bluuebunny/crossref_metadata_2025 huggingface dataset:
Origins of open-channel noise in the large potassium channel of sarcoplasmic reticulum.
Authors: Hainsworth A H , Levis R A , Eisenberg R S
Published: May 2004

Open-channel noise was studied in the large potassium channel of the sarcoplasmic reticulum (SR). Inside-out patches were excised directly from the SR of split skeletal muscle fibers of lobster, with lobster relaxing ringer (LRR) in bath and pipette. The power spectrum of open-channel noise is very low and approximately flat in the 100 Hz-10 kHz frequency range. At 20 degrees C, with an applied voltage of 50 mV, the mean single-channel current (i) is 9 pA (mean single-channel conductance = 180 pS) and the mean power spectral density 1.1 x 10(-29) A2/Hz. The latter increases nonlinearly with (i), showing a progressively steeper dependence as (i) increases. At 20 mV, the mean power spectral density is almost independent of (i) and approximately 1.4 times that of the Johnson noise calculated for the equivalent ideal resistor with zero net current; at 70 mV it increases approximately in proportion to (i)2. The mean power spectral density has a weak temperature dependence, very similar to that of (i), and both are well described by a Q10 of 1.3 throughout the range 3-40 degrees C. Discrete ion transport events are thought to account for a significant fraction of the measured open-channel noise, probably approximately 30-50% at 50 mV. Brief interruptions of the single-channel current, due either to blockage of the open channel by an extrinsic aqueous species, or to intrinsic conformational changes in the channel molecule itself, were a possible additional source of open-channel noise. Experiments in modified bathing solutions indicate, however, that open-channel noise is not affected by any of the identified aqueous species present in LRR. In particular, magnesium ions, the species thought most likely to cause brief blockages, and calcium and hydrogen ions, have no detectable effect. This channel's openings exhibit many brief closings and substrates, due to intrinsic gating of the channel. Unresolved brief full closings are calculated to make a negligible contribution (< 1%) to the measured power spectral density. The only significant source of noise due to band width-limited missed events is brief, frequent 80% substrates (mean duration 20 microseconds, mean frequency 1,000 s-1) which account for a small part of the measured power spectral density (approximately 14%, at 50 mV, 20 degrees C). We conclude that a large fraction of the measured open-channel noise results from intrinsic conductance fluctuations, with a corner frequency higher than the resolution of our recordings, in the range 10(4)-10(7) Hz.(ABSTRACT TRUNCATED AT 400 WORDS)
Differences in calcium homeostasis between retinal rod and cone photoreceptors revealed by the effects of voltage on the cGMP-gated conductance in intact cells.
Authors: Miller J L , Korenbrot J I
Published: May 2004

We measured currents under voltage clamp in intact retinal rod photoreceptors with tight seal electrodes in the perforated patch mode. In the dark, membrane depolarization to voltages > or = +20 mV activates a time- and voltage-dependent outward current in the outer segment. This dark voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course that is voltage dependent. DVAC reaches its maximum enhancement of approximately 30% in 4-6 s at +60 mV. DVAC is entirely suppressed by light and its current-voltage curve and reversal potential are the same as those of the photocurrent. Therefore, DVAC arises from the opening in darkness of the cGMP-gated channels of the outer segment. DVAC is blocked by BAPTA loaded into the cell's cytoplasm and is enhanced by lowering extracellular Ca2+ concentration. Because the cGMP-gated channels are not directly gated by voltage and because BAPTA blocks DVAC, we suggest this signal arises from a voltage-dependent decrease in cytoplasmic Ca2+ concentration that, in turn, activates guanylyl cyclase and causes cGMP synthesis. In rods loaded with high cytoplasmic Na+, membrane depolarization in darkness to voltages > or = +20 mV inactivates the outward current in the outer segment with an exponential time course. We call this DVIC (dark, voltage-inactivated current). DVIC reflects voltage-dependent closing of the cGMP-gated channel in the dark. DVIC, too, is blocked by cytoplasmic BAPTA, and it arises from a voltage-dependent rise in cytoplasmic Ca2+ in darkness, which occurs only if cytoplasmic Na is high. We develop a quantitative model to calculate the rate and extent of the voltage-dependent change in cytoplasmic Ca2+ concentration in a normal rod. We assume that this concentration is controlled by the balance between Ca2+ influx through the cGMP-gated channels and its efflux through a Na+/Ca2+, K+ exchanger. Lowered cytoplasmic Ca2+ is linked to guanylyl cyclase activation with characteristics determined from biochemical studies. The model considers the cytoplasmic buffering of both Ca2+ and cGMP. Simulated data generated by the model fit well DVAC measured in rods and also DVAC previously measured in cones. DVAC in cones is larger in magnitude and faster in time course than that in rods. The successful fit of DVAC by the model leads us to suggest that the activity and Ca2+ dependence of the enzymes of transduction are not different in rods and cones, but the quantitative features of Ca2+ homeostasis in the outer segment of the two receptor types differ profoundly.(ABSTRACT TRUNCATED AT 400 WORDS)
Differential effects of pertussis toxin on the muscarinic regulation of Ca2+ and K+ currents in frog cardiac myocytes.
Authors: Li Y , Hanf R , Otero A S , Fischmeister R , Szabo G
Published: May 2004

