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D D Loo

Publications and source records attributed to D D Loo.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of potassium and chloride channels in the basolateral membrane of bovine nonpigmented ciliary epithelial cells.

PURPOSE: The pigmented epithelial (PE) and the nonpigmented epithelial (NPE) cells of the ciliary body may function as a syncytium for secretion of the aqueous humor, with solute and water entering through the PE and leaving through the NPE cell. Secretion across the basolateral membrane of the NPE cell was postulated to depend on coupling sodium extrusion through the NaK-ATPase to efflux through Cl channels. In the current study, the authors used single-channel patch clamp techniques to characterize the ion channels present in the basolateral membrane of the NPE cell. METHODS: Mild enzymatic digestion of the bovine ciliary process was used to obtain pairs or clusters of NPE cells that were coupled to their neighbor PE cells. Cells were dispersed immediately onto a coverslip, bathed in a HEPES-buffered saline, and imaged with an inverted microscope. With the anatomic relationship between cells still intact, a patch-pipette electrode was applied to the basolateral surface of the NPE cell. Single-channel currents were then characterized in 52 cells by using either the cell-attached or excised versions of the patch-clamp technique. RESULTS: A calcium-dependent "maxi" K channel was found in approximately 33% of the patches. It was activated by depolarizing voltage steps in cell-attached patches and was relatively inactive near the resting potential. When excised from the cell, it was activated by high levels of bath calcium and, in symmetrical K solutions (150 mM), showed a linear current-voltage (I-V) relationship with a slope conductance of approximately 150 pS. This channel was highly selective to K over Na and was blocked by barium (2 mM) or quinine (1 mM). In cell-attached recordings, a smaller conductance Cl channel was observed in 73% of the patches. The channel currents were inward at resting potential and outward with depolarizing voltage steps, with a pipette reversal potential of approximately -23 mV. The current-voltage relationship of this channel was nearly linear with a slope conductance of approximately 24 pS. When pipette Na and K were replaced with N-methyl-D-glucamine, this channel produced an inward current showing a high selectivity for Cl. This Cl-selective channel was activated by dibutyryl-cyclic adenosine monophosphate (cAMP) but not by elevation of intracellular calcium with ionomycin or by anisosmotic cell swelling. CONCLUSIONS: Two ion channels were found in the NPE basolateral membrane: One was K selective, the other was Cl selective. The cAMP-activated Cl channel may be important in supplying the counterion for Na extrusion across the basolateral surface of the NPE cell, whereas the calcium-dependent maxi K channel may be useful for membrane hyperpolarization to increase the driving force for Cl exit.

Animals↗
PubMed1994

SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation.

Recently a member of the Na+/glucose (SGLT1) gene family of cotransporters was isolated from a pig renal cell line and was thought to be the neutral amino acid transporter System A. This cDNA (Kong, C. T., Yet, S. F., and Lever, J. E. (1993) J. Biol. Chem. 268, 1509-1512) encodes a 660-amino acid protein with 76% identity to SGLT1. To confirm and extend the kinetic characterization of SAAT1, we have expressed this clone in Xenopus oocytes and measured transport using both radiotracer and electrophysiological techniques. SAAT1 did not stimulate either 50 microM 2-(methylamino)isobutyrate uptake or 2-(methylamino)isobutyrate-evoked inward Na+ currents, but instead stimulated 50 microM alpha MG (alpha-methyl-D-glucopyranoside) uptake 27-fold from 2 +/- 1 pmol.h-1/oocyte (n = 9) to 55 +/- 6 pmol.h-1/oocyte (n = 9) and alpha MG-evoked inward Na+ currents (I) by up to 1000 nA/oocyte. The apparent affinity constant for alpha MG (K alpha MG 0.5) was approximately 2 mM and was independent of membrane potential from -30 to -150 mV but was voltage-sensitive between -30 and +30 mV. The relative sugar specificity for the transporter was alpha MG > or = D-glucose >> D-galactose >>> 3-O-methyl-D-glucopyranose, L-glucose. The sugar-evoked currents were Na(+)-dependent (KNa 0.5 approximately 10 mM at -50 mV) and the Hill coefficient was 1. KNa 0.5 decreased with hyperpolarization of the membrane from -50 to -150 mV. Phlorizin inhibited the alpha MG-evoked current with apparent Ki of 18 microM at -50 mV. We conclude that the SAAT1 cDNA encodes a renal low affinity Na+(1)/glucose(1) cotransporter and propose that pig SAAT1 be renamed pSGLT2.

