Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO3 cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5

Others have shown that H2DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO3 exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (−160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK ( wt, ∼40 μM) < NKMIK ≅ NKMIN ≅ KKMIN < KNMIN ≅ KNMIK < NNMIK < NNMIN (∼400 μM) < DDMID < EEMIE (∼800 μM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80–91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.

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Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney

The Journal of General Physiology ◽

10.1085/jgp.115.5.533 ◽

2000 ◽

Vol 115 (5) ◽

pp. 533-546 ◽

Cited By ~ 29

Author(s):

Irina I. Grichtchenko ◽

Michael F. Romero ◽

Walter F. Boron

Keyword(s):

Membrane Potential ◽

Voltage Clamp ◽

Xenopus Oocytes ◽

Rat Kidney ◽

Outward Current ◽

Membrane Vesicles ◽

Disulfonic Acid ◽

Ambystoma Tigrinum ◽

Ph Titration ◽

Electrode Voltage

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

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The role of Ca2+ in the protoveratrine-induced release of γ-aminobutyrate from rat brain slices

Biochemical Journal ◽

10.1042/bj1900333 ◽

1980 ◽

Vol 190 (2) ◽

pp. 333-339 ◽

Cited By ~ 19

Author(s):

M C W Minchin

Keyword(s):

Cerebral Cortex ◽

Rat Brain ◽

Dose Response ◽

Transmitter Release ◽

Response Curve ◽

Brain Slices ◽

Veratrum Alkaloids ◽

Similar Dose ◽

22Na Uptake

1. Protoveratrine A increased the release of gamma-amino[3H]butyrate from small slices of rat cerebral cortex. This effect increased with increasing protoveratrine concentration, reaching a maximum at 100 microM. 2. Removal of Ca2+ from the superfusing medium did not change the increase in release due to 10 microM-protoveratrine; however, the Ca2+ antagonists, compound D-600, La3+, Mn2+, Mg2+ and also high Ca2+ concentration inhibited the effect of the alkaloid, as did procaine. 3. Protoveratrine A increased the uptake of 22Na+ into the slices with a similar dose-response curve to that found for gamma-aminobutyrate release. For the most part, the substances that inhibited protoveratrine-stimulated gamma-aminobutyrate release also inhibited 22Na+ uptake, although the correlation was not perfect. 4. Although extracellular Ca2+ is not required for protoveratrine-induced gamma-aminobutyrate release, an increase in Na+ influx that is susceptible to inhibition by some Ca2+ antagonists does appear to be associated with this phenomenon. However, the possibility remains that changes in the free intracellular Ca2+ concentration may be important for transmitter release induced by depolarizing veratrum alkaloids.

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Role of oxygen in the production of human decompression sickness

Journal of Applied Physiology ◽

10.1152/jappl.1987.63.6.2380 ◽

1987 ◽

Vol 63 (6) ◽

pp. 2380-2387 ◽

Cited By ~ 15

Author(s):

P. K. Weathersby ◽

B. L. Hart ◽

E. T. Flynn ◽

W. F. Walker

Keyword(s):

Dose Response ◽

Response Curve ◽

Decompression Sickness ◽

Animal Experiments ◽

Inert Gases ◽

Relative Risks ◽

Data Variability ◽

Increase Risk ◽

High O2

In the calculation of decompression schedules, it is commonly assumed that only the inert gas needs to be considered; all inspired O2 is ignored. Animal experiments have shown that high O2 can increase risk of serious decompression sickness (DCS). A trial was performed to assess the relative risks of O2 and N2 in human no-decompression dives. Controlled dives (477) of 30- to 240-min duration were performed with subjects breathing mixtures with low (0.21–0.38 ATA) or high (1.0–1.5 ATA) Po2. Depths were chosen by a sequential dose-response format. Only 11 cases of DCS and 18 cases of marginal symptoms were recorded despite exceeding the presently accepted no-decompression limits by greater than 20%. Analysis by maximum likelihood showed a shallow dose-response curve for increasing depth. O2 was estimated to have zero influence on DCS risk, although data variability still allows a slight chance that O2 could be 40% as effective as N2 in producing a risk of DCS. Consideration of only inert gases is thus justified in calculating human decompression tables.

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Immunoassay of low concentrations of albumin in urine by latex particle counting.

