactivation produced contractures that were maximal in soleus fibers but submaximal in e.d.l.: a relatively similar depression was recorded in maximal (200 mM K) and submaximal (60 and 80 mM K) contracture tension. A steady state "pedestal" of tension was observed with maintained depolarization after K contracture relaxation and was larger in soleus than in e.d.l. fibers. The pedestal tension was attributed to the overlap between the activation and inactivation curves for tension vs. membrane potential, which was greater in soleus than in e.d.l. fibers. The K contracture results were confirmed with the two-microelectrode voltage clamp: the contraction threshold increased to more positive potentials at holding potentials of -50 mV in e.d.l. or -40 mV in soleus. At holding potentials of -30 mV in e.d.l. or 0 mV in soleus, contraction could not be evoked by 15-ms pulses to +20 mV. Both K contracture and voltage-clamp experiments revealed that activation in soleus fibers occurred with a smaller transient depolarization and was maintained with greater steady state depolarization than in e.d.l. fibers. The K contracture and voltage-clamp results are described by a model in which contraction depends on the formation of a threshold concentration of activator from a voltage-sensitive molecule that can exist in the precursor, activator, or inactive states.
Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells.
Authors: Pappone P A , Lucero M T
Published: May 2004

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.
Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum.
Authors: Smith J S , Imagawa T , Ma J , Fill M , Campbell K P , Coronado R
Published: May 2004

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified as a single 450,000-dalton polypeptide from CHAPS-solubilized triads using immunoaffinity chromatography. The purified receptor had a [3H]ryanodine-binding capacity (Bmax) of 490 pmol/mg and a binding affinity (Kd) of 7.0 nM. Using planar bilayer recording techniques, we show that the purified receptor forms cationic channels selective for divalent ions. Ryanodine receptor channels were identical to the Ca-release channels described in native sarcoplasmic reticulum using the same techniques. In the present work, four criteria were used to establish this identity: (a) activation of channels by micromolar Ca and millimolar ATP and inhibition by micromolar ruthenium red, (b) a main channel conductance of 110 +/- 10 pS in 54 mM trans Ca, (c) a long-term open state of lower unitary conductance induced by ryanodine concentrations as low as 20 nM, and (d) a permeability ratio PCa/PTris approximately equal to 14. In addition, we show that the purified ryanodine receptor channel displays a saturable conductance in both monovalent and divalent cation solutions (gamma max for K and Ca = 1 nS and 172 pS, respectively). In the absence of Ca, channels had a broad selectivity for monovalent cations, but in the presence of Ca, they were selectively permeable to Ca against K by a permeability ratio PCa/PK approximately equal to 6. Receptor channels displayed several equivalent conductance levels, which suggest an oligomeric pore structure. We conclude that the 450,000-dalton polypeptide ryanodine receptor is the Ca-release channel of the sarcoplasmic reticulum and is the target site of ruthenium red and ryanodine.
Structural states in the Z band of skeletal muscle correlate with states of active and passive tension.
Authors: Goldstein M A , Michael L H , Schroeter J P , Sass R L
Published: May 2004

In skeletal muscle Z bands, the ends of the thin contractile filaments interdigitate in a tetragonal array of axial filaments held together by periodically cross-connecting Z filaments. Changes in these two sets of filaments are responsible for two distinct structural states observed in cross section, the small-square and basketweave forms. We have examined Z bands and A bands in relaxed, tetanized, stretched, and stretched and tetanized rat soleus muscles by electron microscopy and optical diffraction. In relaxed muscle, the A-band spacing decreases with increasing load and sarcomere length, but the Z lattice remains in the small-square form and the Z spacing changes only slightly. In tetanized muscle at sarcomere lengths up to 2.7 micron, the Z lattice assumes the basketweave form and the Z spacing is increased. The increased Z spacing is not the result of sarcomere shortening. Further, passive tension is not sufficient to cause this change in the Z lattice; active tension is necessary.
Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction.
Authors: Mierson S , DeSimone S K , Heck G L , DeSimone J A
Published: May 2004