Amino Acid Transport Systems↗
PubMed1994

Protons drive sugar transport through the Na+/glucose cotransporter (SGLT1).

Na(+)-dependent transporters, such as the Na+/glucose cotransporter (SGLT1) have long been characterized as having an overwhelming preference for Na+ as the essential ion. We have applied electrophysiological measurements to analyze the ability of protons to transport glucose with a cloned transporter, SGLT1, expressed in Xenopus laevis oocytes. Our results show that protons can drive sugar transport through SGLT1 in the absence of Na+ with the following characteristics: 1) the affinity of SGLT1 for H+ is 3 orders of magnitude higher than its affinity for Na+ (3 microM versus 20 mM); 2) H+ supports a higher maximum transport than Na+, suggesting an alteration in rate-limiting processes; and 3) the cation determines the transporter's affinity for sugar (at Vm = -50 mV, the apparent affinity for alpha-methyl-D-glucoside is 0.2 mM in Na+ and 20 mM in H+). The similarity in the kinetics of H(+)- and Na(+)-dependent sugar transport suggests that the transport mechanism for SGLT1 does not depend on the driving ion.

Animals↗
PubMed1994

Sodium/D-glucose cotransporter charge movements involve polar residues.

Na(+)-dependent glucose transporters (SGLT1) exhibit transient carrier currents with a time constant (tau) of 2-20 ms, and the charge transfer (Q) fits the Boltzmann equation. There is a 60-mV negative displacement in the tau/V and Q/V curves between the human and rabbit SGLT1 proteins, and the initial goal was to identify the charges responsible for these differences in kinetics. We have focused on residue 176 in putative transmembrane helix (M4) because this is an aspartic acid in rabbit and asparagine in human. Asp-176 in rabbit SGLT1 was replaced with asparagine and alanine residues, and the wild-type and mutant proteins were expressed in Xenopus laevis oocytes. A two-electrode voltage clamp was used to measure the kinetics of charge transfer. There was no difference between the wild-type and D176N, but there was a 60-mV negative shift in the tau/V and Q/V curves with D176A. This suggests that polar residues at position 176 play an important role in determining charge transfer, probably by electrostatic bonding to a neighboring polar residue in the membrane domain of the protein. The similarity between rabbit SGLT1 and the D176N mutant further indicates that other membrane residues account for the difference between rabbit and human SGLT1. There were only modest changes in the steady-state Na+/glucose cotransport kinetics between wild-type and D176A mutant transporters in the voltage range +50 to -50 mV. Model simulations show that the mutation alters the rate constants for conformational changes of the unloaded transporter. Phlorizin, a specific competitive inhibitor of sugar transport, has a lower affinity for the D176A mutant than for SGLT1. This indicates that polar residues at position 176 hydrogen bond with the -OH group on the B-phenyl ring of the inhibitor.

Amino Acid Sequence↗
PubMed1994

Steady-state and presteady-state kinetics of the H+/hexose cotransporter (STP1) from Arabidopsis thaliana expressed in Xenopus oocytes.

