Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

Electrophysiological Characterization of the Flounder Type II Na+/P i Cotransporter (NaPi-5) Expressed in Xenopus laevis Oocytes

The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na⁺/P _i cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state P _i -induced currents had a voltage-independent apparent K _m for P _i of 0.03 mm and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mm Na⁺. The apparent K _m for Na⁺ at 1 mm P _i was strongly voltage dependent (increasing from 32 mm at −70 mV to 77 mm at −30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na⁺ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mm Na⁺ which were suppressed at saturating P _i (1 mm). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V _0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum P _i -suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec⁻¹. The voltage dependence of the relaxations was P _i -independent, whereas changes in Na⁺ shifted V _0.5 to −60 mV at 25 mm Na⁺. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na⁺. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na⁺ contribute to transmembrane charge movement. Received: 11 March 1997/Revised: 3 June 1997     

Characterization of Fumarate Transport in Helicobacter pylori

The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. The transport of fumarate at micromolar concentrations was saturable with a K _M of 220 ± 21 μm and V _max of 54 ± 2 nmole/min/mg protein at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors, suggesting the presence of one or more fumarate carriers. The release of fumarate from cells was also saturable with a K _M of 464 ± 71 μm and V _max of 22 ± 2 nmol/min/mg protein at 20°C. The rates of fumarate influx at millomolar concentrations increased linearly with permeant concentration, and depended on the age of the cells. The transport system was specific for dicarboxylic acids suggesting that fumarate is taken up via dicarboxylate transporters. Succinate and fumarate appeared to form an antiport system. The properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential inhibitors. The results provided evidence that influx and efflux of fumarate at low concentrations from H. pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion. The anaerobic C₄-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate transporter. Received: 11 December 1997/Revised: 7 May 1998     

Expression and Molecular Characterization of Rat Renal d-Mannose Transport in Xenopus Oocytes

Renal reabsorption appears to play a major role in d-mannose homeostasis. Here we show that in rat kidney, the transport of d-mannose by brush border membrane vesicles from tubular epithelial cells involves an uphill and rheogenic Na-dependent system, which is fully inhibited by d-mannose itself, incompletely inhibited by d-glucose, d-fructose, phloridzin, and phloretin, and noninhibited by l-mannose or disaccharides. In addition, this system exhibits both low capacity (112.9 ± 15.6 pmol/mg/second) and high affinity (0.18 ± 0.04 mm), with a 2:1 stoichiometry for the Na:d-mannose interaction, and low affinity for sodium (16.6 ± 3.67 mm). We also show expression of d-mannose transport by Xenopus laevis oocytes injected with rat renal polyA⁺ RNA. Kinetic analysis of the expressed transport was performed after RNA enrichment by fractionation through a sucrose density gradient and was shown to be identical to that measured in membrane vesicles. The RNA species encoding the expressed transport has a small mean size, 1 kb approximately, and shows no homology with the SGLT family of Na-dependent d-glucose transporters, as shown by low stringent RT-PCR and northern analysis. The expressed transport is specific for d-mannose, since in spite of a significant inhibition by d-glucose and d-fructose, neither of these two substrates was transported above the level of the water-injected oocytes. Received: 29 February 2000/Revised: 25 August 2000     

Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes

When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V _max in Na⁺ activation curves and l-leucine kinetics. The kinetic parameters are K _mNa = 4.6 ± 2 mm, V _maxNa = 14.8 ± 1.7 pmol/oocyte/5 min for pH 8.0 and K _mNa = 2.8 ± 0.7 mm, V _maxNa = 31.3 ± 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K _m = 120.4 ± 24.2 μm, V _max = 23.2 ± 1.4 pmol/oocyte/5 min at pH 8.0 and K _m = 81.3 ± 24.2 μm, V _max = 65.6 ± 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted α-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification. Received: 27 April 1999/Revised: 10 January 2000     

Vacuolar Chloride Transport in Mesembryanthemum crystallinum L. Measured Using the Fluorescent Dye Lucigenin