Clinical Chemistry ◽

10.1093/clinchem/34.2.416 ◽

1988 ◽

Vol 34 (2) ◽

pp. 416-418 ◽

Cited By ~ 15

Author(s):

C L Cambiaso ◽

D Collet-Cassart ◽

M Lievens

Keyword(s):

Detection Limit ◽

Dose Response ◽

Response Curve ◽

Latex Particle ◽

Urinary Albumin ◽

Diagnostic Systems ◽

Particle Counting ◽

Low Concentrations ◽

The Impact

Abstract We describe here a nonisotopic immunoassay, based on particle-counting technology, for the determination of urinary albumin. The assay takes only 35 min and has been fully automated on the IMPACT (Acade Diagnostic Systems, Brussels, Belgium) machine. The system measures albumin within a linear range between 6.25 and 50 mg/L and has a detection limit of 0.4 mg/L. Analytical recoveries at three concentrations ranged between 96% and 102%. Within-run precision ranged from 1.6% to 9.5%. The method was compared with a commercial nephelometric immunoassay system and a correlation coefficient of 0.996 was found for 216 urine samples. No antigen excess affects the shape of the curve in our system, whereas in nephelometry a 3 g/L solution of albumin starts to decrease the dose-response curve.

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Role of Endothelial K + Channels in NO-Dependent Vasorelaxant Responses to Acetylcholine in Rat Aorta.

Hypertension ◽

10.1161/hyp.36.suppl_1.698-b ◽

2000 ◽

Vol 36 (suppl_1) ◽

pp. 698-698

Author(s):

John Quilley ◽

Yue Qiu

Keyword(s):

Dose Response ◽

Response Curve ◽

Rat Aorta ◽

Dose Response Curve ◽

Response Curves ◽

K Channels ◽

Voltage Dependent ◽

The Right ◽

Stimulation Of

P30 Endothelium-dependent vasorelaxant responses to acetylcholine (Ach) in rat aorta are mediated solely by NO. Rings precontracted with U46619 were used to investigate the role of endothelial K + channels. Thus, any effect of K + channel inhibitors on Ach responses in the absence of an effect on those to nitroprusside (NP) can be attributed to interference with Ach-induced stimulation of NO. Vasorelaxant responses to Ach (log EC 50 -7.29M) were abolished by removal of the endothelium or inhibition of NO synthesis with nitroarginine (100μM) which potentiated responses to NP (log EC 50 -9.41M vs -8.47M for control). In the presence of TEA (10mM) to inhibit K + channels, the dose-response curve for Ach, but not NP, was shifted to the right (log EC 50 -6.06). Elevation of extracellular K + (25mM KCl)also shifted the dose-response curve for Ach to the right. Inhibitors of specific types of K + channels: BaCl 2 (30μM), apamin (100nM), glibenclamide (10μM), charybdotoxin (50nM) and iberiotoxin (100nM) were without effect on dose-response curves to either Ach or NP. However, the combination of apamin (100nM) and charybdotoxin (50nM) but not apamin plus iberiotoxin, reduced relaxant responses to Ach (log EC 50 -6.95M) without affecting those to NP.These results confirm that Ach-induced relaxation of rat aorta is mediated entirely by endothelium-derived NO, the release of which apparently involves hyperpolarization of the endothelium. This effect is dependent on activation of a K + channel that is blocked by a combination of apamin/charybdotoxin but neither agent alone, possibly indicating characteristics of both Ca 2+ - activated and voltage-dependent K + channels.

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Studies of the role of gamma-aminobutyric acid in the hypothalamic control of feed intake in sheep

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y85-214 ◽

1985 ◽

Vol 63 (10) ◽

pp. 1297-1301 ◽

Cited By ~ 13

Author(s):

C. L. Girard ◽

J. R. Seoane ◽

J. J. Matte

Keyword(s):

Dose Response ◽

Feed Intake ◽

Gaba Receptors ◽

Response Curve ◽

Aminobutyric Acid ◽

Gamma Aminobutyric Acid ◽

Gaba Antagonists ◽

Gaba Agonist ◽

Hypothalamic Control

Fourteen sheep were used to study the role of gamma-aminobutyric acid (GABA) on the hypothalamic control of feed intake. Injections (1 μL) of pentobarbital (262 nmol) into preoptic and paraventricular areas induced feeding in satiated sheep. Injections of GABA into the same loci gave variable results, probably because the neuronal and glial uptake of GABA limits its effects. Muscimol, a GABA agonist with a higher affinity for postsynaptic GABA receptors than GABA, injected at doses from 0 to 0.750 nmol, gave a cubic dose–response curve; the highest feed intake was measured at 0.5 nmol. The response induced by muscimol was blocked by preinjections of two GABA antagonists, picrotoxin and bicuculline, with picrotoxin being more effective than bicuculline. Muscimol responsive loci were identified mainly in the preoptic, paraventricular, and anterior hypothalamus. The data suggests that neurons sensitive to gamma-aminobutyric acid may be implicated in the control of feed intake in sheep.