The ability of acetylcholine (ACh) to inhibit beta-agonist stimulated calcium current was compared to its ability to activate the inwardly rectifying potassium current IK(ACh) in frog atrial myocytes. As suggested by previous studies, ACh inhibited the calcium current at concentrations (EC50 = 8 nM) significantly lower than those required for the activation of IK(ACh) (EC50 = 101 nM). The pharmacological profiles of the two responses suggest that despite the differences in agonist sensitivity, both are mediated by the same (m2) type of muscarinic receptors. Intracellular application of GDP beta S, an inhibitor of G protein function, completely abolished both responses, implying that both actions of ACh are coupled to effectors by G proteins. In contrast, intracellular application of pertussis toxin (PTX) shifted to higher concentrations (EC50 = 170 nM) but did not abolish inhibition of the calcium current by ACh even though the block of the IK(ACh) response was complete. Increasingly large PTX concentrations and/or prolonged PTX treatments revealed a limiting, PTX-resistant inhibitory component that appears to be mediated by a PTX-insensitive G protein distinct from that mediating IK(ACh). For the PTX-sensitive components, the different agonist dependencies of IK(ACh) activation and calcium current inhibition may imply that different G proteins mediate each response although alternate possibilities involving the same G protein either functionally sequestered and/or differentially affected by interactions with effectors, can not be ruled out.
Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.
Authors: Freedman J C , Novak T S , Bisognano J D , Pratap P R
Published: May 2004

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.
A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina.
Authors: Malchow R P , Qian H , Ripps H
Published: May 2004

The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage-dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein-dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels.
Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta cells.
Authors: Tsuura Y , Ishida H , Hayashi S , Sakamoto K , Horie M , Seino Y
Published: May 2004