There is good evidence indicating that ion-transport pathways in the apical regions of lingual epithelial cells, including taste bud cells, may play a role in salt taste reception. In this article, we present evidence that, in the case of the dog, there also exists a sugar-activated ion-transport pathway that is linked to sugar taste transduction. Evidence was drawn from two parallel lines of experiments: (a) ion-transport studies on the isolated canine lingual epithelium, and (b) recordings from the canine chorda tympani. The results in vitro showed that both mono- and disaccharides in the mucosal bath stimulate a dose-dependent increase in the short-circuit current over the concentration range coincident with mammalian sugar taste responses. Transepithelial current evoked by glucose, fructose, or sucrose in either 30 mM NaCl or in Krebs-Henseleit buffer (K-H) was partially blocked by amiloride. Among current carriers activated by saccharides, the current response was greater with Na than with K. Ion flux measurements in K-H during stimulation with 3-O-methylglucose showed that the sugar-evoked current was due to an increase in the Na influx. Ouabain or amiloride reduced the sugar-evoked Na influx without effect on sugar transport as measured with tritiated 3-O-methylglucose. Amiloride inhibited the canine chorda tympani response to 0.5 M NaCl by 70-80% and the response to 0.5 M KCl by approximately 40%. This agreed with the percent inhibition by amiloride of the short-circuit current supported in vitro by NaCl and KCl. Amiloride also partially inhibited the chorda tympani responses to sucrose and to fructose. The results indicate that in the dog: (a) the ion transporter subserving Na taste also subserves part of the response to K, and (b) a sugar-activated, Na-preferring ion-transport system is one mechanism mediating sugar taste transduction. Results in the literature indicate a similar sweet taste mechanism for humans.
Muscarinic activation of ionic currents measured by a new whole-cell recording method.
Authors: Horn R , Marty A
Published: May 2004

A new method is described as an alternative to whole-cell recording in order to prevent "wash-out" of the muscarinic response to acetylcholine (ACh) in rat lacrimal gland cells. The membrane of a cell-attached patch is permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without the loss or alteration of cytoplasmic compounds necessary for the maintenance of the response to ACh. With normal whole-cell recording in these cells, the response to ACh, seen as the activation of Ca-activated K and Cl currents, lasts for approximately 5 min. With the nystatin method, the response is not diminished after 1 h. Nystatin, applied extracellularly, is shown to cause a rapid and reversible increase of membrane conductance to cations. In the absence of wash-out, we were able to obtain dose-response curves for the effect of ACh on Ca-activated K currents. An increase of [ACh] caused an increase in the K current, with apparent saturation at concentrations above approximately 1 microM ACh. The delay between ACh application and the activation of K current was inversely related to [ACh] and reached a minimum value of 0.7-1.0 s at high [ACh].
Modification of a carboxyl group that appears to cross the permeability barrier in the red blood cell anion transporter.
Authors: Jennings M L , Al-Rhaiyel S
Published: May 2004

A recently developed method for converting protein carboxyl groups to alcohols has been used to examine the functional role of carboxyl groups in the red blood cell inorganic anion-transport protein (band 3). A major goal of the work was to investigate the carboxyl group that is protonated during the proton-sulfate cotransport that takes place during net chloride-sulfate exchange. Three kinds of evidence indicate that the chemical modification (Woodward's reagent K followed by borohydride) converts this carboxyl to an alcohol. First, monovalent anion exchange is inhibited irreversibly. Second, the modification stimulates sulfate influx into chloride-loaded cells and nearly eliminates the extracellular pH dependence of the sulfate influx. (The stimulated sulfate influx in the modified cells is inhibitable by stilbenedisulfonate.) Third, the proton influx normally associated with chloride-sulfate exchange is inhibited by the modification. These results would all be expected if the titratable carboxyl group were converted into the untitratable, neutral alcohol. In addition to altering the extracellular pH dependence of sulfate influx, the chemical modification removes the intracellular pH dependence of sulfate efflux. The modification is performed under conditions in which the reagent does not cross the permeability barrier. The large effect on the intracellular pH dependence of sulfate transport suggests that a single carboxyl group can at different times be in contact with the aqueous medium on each side of the permeability barrier.
Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger.
Authors: Green J , Yamaguchi D T , Kleeman C R , Muallem S
Published: May 2004

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.
Digitonin-permeabilized colonic cell layers. Demonstration of calcium-activated basolateral K+ and Cl- conductances.
Authors: Chang D , Dawson D C
Published: May 2004

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.
Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions.
Authors: Nakhoul N L , Lopes A G , Chaillet J R , Boron W F
Published: May 2004

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.
Acetate transport in the S3 segment of the rabbit proximal tubule and its effect on intracellular pH.
Authors: Nakhoul N L , Boron W F
Published: May 2004