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Role of potassium channels in hypoxic relaxation of porcine bronchi in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.266.3.l232 ◽

1994 ◽

Vol 266 (3) ◽

pp. L232-L237

Author(s):

K. S. Lindeman ◽

L. B. Fernandes ◽

T. L. Croxton ◽

C. A. Hirshman

Keyword(s):

Smooth Muscle ◽

Dose Response ◽

Katp Channel ◽

Response Curve ◽

Channel Blocker ◽

Katp Channels ◽

Response Curves ◽

Third Order

To elucidate the mechanism of hypoxic relaxation of airway smooth muscle in vitro, we investigated the role of adenosine triphosphate-sensitive potassium (KATP) channels in this response. Second- and third-order porcine bronchial rings were suspended in 10-ml organ baths containing Krebs-Henseleit solution. To demonstrate the presence of KATP channels in this tissue, bronchial rings were contracted with carbachol (1 microM) in the presence of glibenclamide (100 microM), a KATP channel blocker, or the vehicle dimethyl sulfoxide (DMSO) (0.1 ml), and dose-response curves to levcromakalim (a KATP channel opener) or isoproterenol were constructed. In separate experiments, either glibenclamide or DMSO was added to the chamber and rings were contracted with carbachol (1 microM) in the presence of 95% O2-5% CO2. At the plateau, airways were relaxed with either isoproterenol (0.1 or 0.3 microM) or hypoxia (50, 28, or 0% O2, with constant 5% CO2). Glibenclamide, when compared with DMSO, shifted the dose-response curve to levcromakalim, but not to isoproterenol. Glibenclamide attenuated hypoxic relaxation in rings exposed to 50% O2 (from 35 +/- 4% to 23 +/- 3%, n = 6, P < 0.001) and increased the time to 63% relaxation in rings exposed to 50% O2 or to 28% O2. Responses in rings exposed to 0% O2 or to isoproterenol (0.1 or 0.3 microM) were not significantly altered. The ability of glibenclamide to attenuate the maximum response to 50% O2 and to increase the time to 63% relaxation during exposure to 50 or 28% O2 suggests that one component of hypoxic bronchodilation during moderate degrees of hypoxia is opening of KATP channels.

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Mechanisms of ammonia and ammonium transport by rhesus-associated glycoproteins

AJP Cell Physiology ◽

10.1152/ajpcell.00085.2015 ◽

2015 ◽

Vol 309 (11) ◽

pp. C747-C758 ◽

Cited By ~ 24

Author(s):

Tolga Caner ◽

Solange Abdulnour-Nakhoul ◽

Karen Brown ◽

M. Toriqul Islam ◽

L. Lee Hamm ◽

...

Keyword(s):

Xenopus Oocytes ◽

Collecting Duct ◽

Unique Property ◽

Ammonium Transport ◽

Surface Ph ◽

Methyl Amine ◽

Electrode Voltage ◽

Rh Proteins ◽

Rh Glycoproteins

In this study we characterized ammonia and ammonium (NH3/NH4+) transport by the rhesus-associated (Rh) glycoproteins RhAG, Rhbg, and Rhcg expressed in Xenopus oocytes. We used ion-selective microelectrodes and two-electrode voltage clamp to measure changes in intracellular pH, surface pH, and whole cell currents induced by NH3/NH4+ and methyl amine/ammonium (MA/MA+). These measurements allowed us to define signal-specific signatures to distinguish NH3 from NH4+ transport and to determine how transport of NH3 and NH4+ differs among RhAG, Rhbg, and Rhcg. Our data indicate that expression of Rh glycoproteins in oocytes generally enhanced NH3/NH4+ transport and that cellular changes induced by transport of MA/MA+ by Rh proteins were different from those induced by transport of NH3/NH4+. Our results support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4+ and neutral NH3 species; 2) transport of NH4+ is electrogenic; 3) like Rhbg, RhAG transport of NH4+ masks NH3 transport; and 4) Rhcg is likely to be a predominantly NH3 transporter, with no evidence of enhanced NH4+ transport by this transporter. The dual role of Rh proteins as NH3 and NH4+ transporters is a unique property and may be critical in understanding how transepithelial secretion of NH3/NH4+ occurs in the renal collecting duct.

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Enhanced Shear Force Responsiveness of Epithelial Na+ Channel’s (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix

International Journal of Molecular Sciences ◽

10.3390/ijms22052500 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2500

Author(s):

Daniel Barth ◽

Fenja Knoepp ◽

Martin Fronius

Keyword(s):

Shear Force ◽

Xenopus Oocytes ◽

Site Directed Mutagenesis ◽

Na Channel ◽

Cell Matrix ◽

Core Feature ◽

Electrode Voltage ◽

Ecm Degradation ◽

Epithelial Na Channel

Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC—which is less responsive to shear force (SF)—increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with β and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC’s SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.

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