We have investigated the steady-state and presteady-state kinetics of the cloned H+/hexose cotransporter from Arabidopsis thaliana (STP1) expressed in Xenopus oocytes using the two-electrode voltage-clamp method. Steady-state sugar-dependent currents were measured between -150 and +50 mV as a function of external [3-O-methyl-D-glucose] (3OMG) and [H+]. At pH 6.5 (316 nM H+) the maximal current for sugar, i3OMGmax, was voltage-dependent, increasing from 40 nA at -30 mV to 95 nA at -150 mV. The apparent affinity of sugar, K3OMG0.5, at pH 6.5 decreased from 30 microM at -30 mV to 11 microM at -70 mV and was then voltage-independent between -70 and -150 mV. Increasing the extracellular [H+] to 3160 nM (pH 5.5) increased i3OMGmax to 65 nA at -30 mV and 212 nA at -150 mV. K3OMG0.5 at pH 5.5 also increased and was voltage-dependent: 15 microM at -50 mV rising to 25 microM at -150 mV. At pH 6.5 and 5.5, the Hill coefficient n for 3OMG was voltage-independent and averaged 1.2 and 1.4, respectively. At saturating [3OMG] iHmax was voltage-dependent and KH0.5 was voltage-independent, averaging 370 nM (pH 6.4). The Hill coefficient n for H+ was voltage-independent and averaged 1. The sugar specificity of STP1 was D-mannose > or = 2-deoxyglucose > D-galactose > or = 3OMG > D-xylose > D-glucose > D-fucose > D-fructose > L-glucose > L-arabinose > D-arabinose, demonstrating that STP1 has a low substrate specificity. Transient currents recorded after rapid steps in membrane potential relaxed with time constants tau, between 3 and 14 ms. The charge movement Q (the integral of the current transients) fitted to a Boltzmann relation with maximal charge Qmax of 3.4 nanocoulombs and an apparent valence z approximately 1 corresponding to a transporter density of 2 x 10(10)/oocyte. Potential for 50% Qmax (V0.5) was -28 mV. At saturating 3OMG and at low external [H+] (pH 7.5), the transient STP1 currents were eliminated. The presteady-state data indicate that STP1 can bind H+ in the absence of sugar, and the steady-state data suggest that H+/hexose cotransport occurs via a sequential mechanism.

Animals↗
PubMed1994

Phenylglucosides and the Na+/glucose cotransporter (SGLT1): analysis of interactions.

Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na(+)-dependent uptake of 14C-alpha-methyl-D-glucopyranoside (alpha MDG) with apparent Kis in the range 0.1 to 20 mM, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na(+)-loaded transporter. Computational chemistry indicated that all these beta-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the -NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.

Animals↗
PubMed1994

'Active' sugar transport in eukaryotes.

Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Regulation of mouse choroid plexus apical Cl- and K+ channels by serotonin.

Using patch-clamp techniques, we have characterized ion channels in the apical membrane of the mouse choroid plexus epithelium and have examined the effect of serotonin on these channels. When the pipette contained 140 mM KCl and the bath contained NaCl Ringer solution, cell-attached patches revealed both Cl- and K+ channels. The Cl- channel was activated by hyperpolarizing membrane potentials, and 70% were also activated by large depolarizing potentials (pipette potential, Vp, more negative than -40 mV). The channel exhibited linear current-voltage (I-V) relations with a conductance of 4 +/- 1 pS (n = 30), and a reversal potential at Vp = -14 +/- 1 mV (n = 30). The majority of the K+ channels (84%) were activated by depolarizing membrane potentials. These exhibited linear I-V relations with a conductance of 18 +/- 1 pS (n = 10) and a reversal potential at Vp = -51 +/- 8 mV (n = 10). Serotonin (10(-6) M) increased the open probability (Po) of active Cl- channels (n = 20) by an order of magnitude at the resting potential (Vp = 0 mV) as well as activating previously silent Cl- channels. In contrast, complete inhibition of K+ channel activity was observed in the majority of experiments. There was a 30 s delay after exposure of the tissue to serotonin, thereafter the K+ channel was rapidly inhibited (within 1 min) prior to the stimulation of the Cl- channel. Stimulation of the Cl- channel by serotonin was abolished by mianserin (10(-3) M). We conclude that serotonin exerts its effect on apical Cl- channels via the 5-HT1c receptor. The modulation of these channels by serotonin may be important to CSF secretion and its regulation.

Animals↗
PubMed1993

Relaxation kinetics of the Na+/glucose cotransporter.

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Qmax of approximately 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Qmax. Lowering external Na+ from 100 to 10 mM reduced Qmax 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Qmax was independent of, but tau was dependent on, temperature (a 10 degrees C increase increased tau by a factor of approximately 2.5 at -50 mV). Addition of sugar or phlorizin reduced Qmax. Analyses of hSGLT1 pre-steady-state kinetics indicate that transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qmax provides a measure of transporter density (approximately 10(4)/microns 2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.