We monitored intracellular pH (pHi) in isolated perfused S3 segments of the rabbit proximal tubule, and studied the effect of acetate (Ac-) transport on pHi. pHi was calculated from the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein trapped intracellularly. All solutions were nominally HCO3(-)-free. Removal of 10 mM Ac- from bath and lumen caused pHi to rapidly rise by approximately 0.2, and then to decline more slowly to a value approximately 0.35 below the initial one. Removal of only luminal Ac- caused pHi changes very similar to those resulting from bilateral removal of Ac-. When Ac- was removed from bath only, pHi rose rapidly at first, and then continued to rise more slowly. Readdition of Ac- to bath caused pHi to rapidly fall to a value slightly higher than the one prevailing before the removal of Ac- from the bath. In experiments in which Ac- was first removed from both bath and lumen, readdition of 10 mM Ac- to only lumen caused a rapid but small acidification, followed by a slower alkalinization that brought the pHi near the value that prevailed before the bilateral removal of Ac-. The alkalinizing effects caused by the readdition of 10 or 0.5 mM Ac- were indistinguishable. When Ac- was returned to only lumen in the absence of luminal Na+, there was a small and rapid pHi decrease, but no pHi recovery. Removal of Na+ from bath did not affect the pHi transients caused by the addition of Ac- to lumen. In experiments in which Ac- was first removed bilaterally, readdition of Ac- to only bath caused a large and sustained drop in pHi, whereas the subsequent removal of Ac- from the bath caused a slight alkalinization. These pHi changes caused by readdition or removal of Ac- from baths were unaffected by the absence of external Na+. We conclude that there is a Na+/Ac- cotransporter at the luminal membrane, and pathways for acetic acid transport at both luminal and basolateral membranes. The net effect of Ac- transport on pHi is to alkalinize the cell as a result of the luminal entry of Na+/Ac-, which is followed by the luminal and basolateral exit of acetic acid.
Sodium channel subconductance levels measured with a new variance-mean analysis.
Authors: Patlak J B
Published: May 2004

The currents through single Na+ channels were recorded from dissociated cells of the flexor digitorum brevis muscle of the mouse. At 15 degrees C the prolonged bursts of Na+ channel openings produced by application of the drug DPI 201-106 had brief sojourns to subconductance levels. The subconductance events were relatively rare and brief, but could be identified using a new technique that sorts amplitude estimates based on their variance. The resulting "levels histogram" had a resolution of the conductance levels during channel activity that was superior to that of standard amplitude histograms. Cooling the preparation to 0 degrees C prolonged the subconductance events, and permitted further quantitative analysis of their amplitudes, as well as clear observations of single-channel subconductance events from untreated Na+ channels. In all cases the results were similar: a subconductance level, with an amplitude of roughly 35% of the fully open conductance and similar reversal potential, was present in both drug-treated and normal Na+ channels. Drug-treated channels spent approximately 3-6% of their total open time in the subconductance state over a range of potentials that caused the open probability to vary between 0.1 and 0.9. The summed levels histograms from many channels had a distinctive form, with broader, asymmetrical open and substate distributions compared with those of the closed state. Individual subconductance events to levels other than the most common 35% were also observed. I conclude that subconductance events are a normal subset of the open state of Na+ channels, whether or not they are drug treated. The subconductance events may represent a conformational alteration of the channel that occurs when it conducts ions.
Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers.
Authors: Cukierman S , Zinkand W C , French R J , Krueger B K
Published: May 2004

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.
Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism.
Authors: Shoemaker D G , Bender C A , Gunn R B
Published: May 2004

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0-1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene-2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)
pH homeostasis in promyelocytic leukemic HL60 cells.
Authors: Restrepo D , Kozody D J , Spinelli L J , Knauf P A
Published: May 2004

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.
Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells.
Authors: Kalman D , O'Lague P H , Erxleben C , Armstrong D L
Published: May 2004

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.
Dependency on light and vitamin A derivatives of the biogenesis of 3-hydroxyretinal and visual pigment in the compound eyes of Drosophila melanogaster.
Authors: Isono K , Tanimura T , Oda Y , Tsukahara Y
Published: May 2004

When the fruitfly, Drosophila melanogaster, was reared on media deficient in carotenoids and retinoids, the level of 3-hydroxyretinal (the chromophore of fly rhodopsin) in the retina decreased to less than 1% compared with normal flies. The level of 3-hydroxyretinal increased markedly in flies that were given a diet supplemented with retinoids or carotenoids. The retinas of flies fed on all-trans retinoids and maintained in the dark predominantly contained the all-trans form of 3-hydroxyretinal, and showed no increase in the level of either the 11-cis isomer or the visual pigment. Subsequent illumination of the flies converted substantial amounts of all-trans 3-hydroxyretinal to its 11-cis isomer. The action spectrum of the conversion by illumination showed the optimum wavelength to be approximately 420 nm, which is significantly greater than the absorption maximum of free, all-trans 3-hydroxyretinal. Flies that were fed on carotenoids showed a rapid increase of the levels of 11-cis 3-hydroxyretinal and of visual pigment in the absence of light.
In vivo environmental temperature and the in vitro pattern of luminal acidification in turtle bladders. Evidence for HCO3 ion reabsorption.
Authors: Schilb T P , Durham J H , Brodsky W A
Published: May 2004