To study vacuolar chloride (Cl⁻) transport in the halophilic plant Mesembryanthemum crystallinum L., Cl⁻ uptake into isolated tonoplast vesicles was measured using the Cl⁻-sensitive fluorescent dye lucigenin (N,N′-dimethyl-9,9′-bisacridinium dinitrate). Lucigenin was used at excitation and emission wavelengths of 433 nm and 506 nm, respectively, and showed a high sensitivity towards Cl⁻, with a Stern-Volmer constant of 173 m ⁻¹ in standard assay buffer. While lucigenin fluorescence was strongly quenched by all halides, it was only weakly quenched, if at all, by other anions. However, the fluorescence intensity and Cl⁻-sensitivity of lucigenin was shown to be strongly affected by alkaline pH and was dependent on the conjugate base used as the buffering ion. Chloride transport into tonoplast vesicles of M. crystallinum loaded with 10 mm lucigenin showed saturation-type kinetics with an apparent K _m of 17.2 mm and a V _max of 4.8 mm min⁻¹. Vacuolar Cl⁻ transport was not affected by sulfate, malate, or nitrate. In the presence of 250 μm p-chloromercuribenzene sulfonate, a known anion-transport inhibitor, vacuolar Cl⁻ transport was actually significantly increased by 24%. To determine absolute fluxes of Cl⁻ using this method, the average surface to volume ratio of the tonoplast vesicles was measured by electron microscopy to be 1.13 × 10⁷ m⁻¹. After correcting for a 4.4-fold lower apparent Stern-Volmer constant for intravesicular lucigenin, a maximum rate of Cl⁻ transport of 31 nmol m⁻² sec⁻¹ was calculated, in good agreement with values obtained for the plant vacuolar membrane using other techniques. Received: 18 February 2000/Revised: 30 June 2000     

GABA Concentration Sets the Conductance of Delayed GABAA Channels in Outside-out Patches from Rat Hippocampal Neurons

GABA_A channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC ₅₀ value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 μm diazepam, the GABA EC ₅₀ decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC ₅₀ was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABA_A receptor channel function is more complex than previously thought. Received: 23 October 2000/Revised: 27 February 2001     

Mg-Dependent, Zn-ATPase: Enzymatic Characteristics, Ion Specificities and Tissue Distribution

Mucosal crude microsomes, prepared from proximal rat small intestine, exhibited significant Mg-dependent, Zn-ATPase activity; V _max = 23 μmoles Pi/mg protein/hr, K _m = 160 nm, and Hill Coefficient, n= 1.5. Partial purification (∼10-fold) was achieved by detergent extraction, and centrifugation through 250 mm sucrose: V _max = 268 units, K _m = 1 nm, and n= 6. In partially purified preparations, the assay was linear with time to 60 min, and with protein concentration to 1 μg/300 μl. Activities at pH 8 and 8.5 were higher than at pH 7.2. The ATP K _m was 0.7 mm, with an optimal ATP/Mg ratio of ∼2. Ca elicited ATPase activity but did not augment the Zn-dependent activity. In partially purified preparations, the homologous salts of Co, Cd, Cu, and Mn exhibited no detectable activity. Vanadate inhibition studies yielded two component kinetics with a K _i of 12 μm for the first component, and 96 μm for the second component, in partially purified preparations. Tissue distribution analyses revealed gradients of activity. In the proximal half of the small intestine, Mg/Zn activity increased progressively from crypt to villus tip. In long axis studies, this activity decreased progressively from proximal to distal small bowel. Received: 12 September 2000/Revised 6 January 2001     

The H+ Pump in Frog Skin (Rana esculenta): Identification and Localization of a V-ATPase

We here report on studies on the frog skin epithelium to identify the nature of its excretory H⁺ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A₁ (10 μm) blocked H⁺ excretion (69 ± 8% inhibition) and therefore Na⁺ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na₂SO₄ solution, ``low-Na<sup>+</sup> conditions' under which H<sup>+</sup> and Na<sup>+</sup> fluxes are coupled 1:1. The electrogenic outward H<sup>+</sup> current measured in absence of Na<sup>+</sup> transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A<sub>1</sub> or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na<sup>+</sup> transport (short-circuit current), which develops with apical solutions containing 115 mm Na<sup>+</sup> (``high-Na⁺ conditions'), demonstrating a specific action on H⁺ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H⁺ excretion is mediated by a V-ATPase being the electrogenic H⁺ pump in frog skin. Received: 21 May 1996/Revised: 24 December 1996     

Effects of Barbiturates on Facilitative Glucose Transporters are Pharmacologically Specific and Isoform Selective

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [³H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC₅₀s of 0.2–2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all ≤10 mm) do not significantly inhibit glucose transport. The interaction of these three classes of anesthetics with purified GLUT-1 was evaluated by quenching of intrinsic protein fluorescence and displayed similar specificities and characteristics. The ability of barbiturates to inhibit other facilitative glucose transporters was determined in cell types expressing predominantly one isoform. Pentobarbital inhibits [³H]-2-deoxyglucose and [¹⁴C]-3-O-methyl-glucose uptake in cells expressing GLUT-1, GLUT-2, and GLUT-3 with IC₅₀s of ∼1 mm. In contrast, GLUT-4 expressed in insulin-stimulated rat adipocytes was much less sensitive than the other isoforms to inhibition by pentobarbital (IC₅₀ of >10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms. Received: 10 November 1998/Revised: 3 February 1999     