Nitric oxide (NO) is known to be a potent messenger in the intracellular signal transduction system in many tissues. In pancreatic beta cells, NO has been reported to be formed from L-arginine through NO synthase. To elucidate the effect of NO on insulin secretion and to investigate the intracellular mechanism of its effect, we have used sodium nitroprusside (SNP) as a NO donor. SNP inhibited glucose-induced insulin secretion in a dose-dependent manner, and its effect was reversed by hemoglobin, a known NO scavenger. However, glyceraldehyde-induced insulin secretion was not affected by SNP. Since the closure of ATP-sensitive K+ channels (KATP channel) has been established as a key step in glucose-induced insulin secretion, we have directly assessed the effect of SNP on KATP channel activity using the patch clamp technique. The KATP channel activity reduced by glucose was found to be reversibly activated by the addition of SNP, and this activation was able to be similarly reproduced by applying S-Nitroso-N-acetyl-DL-penicillamine (SNAP), another NO generator. Furthermore, these activating effects were completely eliminated by hemoglobin, in accordance with the reversibility in inhibition of glucose-induced insulin release. However, SNP could not affect the KATP channel suppression by ATP applied to the inside of the plasma membrane. The activation of the KATP channel by NO, therefore, seems to be due to the decreased ATP production attributable to impairment of glucose metabolism in beta cells. Since SNP exhibited no effect on glyceraldehyde-induced KATP channel inhibition, NO may disturb a glycolytic step before glyceraldehyde-3-phosphate. The KATP channel activation by 2-deoxyglucose through presumable ATP consumption due to its phosphorylation by glucokinase was, however, not affected even in the presence of SNP. But in the permeabilized beta cells made by exposure to a low concentration (0.02 U/ml) of streptolysin O (open cell-attached configuration), SNP reopens KATP channels which have been eliminated by fructose-6-phosphate, while this effect was not observed in the KATP channels inhibited by fructose-1,6-bisphosphate. On the other hand, in rat ventricular myocyte KATP channels were not activated by SNP even under a low concentration of glucose. From these observations, the inhibition of phosphofructokinase activity is probably the site responsible for the impairment of glucose metabolism induced by NO in pancreatic beta cells. NO, therefore, seems to be a factor in the deterioration of glucose-induced insulin secretion from pancreatic beta cells through a unique intracellular mechanism.
Swelling-activated chloride channels in multidrug-sensitive and -resistant cells.
Authors: Ehring G R , Osipchuk Y V , Cahalan M D
Published: May 2004

Resistance to chemotherapeutic agents in neoplastic cells is often mediated by expression of P-glycoprotein, which functions as a drug-efflux pump for a broad range of substrates. We have used a combination of patch clamp and video-imaging techniques to examine the expression and drug-efflux function of P-glycoprotein and to determine the possible correlation with swelling-activated chloride channels in drug-sensitive and -resistant cell lines. Two pairs of cell lines were used in these experiments: (a) control NIH-3T3 cells and a corresponding MDR1-transfectant; and (b) control 8226 myeloma cells and a derivative cell line selected for resistance to chemotherapeutic agents. Control cells lacked detectable P-glycoprotein expression based on Western blotting, immunofluorescence staining with a specific monoclonal antibody, and a functional assay of rhodamine-123 (R123) efflux. Resistant cells expressed P-glycoprotein at high levels and rapidly exported R123. During whole-cell recording using either hyperosmotic pipette solution or hypoosmotic Ringer solution, cell swelling was accompanied by Cl- channel opening in all four cell lines. The rates of induction, biophysical properties and magnitudes of Cl conductance (gCl) were indistinguishable between control and corresponding multidrug-resistant cells: gCl reached 0.96 +/- 0.31 (n = 14) and 0.83 +/- 0.31 nS/pF (mean +/- SD; n = 31) in NIH-3T3 and NIH-3T3/MDR cells, respectively; and 0.31 +/- 0.20 (n = 9) and 0.37 +/- 0.22 nS/pF (n = 7) in 8226 and 8226/Dox40 cells, respectively. gCl exhibited moderate outward rectification in symmetrical Cl- solutions, with a rectification ratio of 1.4 at +/- 50 mV. Cl- channels slowly closed during strong depolarization beyond +60 mV. Using video-imaging techniques with SPQ as a fluorescent probe, we monitored Cl(-)-channel opening in intact drug-sensitive and -resistant cells. gCl, measured either with whole-cell recording or SPQ imaging, was blocked by DIDS (voltage-dependent Kd < 50 microM at +40 mV), NPPB (Kd approximately 30 microM), and tamoxifen (complete and irreversible block approximately 10 microM). None of these blockers inhibited R123 efflux. NPPB accelerated R123 efflux, an effect that was mimicked by CCP, a mitochondrial uncoupler. In contrast, verapamil selectively blocked R123 efflux (Kd = 0.3 to 0.5 microM); 10 microM left gCl unaltered. Induction of gCl was not affected by vincristine or doxorubicin in the pipette solution. Moreover, the rate of R123 efflux did not change during cell swelling. We conclude that P-glycoprotein and swelling-activated chloride channels function independently and are separable by expression and by pharmacological sensitivities.
Swelling-activated and isoprenaline-activated chloride currents in guinea pig cardiac myocytes have distinct electrophysiology and pharmacology.
Authors: Vandenberg J I , Yoshida A , Kirk K , Powell T
Published: May 2004