Animals↗
PubMed1993

Whole-cell currents in isolated resting Necturus gastric oxynticopeptic cells.

1. Necturus gastric mucosa secretes Cl- actively across the gastric glands which are composed almost entirely of acid- and enzyme-secreting oxynticopeptic cells. Single channel studies on Necturus oxynticopeptic cells have shown that the basolateral membrane possesses multiple K(+)-selective channels but no observable Cl- channels while the apical membrane has Cl- channels but no observable K+ channels. To relate these channel properties to the conductance of the whole cell we have investigated the macroscopic membrane currents with conventional whole-cell patch-clamp techniques. 2. When bathed in amphibian Ringer solution, gastric oxynticopeptic cells had a membrane resistance of 47.8 +/- 2.8 M omega and a membrane capacitance of 75.5 +/- 2.7 pF (n = 82). This gave a specific membrane resistance of 3260 +/- 160 omega cm2 (n = 82). Reversal potentials of the oxynticopeptic cells were -13.8 +/- 1.2 mV (n = 45) for an intracellular Cl- concentration ([Cl-]i) of 42 mM and were significantly more negative -24.4 +/- 3.1 mV (n = 31, P < 0.001) for [Cl-]i = 22 mM. 3. In the absence of ATP in the pipette solution, there was an 80% reduction of the whole-cell current with a typical half-time (t1/2) of 5 min. The run-down was not observed when the pipette solution contained 4 mM ATP. 4. A slow and voltage-independent inhibition of 80% of the whole-cell currents occurred after addition of NPPB (35 microM). Ba2+ (10 mM) produced a reversible inhibition of 20% of the total current. Together, 35 microM NPPB and 10 mM Ba2+ eliminated 95% of the whole-cell currents. These data suggest that in the resting oxynticopeptic cells Cl- carried the major fraction of the current while K+ ions carried only a small fraction. 5. Total replacement of Cl- in the pipette and bath solution by gluconate- increased the membrane resistance to 751 +/- 104 M omega (n = 53) and shifted the reversal potential to -38.1 +/- 2.8 mV (n = 53). 6. Increasing the bath K+ concentration from 6 to 91 mM activated a current which had a high selectivity for K+ over choline+, Li+, Na+, Rb+ and Cs+ and was independent of Cl-. The activation of this K+ current (IK*) by high external K+ was not seen with ATP-free pipette solution. 7. Ba2+ or Cs+ had a voltage-dependent blocking effect of this inward K+ current. Ouabain (1 mM) or SCH 28080 (200 microM), specific inhibitors of the Na+,K(+)-ATPase and H+,K(+)-ATPase, had no effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

Electrogenic properties of the cloned Na+/glucose cotransporter: I. Voltage-clamp studies.

The cloned rabbit intestinal Na+/glucose cotransporter was expressed in Xenopus laevis oocytes. Presteady-state and steady-state currents associated with cotransporter activity were measured with the two-electrode voltage-clamp technique. Steady-state sugar-dependent currents were measured between -150 and +90 mV as a function of external Na+ ([Na]o) and alpha-methyl-D-glucopyranoside concentrations ([alpha MDG]o). K alpha MDG0.5 was found to be dependent upon [Na]o and the membrane potential. At Vm = -50 mV, increasing [Na]o from 10 to 100 mM decreased K alpha MDG0.5 from 1.5 mM to 180 microM. Increasing membrane potential toward negative values decreased K alpha MDG0.5 at nonsaturating [Na]o. For instance, at 10 mM [Na]o, K alpha MDG0.5 decreased from 1.5 mM to 360 microM on increasing the membrane potential from -50 to -150 mV. The i alpha MDGmax was relatively insensitive to [Na]o between 10 and 100 mM and weakly voltage dependent (e-fold increase per 140 mV). KNa0.5 and iNamax were found to be dependent upon membrane potential and [sugar]o. In the presence of 1 mM [alpha MDG]o, KNa0.5 decreased from 50 to 5 mM between 0 and -150 mV and iNamax increased twofold between -30 and -200 mV. The voltage dependence of KNa0.5 is consistent with an effect of potential on Na+ binding (Na(+)-well effect), whereas the voltage dependence of iNamax is compatible with the translocation step being voltage dependent. It is concluded that voltage influences both Na+ binding and translocation. Presteady-state currents were observed for depolarization pulses in the presence of 100 mM [Na]o. The transient current relaxed with a half time of approximately 10 msec, and both the half time and magnitude of the transient varied with the holding potential and the size of depolarization pulse. Presteady-state currents were not observed after the addition of phlorizin or alpha MDG to the external Na+ solution and were not observed for water-injected control oocytes. We conclude that presteady-state currents are due to the activity of the carrier and that they may give a novel insight to the transport mechanism of the Na+/glucose cotransporter.