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na⁺-dependent with K _m = 5 μm and V _max= 0.7 nmoles · min⁻¹· mg protein⁻¹. Influx is sigmoidal as f[Na⁺] with _Na+ K _m ∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in V _max, with no change in K _m . At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the Δμ_Na+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na⁺-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells. Received: 11 January 2001/Revised: 26 March 2001     

Veratridine-Mediated Ca2+ Dynamics and Exocytosis in Paramecium Cells

We analyzed [Ca²⁺] _i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935–945; Plattner et al., 1994. J. Membrane Biol. 158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca<sup>2+</sup>] <sub>i</sub> transients with moderate [Ca<sup>2+</sup>] <sub>o</sub> in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm (c.f. [Ca<sup>2+</sup>] <sup>rest</sup> <sub>i</sub> =∼50 to 100 nm), veratridine produced only a small cortical [Ca<sup>2+</sup>] <sub>i</sub> transient. This increased in size and spatial distribution at [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca<sup>2+</sup>] <sub>o</sub> = 10 mm, [Ca<sup>2+</sup>] <sub>i</sub> transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca<sup>2+</sup>] <sub>o</sub> = 30 nm, the restricted residual response observed is due to Ca<sup>2+</sup> mobilization from subplasmalemmal stores. (ii) With moderate [Ca<sup>2+</sup>] <sub>o</sub> = 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca<sup>2+</sup> <sub>o</sub> influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca<sup>2+</sup> spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca²⁺] _i increase. (iii) With unusually high [Ca²⁺] _o , mobilization of cortical stores and/or Ca²⁺ _o influx may be impeded by the known membrane stabilizing effect of Ca²⁺ _o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein. (v) Finally, Mn²⁺ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific Me²⁺ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine is of particular and continous interest. Received: 8 December 1998/Revised: 2 March 1999     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

Heart and T-Lymphocyte Cell Surfaces Both Exhibit Positive Cooperativity in Binding a Membrane-Lytic Toxin

Cobra venom cytotoxins (CTX) have been shown to disrupt cells as different as immunocytes, skeletal myocytes, erythrocytes and tumor cells. Nevertheless, even subpopulations of tumor cells are differentially susceptible to CTX by an order of magnitude. In the present study, our objective was to compare CTX-specific binding with cytolytic potency for two disparate cell types in vitro. We investigated the lytic activity of cytotoxin-III from Naja naja atra (NNA, fraction D) using heart cells and human leukemic T-cells (CEM cells). For both cell types, 50% cytolysis, assessed by tetrazolium dye conversion, occurred with μm concentrations of toxin (EC₅₀= 2.2 μm). We examined the binding of radiolabeled CTX III to both heart cells and CEM cells and found the apparent dissociation constant (K _Dapp) to be 0.69 μm and 0.75 μm, for CEM and heart cells respectively. The B _max for the CEM cells was 1.0 fmoles/cell and that for heart cells was 5.2 fmoles/cell, both exhibiting positive cooperativity between the sites (Hill coefficients 1.4, T-cells; 1.6, heart). Relatively modest dissociation constants plus high numbers of binding sites per cell are consistent with a model of CTX binding to plasma membranes by interaction with phospholipids in the bilayer. Our results suggest that the lytic activity of this cytotoxin follows its binding to a population of sites on the cells in a cooperative fashion. Received: 8 May 1995/Revised: 17 November 1995     

Role of Cl− in Electrogenic H+ Secretion by Cortical Distal Tubule

The presence of an electrogenic H⁺-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (V _t ) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 × 10⁻⁷ m bafilomycin or 4.6 × 10⁻⁸ m concanamycin were added. No significant increase in lumen-negative V _t upon perfusion with these inhibitors as compared to control, was observed as well as when 10⁻³ m amiloride, 10⁻⁵ m benzamil or 3 mm Ba²⁺ were perfused alone or in combination. The effect of an inhibition of electrogenic H⁺ secretion, i.e., increase in lumen-negative V _t by 2–4 mV, was observed only when Cl⁻ channels were blocked by 10⁻⁵ m 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 ± 0.14 (n= 8) to 0.62 ± 0.03 (8) nmol.cm⁻².sec⁻¹ as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H⁺ ATPase in the establishment of cortical late distal tubule V _t is minor in physiological conditions, but can be demonstrated after blocking Cl⁻ channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H⁺ secretion in this segment is reduced by a Cl⁻ channel blocker, supporting coupling of H⁺-ATPase with Cl⁻ transport. Received: 6 July 1996/Revised: 27 December 1996     

Rectification of the Olfactory Cyclic Nucleotide-Gated Channel by Intracellular Polyamines