We have used the whole-cell patch clamp recording technique to characterize a swelling-activated chloride current in guinea pig atrial and ventricular myocytes and to compare the electrophysiological and pharmacological properties of this current with the isoprenaline-activated chloride current in the same cell types. Osmotic swelling of guinea pig cardiac myocytes caused activation of an outwardly rectifying, anion-selective current with a conductance and permeability sequence of I- approximately NO3- > Br- > Cl- > Asp-. This current was inhibited by tamoxifen, 4,4'-diisothiocyano-stilbene-2,2'-disulphonate and anthracene-9-carboxylic acid, in decreasing order of potency. The isoprenaline-activated anion current, like the swelling-activated current, had a higher permeability to I- relative to Cl-, but it had a markedly reduced conductance for I- compared to Cl-. The isoprenaline-activated current was insensitive to inhibition by tamoxifen, 4,4'-diisothiocyanostilbene-2,2'-disulphonate and anthracene-9-carboxylic acid. The swelling-activated current could be elicited in > 90% atrial myocytes studied but only 34% ventricular myocytes. Conversely, the isoprenaline-activated current was elicited in < 10% atrial myocytes and > 90% ventricular myocytes. In those ventricular myocytes where it was possible to elicit swelling-activated and isoprenaline-activated currents simultaneously, the currents retained the same distinguishing characteristics as when they were elicited in isolation. Thus, while guinea pig atrial cells appear to preferentially express swelling-activated chloride channels and guinea pig ventricular myocytes preferentially express isoprenaline-activated chloride channels, the presence of these two channel types are not necessarily mutually exclusive. This raises the possibility that there may be coordinated regulation of the expression of different Cl- channels within the heart.
Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes.
Authors: Jennings M L
Published: May 2004

One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na-gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.
The calcium-activated potassium channels of turtle hair cells.
Authors: Art J J , Wu Y C , Fettiplace R
Published: May 2004

A major factor determining the electrical resonant frequency of turtle cochlear hair cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987. Journal of Physiology. 385:207-242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of approximately 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single-channel current at -50 mV in 4 microM Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of approximately 2 microM at +50 mV and 12 microM at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 microM. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were approximately 5 times more Ca-sensitive than BK channels at -50 mV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.
Plasticity in canine airway smooth muscle.
Authors: Pratusevich V R , Seow C Y , Ford L E
Published: May 2004

The large volume changes of some hollow viscera require a greater length range for the smooth muscle of their walls than can be accommodated by a fixed array of sliding filaments. A possible explanation is that smooth muscles adapt to length changes by forming variable numbers of contractile units in series. To test for such plasticity we examined the muscle length dependence of shortening velocity and compliance, both of which will vary directly with the number of thick filaments in series. Dog tracheal smooth muscle was studied because its cells are arrayed in long, straight, parallel bundles that span the length of the preparation. In experiments where muscle length was changed, both compliance and velocity showed a strong dependence on muscle length, varying by 1.7-fold and 2.2-fold, respectively, over a threefold range of length. The variation in isometric force was substantially less, ranging from a 1.2- to 1.3-fold in two series of experiments where length was varied by twofold to an insignificant 4% variation in a third series where a threefold length range was studied. Tetanic force was below its steady level after both stretches and releases, and increased to a steady level with 5-6 tetani at 5 min intervals. These results suggest strongly that the number of contractile units in series varies directly with the adapted muscle length. Temporary force depression after a length change would occur if the change transiently moved the filaments from their optimum overlap. The relative length independence of the adapted force is explained by the reforming of the filament lattice to produce optimum force development, with commensurate changes of velocity and compliance.
Measurement of cytosolic Ca2+ concentration in Limulus ventral photoreceptors using fluorescent dyes.
Authors: Ukhanov K Y , Flores T M , Hsiao H S , Mohapatra P , Pitts C H , Payne R
Published: May 2004