Animals↗
PubMed1992

Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions.

The results of the accompanying electrophysiological study of the cloned Na+/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Mémbrane Biol. 125:49-62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model ("mirror" symmetry) with a Na+:alpha MDG stoichiometry of 2. Carrier translocation in the membrane as well as Na+ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na+ binding/dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na+ and voltage dependence of Ksugar0.5 and isugarmax, (iii) sugar and voltage dependence of KNa0.5 and iNamax, (iv) presteady-state currents and their dependence on external Na+, alpha MDG and membrane potential, and (v) and carrier Na+ leak current. We conclude that the main voltage effect is on carrier translocation. Na+ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na+ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, [Na]o and [alpha MDG]o. At 0 mV and at saturating [Na]o and [alpha MDG]o, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec-1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na+ dissociation becomes increasingly rate limiting. However, as [Na]o is decreased to less than 10 mM, the rate-limiting step is the external Na+ ions binding in the 0 to -150 mV potential range. At 0 mV, the external Na+ dissociation constant KNa' is 80 mM and decreases to 24 mM at -150 mV. The external sugar dissociation constant KNaS' is estimated to be 200 microM and voltage independent. Finally, the internal leak pathway (CNa2 translocation) is insignificant.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1992

The Na+/glucose cotransporter (SGLT1).

An important class of Na+ transport proteins is the cotransporters. They exist in bacteria and animal cells and are responsible for the "active" accumulation of sugars, amino acids, carboxylic acids and some ions, e.g., I-, Cl-, and PO-4, in cells. In the small intestine and renal proximal tubule the cotransporters play an important role in the transport of salt and water across the epithelia. The most well known and best characterized Na+ cotransporter is the intestinal brush border Na+/glucose cotransporter. We have cloned, sequenced, and expressed both the rabbit and human Na+/glucose cotransporters. The cDNAs code for 73kDa proteins with 662-664 residues (86% identity). Secondary structure analysis suggests a 12 membrane-spanning helical model with the N- and C-termini in the cytoplasm. A single N-linked glycosylation site is utilized at Asn248. These sugars are not required for function. Two essential residues for functional expression in oocytes have been identified, Asp28 and Arg300. In two sisters with glucose-galactose malabsorption the transport defect is caused by a missense mutation changing Asp28 to Asn28, and we have found that changing Arg300 to Cys300 eliminated transport. Current research is directed to finding residues and domains essential for ligand binding and transport, and we are using electrophysiological techniques to correlate structure and function.

Amino Acid Sequence↗
PubMed1991

Voltage-clamp studies of the Na+/glucose cotransporter cloned from rabbit small intestine.

Inward Na+ currents associated with the cloned intestinal Na+/glucose cotransporter expressed in Xenopus oocytes have been studied using the two-microelectrode voltage-clamp method. The steady-state current/voltage relations showed voltage-dependent (Vm from +20 to -75 mV) and relatively voltage-independent (Vm from -75 to -150 mV) regions. The apparent Imax for Na+ and glucose increased with negative membrane potentials, and the apparent K0.5 for glucose (K(Glc)0.5) depended on Vm and [Na]o. Increasing [Na]o from 7 to 110 mmol/l had the same effect in decreasing K(Glc)0.5 from 0.44 to 0.03 mmol/l as increasing the Vm from -40 to -150 mV. The I/V curves under saturating conditions (20 mmol/l external sugars and 110 mmol/l [Na]o) were identical for D-glucose, D-galactose, alpha-methyl D-glucopyranoside and 3-O-methyl D-glucoside. The specificity of the cotransporter for sugars was: D-glucose, D-galactose, alpha-methyl D-glucopyranoside greater than 3-O-methyl D-glucoside much greater than D-xylose greater than D-allose much greater than D-mannose. Ki for phlorizin (approximately 10 mumol/l) was independent of Vm at saturating [Na]o. We conclude that a variety of sugars are transported by the cloned Na+/glucose cotransporter at the same maximal rate and that membrane potential affects both the maximal current and the apparent K0.5 of the cotransporter for Na+ and glucose.