Polyamine-induced inward rectification of cyclic nucleotide-gated channels was studied in inside-out patches from rat olfactory neurons. The polyamines, spermine, spermidine and putrescine, induced an `instantaneous' voltage-dependent inhibition with K <sub>d</sub> values at 0 mV of 39, 121 μm and 2.7 mm, respectively. Hill coefficients for inhibition were significantly < 1, suggesting an allosteric inhibitory mechanism. The Woodhull model for voltage-dependent block predicted that all 3 polyamines bound to a site 1/3 of the electrical distance through the membrane from the internal side. Instantaneous inhibition was relieved at positive potentials, implying significant polyamine permeation. Spermine also induced exponential current relaxations to a `steady-state' impermeant level. This inhibition was also mediated by a binding site 1/3 of the electrical distance through the pore, but with a K _d of 2.6 mm. Spermine inhibition was explained by postulating two spermine binding sites at a similar depth. Occupation of the first site occurs rapidly and with high affinity, but once a spermine molecule has bound, it inhibits spermine occupation of the second binding site via electrostatic repulsion. This repulsion is overcome at higher membrane potentials, but results in a lower apparent binding affinity for the second spermine molecule. The on-rate constant for the second spermine binding saturated at a low rate (∼200 sec⁻¹ at +120 mV), providing further evidence for an allosteric mechanism. Polyamine-induced inward rectification was significant at physiological concentrations. Received: 17 February 1999/Revised: 27 April 1999     

Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium

Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies of the Na/K ATPase. RNase protection assays showed that all cells express α₁- and α₂-isoforms of the Na/K pump but not the α₃-isoform, however the α₂-isoform dominates in anterior cells whereas the α₁-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the Na/K pump current (I _P ), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I _P blockade data indicate the α₁-isoform has a dissociation constant of 100 μm DHO whereas for the α₂-isoform it is 0.75 μm DHO. Both α₁- and α₂-isoforms are half maximally activated at an intracellular Na⁺-concentration of 9 mm. The α₁-isoform is half maximally activated at an extracellular K⁺-concentration of 3.9 mm whereas for the α₂-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α₁-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α₂-isoform. Under normal physiological conditions, I _P in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer to the area of cell membrane assuming a capacitance of around 1 μF/cm². Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm² at the equator vs. 0.5 μA/cm² at the anterior pole. Received: 17 May 2000/Revised: 11 August 2000     

Nonselective Cation and BK Channels in Apical Membrane of Outer Sulcus Epithelial Cells

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K _Ca ) channel and (iii) a small K⁺ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na⁺ and K⁺, but not permeable to Cl⁻ or N-methyl-d-glucamine. Channel activity required the presence of Ca²⁺ at the cytosolic face, but was detected at Ca²⁺ concentrations as low as 10⁻⁷ m (open probability (P _o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P _o of the NSC channel from both the external and cytosolic side (IC₅₀∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K⁺ could be fitted to the I-V relationship in asymmetrical K⁺ and Na⁺ solutions. The channel was impermeable to Cl⁻ and N-methyl-d-glucamine. P _o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca²⁺. TEA (20 mm), charybdotoxin (100 nm) and Ba²⁺ (1 mm) but not amiloride (1 mm) reduced P _o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P _o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca²⁺ activated, voltage-insensitive and involved in both constitutive K⁺ and Na⁺ reabsorption from endolymph while the BK channel might participate in the K⁺ pathway under stimulated conditions that produce an elevated intracellular Ca²⁺ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999     

Relationships Between Na+/Glucose Cotransporter (SGLT1) Currents and Fluxes

The relationships between currents generated by the rabbit Na⁺/glucose cotransporter (SGLT1) and the fluxes of Na⁺ and sugar were investigated using Xenopus laevis oocytes expressing SGLT1. In individual voltage-clamped oocytes we measured: (i) the current evoked by 10 mmαMG and the ²²Na⁺ uptake at 10 mm Na⁺; (ii) the currents evoked by 50 to 500 μm [¹⁴C]αMG and the [¹⁴C]αMG uptakes at 100 mm Na⁺; and (iii) phlorizin-sensitive leak currents in the absence of sugar and ²²Na⁺ uptakes at 10 mm Na⁺. We demonstrate that the SGLT1 leak currents are Na⁺ currents, and that the sugar-evoked currents are directly proportional to both αMG and Na⁺ uptakes. The Na⁺/αMG coupling coefficients were estimated to be 1.6 at −70 mV and 1.9 at −110 mV. This suggests that the rabbit SGLT1 Na⁺/αMG stoichiometry for sugar uptake is 2 under fully saturating, zero-trans conditions. Coupling coefficients of less than 2 are expected under nonsaturating conditions due to uncoupled Na⁺ fluxes (slippage). The similarity between the Na⁺ Hill coefficients and the coupling coefficients suggests strong cooperativity between the two Na⁺ binding sites. Received: 6 October 1997/Revised: 5 December 1997