Several Ca-sensitive fluorescent dyes (fura-2, mag-fura-2 and Calcium Green-5N) were used to measure intracellular calcium ion concentration, Cai, accompanying light-induced excitation of Limulus ventral nerve photoreceptors. A ratiometric procedure was developed for quantification of Calcium Green-5N fluorescence. A mixture of Calcium Green-5N and a Ca-insensitive dye, ANTS, was injected in the cell and the fluorescence intensities of both dyes were used to calculate the spatial average of Cai within the light-sensitive R lobe of the photoreceptor. In dark-adapted photoreceptors, the initial Cai was 0.40 +/- 0.22 microM (SD, n = 7) as measured with fura-2. Cai peaked in the light-sensitive R lobe at 700-900 ms after the onset of an intense measuring light step, when the spatial average of Cai within the R lobe reached 68 +/- 14 and 62 +/- 37 microM (SD, n = 5) as measured with mag-fura-2 and Calcium Green-5N, respectively. The rate of Cai rise was calculated to be approximately 350 microM/s under the measuring conditions. The resting level of Mg2+ was estimated to be 1.9 +/- 0.9 mM, calculated from mag-fura-2 measurements. To investigate the effect of adapting light on the initial Cai level in the R lobe, a 1-min step of 420 nm background light was applied before each measurement. The first significant (P < 0.05) change in the initial level of Cai occurred even at the lowest adapting light intensity, which delivered approximately 3 x 10(3) effective photons/s. The relative sensitivity of the light-adapted photoreceptors was linearly related to the relative Cai on a double log plot with slope between -4.3 and -5.3. We were unable to detect a Cai rise preceding the light-activated receptor potential. The Cai rise, measured with Calcium Green-5N, lagged 14 +/- 5 ms (SD, n = 32) behind the onset of the receptor potential at room temperature in normal ASW. In the absence of extracellular Ca2+ and at 10 degrees C, this lag increased to 44 +/- 12 ms (SD, n = 17).
Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.
Authors: McHugh J , Mok W M , Wang G K , Strichartz G
Published: May 2004

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.
A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes.
Authors: Lim N F , Dascal N , Labarca C , Davidson N , Lester H A
Published: May 2004

In many tissues, inwardly rectifying K channels are coupled to seven-helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.
The role of Mg2+ in the inactivation of inwardly rectifying K+ channels in aortic endothelial cells.
Authors: Elam T R , Lansman J B
Published: May 2004

We have studied the role of Mg2+ in the inactivation of inwardly rectifying K+ channels in vascular endothelial cells. Inactivation was largely eliminated in Mg(2+)-free external solutions and the extent of inactivation was increased by raising Mg2+o. The dose-response relation for the reduction of channel open probability showed that Mg2+o binds to a site (KD = approximately 25 microM at -160 mV) that senses approximately 38% of the potential drop from the external membrane surface. Analysis of the single-channel kinetics showed that Mg2+ produced a class of long-lived closures that separated bursts of openings. Raising Mg2+o reduced the burst duration, but less than expected for an open-channel blocking mechanism. The effects of Mg2+o are antagonized by K+o in manner which suggests that K+ competes with Mg2+ for the inactivation site. Mg2+o also reduced the amplitude of the single-channel current at millimolar concentrations by a rapid block of the open channel. A mechanism is proposed in which Mg2+ binds to the closed channel during hyperpolarization and prevents it from opening until it is occupied by K+.
The olfactory system of migratory adult sea lamprey (Petromyzon marinus) is specifically and acutely sensitive to unique bile acids released by conspecific larvae.
Authors: Li W , Sorensen P W , Gallaher D D
Published: May 2004