In this study, it is shown how to transfer tared aliquots of (HCO3 + CO2)-containing luminal fluids directly into the mercury-sealed chamber of a modified Van Slyke apparatus and how to obtain direct as well as indirect manometric determinations of dissolved CO2 ([CO2]f) in each aliquot of such fluids. It is next shown that the pattern of in vitro luminal acidification in an isolated turtle bladder sac depends upon the prior in vivo ambient temperature to which the donor turtle had become adapted. Under in vivo conditions, the food intake, physical activity, and acid excretion of 32 degrees C-adapted turtles are greater than those of 21 degrees C or 26 degrees C-adapted turtles. Under in vitro conditions of incubating isolated bladder sacs (from 21, 26, and 32 degrees C turtles) in (HCO3 + CO2)-containing Ringer media at a single temperature (21 degrees C), the patterns of luminal acidification are as follows: (a) The rate of depletion of luminal [HCO3] is greatest in bladders from the 32 degrees C-adapted turtles. (b) Concomitant decreases in luminal [CO2]f, [HCO3], and pH (the 'CO2-decreasing patterns' of luminal acidification) develop in all bladders from 32 degrees C turtles, in half of those from 26 degrees C turtles, but in less than one-fifth of those from 21 degrees C-adapted turtles: and (c) a CO2-increasing pattern of luminal acidification is found in most of the bladders from 21 degrees C-adapted turtles. A postulated bicarbonate ion-reabsorbing pump is consistent with all of these patterns of luminal acidification.
Volume-sensitive K influx in human red cell ghosts.
Authors: Sachs J R
Published: May 2004

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.
Cytoplasmic free calcium, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle.
Authors: Himpens B , Matthijs G , Somlyo A V , Butler T M , Somlyo A P
Published: May 2004

The time course of [Ca2+]i, tension, and myosin light chain phosphorylation were determined during prolonged depolarization with high K+ in intact tonic (rabbit pulmonary artery) and phasic (longitudinal layer of guinea pig ileum) smooth muscles. [Ca2+]i was monitored with the 340 nm/380 nm signal ratio of the fluorescent indicator fura-2. The fluorescence ratio had a similar time course in both muscle types during depolarization with 109 mM [K+]o; after a transient peak, there was a decline to 70% of its peak value in tonic smooth muscle, and to 60% in phasic smooth muscle. Tension, however, continued to increase in the pulmonary artery, while in the ileum it declined in parallel with the [Ca2+]i. On changing [K+]o from 109 to 20 mM, tension and [Ca2+]i either remained unchanged or declined in parallel in the pulmonary artery. Phosphorylation of the 20-kD myosin light chain, measured during stimulation of muscle strips with 109 mM [K+]o in another set of experiments, increased from 3% to a peak of 50% in the intact pulmonary artery, and then declined to a steady state value of 23%. In the intact ileum, a very rapid, early transient phosphorylation (up to 50%) at 2-3 s was seen. This transient declined by 30 s to a value that was close to the resting level (7%), while tension remained at 55% of its peak force. A quick release during maintained stimulation induced no detectable change in the [Ca2+]i in either type of smooth muscle. We discuss the possibility that the slowly rising tonic tension in pulmonary artery could be due to cooperativity between phosphorylated and nonphosphorylated crossbridges.
Light-induced losses and dark recovery rates of guanosine 3',5'-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors.
Authors: Cohen A I , Blazynski C
Published: May 2004

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiens, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset. With dim subsecond illuminations these losses were surprisingly large, which suggests a previously underestimated magnification in the cGMP cascade, or a transient substantial inhibition of guanylate cyclase activity in combination with increased cyclic GMP phosphodiesterase activity. Within the subsecond period, significant losses that were proportional to light intensity (2-log-unit range) and duration (60-550 ms) were generally not evident. However, losses significantly proportional to these factors became evident with durations of 1 s or longer. When pieces of retina were first illuminated (10 or 60 ms), then held in darkness for increasing periods before freezing, we observed a continuous loss of cGMP during the early postillumination dark period, followed by a recovery of the total cGMP level. The times for recovery to the preillumination level appear to be significantly longer than times reported for the recovery of the photoreceptor membrane potential after similar light exposures.