Animals↗
PubMed1991

Diversity of K+ channels in the basolateral membrane of resting Necturus oxyntic cells.

Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa of Necturus maculosa. In cell-attached patches with pipette solution containing 100 mM KCl, four major classes of K+ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec for a, and 8 msec for b). Some channels showed subconductance levels. The maximal inward conductance gmax was 31 +/- 5 pS (n = 13) and the reversal potential Erev was at Vp = -34 +/- 6 mV (n = 9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels with gmax = 40 +/- 18 pS (n = 3) and Erev was at Vp = -31 +/- 5 mV (n = 3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1 mM) to the bath solution in cell-attached patches increased the open probability Po of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels with gmax = 21 +/- 3 pS (n = 4) and Erev was at Vp = -24 +/- 9 mV (n = 4). These channels exhibited very high open probability (Po = 0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26 +/- 2 pS (n = 3) and Erev at Vp = -33 +/- 3 mV (n = 3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K+ in the pipette solution replaced by Na+, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K+ over Na+ and Cl-. A variety of K+ channels contributes to the basolateral K+ conductance of resting oxyntic cells.

Animals↗
PubMed1990

The activation of calcium and calcium-activated potassium channels in mammalian colonic smooth muscle by substance P.

1. The regulation of Ca2(+)-activated K+ channels by the agonist substance P in freshly dissociated smooth muscle cells from the rabbit longitudinal colonic muscle was characterized using the patch clamp technique. 2. In the cell-attached recording mode, when pipette and bath solutions contained equal [K+] (126 mM), the Ca2(+)-activated K+ channels showed a linear current-voltage relationship (between -50 mV and 50 mV) with a slope conductance of 210 +/- 35 pS (n = 12). Reversal potential measurements indicated that the channel was highly selective for K+ over Na+ (PK/PNa = 110). 3. Channels were activated by depolarizing membrane voltages and cytosolic Ca2+, and in inside-out patches channel activation depended sigmoidally on voltage and [Ca2+]. The potential for half-activation at a cytosolic [Ca2+] of 5 x 10(-6) M was 0 mV. A tenfold increase in cytosolic Ca2+ resulted in a 60 mV shift of the sigmoidal voltage activation curve to more negative potentials. 4. Threshold concentrations of substance P (10(-12) M), which did not result in cell contraction, caused a prolonged activation of K+ channels. The K+ channels were observed to open in clusters: simultaneous opening of multiple channels was interrupted by complete, prolonged channel closure. 5. Lowering bath [Ca2+] to submicromolar concentrations abolished the effect of substance P. The activation of K+ channels by substance P (10(-12) M) was also inhibited by the dihydropyridine nifedipine (10(-6) M), a blocker of L-type Ca2+ channels. 6. In the whole-cell recording mode, with the pipette solution containing 126 mM-KCl, 0.77 mM-EGTA and 1 mM-ATP, depolarization from a holding potential of -70 mV elicited outward currents which increased to steady-state values. These were K+ currents as they were blocked by TEA (tetraethylammonium, 30 mM) and Ba2+ (1 mM) and were abolished when pipette K+ was replaced by Cs+. 7. The depolarization-activated outward current was not affected by lowering extracellular [Ca2+] or by the Ca2+ channel antagonists Cd2+ (200 microM), nifedipine (10(-6)-10(-5) M) or verapamil (10(-6) M). The current was greatly reduced when the EGTA concentration in the pipette solution was increased from 0.77 to 10 mM. 8. When the pipette solution contained CsCl, membrane depolarization activated inward currents. The peak inward current was identified as current through L-type Ca2+ channels based on its voltage- and time-dependent kinetics, and its modulation by dihydropyridines.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