Larval sea lamprey inhabit freshwater streams and migrate to oceans or lakes to feed after a radical metamorphosis; subsequently, mature adults return to streams to spawn. Previous observations suggested that lamprey utilize the odor of conspecific larvae to select streams for spawning. Here we report biochemical and electrophysiological evidence that this odor is comprised of two unique bile acids released by larvae. High performance liquid chromatography and mass spectrometry demonstrated that larval sea lamprey produce and release two unique bile acids, allocholic acid (ACA) and petromyzonol sulfate (PS). Electro-olfactogram (EOG) recording also demonstrated that the olfactory system of migratory adult sea lamprey is acutely and specifically sensitive to ACA and PS; detection thresholds for these compounds were approximately 10(-12) M. ACA and PS were the most potent of 38 bile acids tested and cross-adaptation experiments suggested that adult sea lamprey have specific olfactory receptor sites associated with independent signal transduction pathways for these bile acids. These receptor sites specifically recognize the key substituents of ACA and PS such as a 5 alpha-hydrogen, three axial hydroxyls, and a C-24 sulfate ester or carboxyl. In conclusion, the unique lamprey bile acids, ACA and PS, are potent and specific stimulants of the adult olfactory system, strongly supporting the hypothesis that these unique bile acids function as migratory pheromones in lamprey.
A kinetically defined Na+/H+ antiporter within a mathematical model of the rat proximal tubule.
Authors: Weinstein A M
Published: May 2004

The luminal membrane antiporter of the proximal tubule has been represented using the kinetic formulation of E. Heinz (1978. Mechanics and Engergetics of Biological Transport. Springer-Verlag, Berlin) with the assumption of equilibrium binding and 1:1 stoichiometry. Competitive binding and transport of NH+4 is included within this model. Ion affinities and permeation velocities were selected in a least-squares fit to the kinetic parameters determined experimentally in renal membrane vesicles (Aronson, P.S., M.A. Suhm, and J. Nee. 1983. Journal of Biological Chemistry. 258:6767-6771). The modifier role of internal H+ to enhance transport beyond the expected kinetics (Aronson, P.S., J. Nee, and M. A. Suhm. 1982. Nature. 299:161-163) is represented as a velocity effect of H+ binding to a single site. This kinetic formulation of the Na+/H+ antiporter was incorporated within a model of the rat proximal tubule (Weinstein, A. M. 1994. American Journal of Physiology. 267:F237-F248) as a replacement for the representation by linear nonequilibrium thermodynamics (NET). The membrane density of the antiporter was selected to yield agreement with the rate of tubular Na+ reabsorption. Simulation of 0.5 cm of tubule predicts that the activity of the Na+/H+ antiporter is the most important force for active secretion of ammonia. Model calculations of metabolic acid-base disturbances are performed and comparison is made among antiporter representations (kinetic model, kinetic model without internal modifier, and NET formulation). It is found that the ability to sharply turn off Na+/H+ exchange in cellular alkalosis substantially eliminates the cell volume increase associated with high HCO3- conditions. In the tubule model, diminished Na+/H+ exchange in alkalosis blunts the axial decrease in luminal HCO3- and thus diminishes paracellular reabsorption of Cl-. In this way, the kinetics of the Na+/H+ antiporter could act to enhance distal delivery of Na+, Cl-, and HCO3- in acute metabolic alkalosis.
Single-channel properties of a volume-sensitive anion conductance. Current activation occurs by abrupt switching of closed channels to an open state.
Authors: Jackson P S , Strange K
Published: May 2004

Swelling-induced loss of organic osmolytes from cells is mediated by an outwardly rectified, volume-sensitive anion channel termed VSOAC (Volume-Sensitive Organic osmolyte/Anion Channel). Similar swelling-activated anion channels have been described in numerous cell types. The unitary conductance and gating kinetics of VSOAC have been uncertain, however. Stationary noise analysis and single-channel measurements have produced estimates for the unitary conductance of swelling-activated, outwardly rectified anion channels that vary by > 15-fold. We used a combination of stationary and nonstationary noise analyses and single-channel measurements to estimate the unitary properties of VSOAC. Current noise was analyzed initially by assuming that graded changes in macroscopic current were due to graded changes in channel open probability. Stationary noise analysis predicts that the unitary conductance of VSOAC is approximately 1 pS at 0 mV. In sharp contrast, nonstationary noise analysis demonstrates that VSOAC is a 40-50 pS channel at +120 mV (approximately 15 pS at 0 mV). Measurement of single-channel events in whole-cell currents and outside-out membrane patches confirmed the nonstationary noise analysis results. The discrepancy between stationary and nonstationary noise analyses and single-channel measurements indicates that swelling-induced current activation is not mediated by a graded increase in channel open probability as assumed initially. Instead, activation of VSOAC appears to involve an abrupt switching of single channels from an OFF state, where channel open probability is zero, to an ON state, where open probability is near unity.
Effects of ionic strength on the regulation of Na/H exchange and K-Cl cotransport in dog red blood cells.
Authors: Parker J C , Dunham P B , Minton A P
Published: May 2004

Dog red cell membranes contain two distinct volume-sensitive transporters: swelling-activated K-Cl cotransport and shrinkage-activated Na/H exchange. Cells were prepared with intracellular salt concentration and weight percentage of cell water (%cw) varied independently by transient permeabilization of the cell membrane to cations. The dependence of transporter-mediated Na and K influxes upon %cw and upon extracellular salt concentration (c(ext)) was measured in cells so prepared. It was found that the critical value of %cw at which transporters are activated, called the set point, is similar for the two transporters, and that the set points for the two transporters decrease similarly with increasing extracellular salt concentration. These findings suggest a common mechanism of regulation of these two transporters. Cellular Na, K, and Cl concentrations were measured as functions of %cw and c(ext). Using these data together with data from the literature for other solute concentrations, empirical expressions were developed to describe the dependence of the intracellular concentrations of all significant small molecule electrolytes, and therefore the intracellular ionic strength, upon %cw and c(ext). A mechanistic model for the dependence of the set point of an individual transporter upon intracellular ionic strength is proposed. According to this model, the set point represents a critical extent of association between the transporter and a postulated soluble regulatory protein, called regulator. Model functions are presented for the calculation of the thermodynamic activity of regulator, and hence extent of regulator-transporter association, as a function of total intracellular protein concentration (or %cw) and ionic strength. The experimentally observed dependence of set point %cw on c(ext) are simulated using these functions and the empirical expressions described above, together with reasonable but not uniquely determined values of model parameters.
Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia.
Authors: Nutter T J , Adams D J
Published: May 2004

Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage clamp. The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ > K+ > Rb+ > Na+ > Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ > Sr2+ > Ba2+ > Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh-evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5-6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and amphibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel.
Intrinsic gating properties of a cloned G protein-activated inward rectifier K+ channel.
Authors: Doupnik C A , Lim N F , Kofuji P , Davidson N , Lester H A
Published: May 2004

The voltage-, time-, and K(+)-dependent properties of a G protein-activated inwardly rectifying K+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K+ (VK = 0 mV), voltage jumps from VK to negative membrane potentials activated inward GIRK1 K+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants tau f approximately 50 ms and tau s approximately 400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from VK. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to VK and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK -20 mV; the equivalent gating charge was approximately 1.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein beta 1 gamma 2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K+ channels.
The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons.
Authors: Lande M B , Donovan J M , Zeidel M L
Published: May 2004

Several barrier epithelia such as renal collecting duct, urinary bladder, and gastric mucosa maintain high osmotic pH and solute gradients between body compartments and the blood by means of apical membranes of exceptionally low permeabilities. Although the mechanisms underlying these low permeabilities have been only poorly defined, low fluidity of the apical membrane has been postulated. The solubility diffusion model predicts that lower membrane fluidity will reduce permeability by reducing the ability of permeant molecules to diffuse through the lipid bilayer. However, little data compare membrane fluidity with permeability properties, and it is unclear whether fluidity determines permeability to all, or only some substances. We therefore studied the permeabilities of a series of artificial large unilamellar vesicles (LUV) of eight different compositions, exhibiting a range of fluidities encountered in biological membranes. Cholesterol and sphingomyelin content and acyl chain saturation were varied to create a range of fluidities. LUV anisotropy was measured as steady state fluorescence polarization of the lipophilic probe DPH. LUV permeabilities were determined by monitoring concentration-dependent or pH-sensitive quenching of entrapped carboxyfluorescein on a stopped-flow fluorimeter. The relation between DPH anisotropy and permeability to water, urea, acetamide, and NH3 was well fit in each instance by single exponential functions (r > 0.96), with lower fluidity corresponding to lower permeability. By contrast, proton permeability correlated only weakly with fluidity. We conclude that membrane fluidity determines permeability to most nonionic substances and that transmembrane proton flux occurs in a manner distinct from flux of other substances.