Mammalian Amino Acid Transport System y+ Revisited: Specificity and Cation Dependence of the Interaction with Neutral Amino Acids

A reevaluation of the specificity of system y⁺, the classical transporter for cationic amino acids is presented. System y⁺ has been defined as a transporter for cationic amino acids that binds neutral amino acids with lower affinity in the presence of Na⁺. The discovery of other transporters for cationic amino has suggested that some properties, originally attributed to system y⁺, may relate to other transport systems. Uncertainty concerns mainly, the affinity for neutral amino acids and the cation dependence of this interaction. Neutral amino acids (13 analogues tested) were found to bind to system y⁺ in human erythrocytes with very low affinity. Inhibition constants (K_iy, mm) ranged between 14.2 mm and >400 mm, and the strength of interaction was similar in the presence of Na⁺, K⁺ or Li⁺ (145 mm). In choline medium, no interaction was detected up to 20 mm of the neutral amino acid. Guanidinium ion (5 mm, osmolarity maintained with choline) potentiated neutral amino acid binding; the effect was most important in the case of l-norvaline which aligned with guanidinium ion is equivalent to arginine. This suggests cooperative interaction at the substrate site. The specificity of system y⁺ was shown to be clearly distinct from that of system y⁺L, a cationic amino acid transporter that accepts neutral amino acids with high affinity in the presence of Na⁺ and which influenced the classical definition of system y⁺. Received: 28 September 1998/Revised: 21 December 1998     

Amino acid transport system y+L of human erythrocytes: Specificity and cation dependence of the translocation step

The transport specificity of system y⁺L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[¹⁴C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na⁺, Li⁺ or K⁺ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na⁺ or Li⁺ were replaced by K⁺. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

System y+L-like Activities Account for High and Low Amino-Acid Transport Phenotypes in Chicken Erythrocytes

The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal population studied (male Leghorn chickens, 6–14 weeks old) was found to consist of two groups presenting either low (LT, 19 individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration of l-lysine of 1 μm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general features of system y⁺L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y⁺L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects, the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective breeding experiments of chicken with high and low amino-acid transport activity. Received: 12 February 2001/Revised: 11 June 2001     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

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     

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     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

Uptake of L-leucine by Trout Red Blood Cells and Peripheral Lymphocytes

The uptake of l-leucine by trout red blood cells and peripheral lymphocytes has been analyzed. The present study shows two functionally different Na⁺-independent systems for apolar branched-chain amino acids. They are designated as L systems because they share some properties with the mammalian L system. The carrier present in red blood cells has low K _m values, is trans-stimulable and not stereospecific for leucine uptake; on the other hand, the system present in lymphocytes is stereospecific for leucine uptake and trans-inhibitable. Both carriers are pH sensitive in a similar fashion at low pHs, but there are important differences at higher pH values (above neutrality). These properties are compared with these of the asc systems previously reported in these cells. Received: 2 June 1995/Revised: 7 March 1996     

Sodium-dependent and -independent Choline Uptake by Type II Epithelial Cells from Rat Lung

The uptake of ³H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium. Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1 μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected: (i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when Na⁺ was replaced by Li⁺, K⁺ or Mg²⁺, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na⁺ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na⁺ concentration. Kinetic properties of the uptake of 0.1 μm ³H-choline in the presence and absence of medium Na⁺ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na⁺; a Na⁺-dependent portion (a mean of 0.52 of the total) had a K _m for choline of 1.5 μm while K _m in the absence of Na⁺ (Li⁺ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave K_i values of 1.7 μm and 5.0 μm HC-3 for the Na⁺-dependent and -independent fractions. The apparent K _m of the Na⁺-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K _m values reported for high-affinity, Na⁺-dependent choline uptake by neuronal cells. Received: 18 February 1997/Revised: 7 December 1997     

Evidence for Involvement of a Zymogen Granule Na+/H+ Exchanger in Enzyme Secretion from Rat Pancreatic Acinar Cells

We have characterized a Na⁺/H⁺ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na⁺/H⁺ exchanger activity was estimated by measuring Na⁺ or Li⁺ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na⁺- or Li⁺-dependent ZG lysis was enhanced by increasing inward to outward directed H⁺ gradients. Na⁺-dependent ZG lysis was not prevented by an inside-positive K⁺ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na⁺ and H⁺ permeabilities and for coupled Na⁺/H⁺ exchange through an electroneutral carrier. Na⁺- and Li⁺-dependent ZG lysis was inhibited by EIPA (EC₅₀∼25 μm) and benzamil (EC₅₀∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na⁺ and Li⁺ salts only, and were inhibited by EIPA, suggesting the presence of a Na⁺/H⁺ exchanger in the membrane. Na⁺ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na⁺/H⁺ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na⁺ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na⁺ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na⁺ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P < 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na⁺ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P < 0.02). In the absence of Na⁺ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na⁺/H⁺ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini. Received: 7 December 1995/Revised: 2 April 1996     

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

In the E₁ state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di- and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br₂-Titu³⁺ is bound with the highest affinity (<μm), among the tested divalent cations Ca²⁺ binds the strongest, and Na⁺ binds with about the same equilibrium dissociation constant as Mg²⁺ (∼0.8 mm). For alkali ions it exhibits binding affinities following the order of Rb⁺≃ K⁺ > Na⁺ > Cs⁺ > Li⁺. The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na⁺ ions the equilibrium dissociation constant is < 0.01 mm. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na⁺, no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mm. Received: 7 August 2000/Revised: 14 November 2000     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

Gating and Conduction Properties of a Sodium-activated Cation Channel from Lobster Olfactory Receptor Neurons

The gating and conduction properties of a channel activated by intracellular Na⁺ were studied by recording unitary currents in inside-out patches excised from lobster olfactory receptor neurons. Channel openings to a single conductance level of 104 pS occurred in bursts. The open probability of the channel increased with increasing concentrations of Na⁺. At 210 mm Na⁺, membrane depolarization increased the open probability e-fold per 36.6 mV. The distribution of channel open times could be fit by a single exponential with a time constant of 4.09 msec at −60 mV and 90 mm Na⁺. The open time constant was not affected by the concentration of Na⁺, but was increased by membrane depolarization. At 180 mm Na⁺ and −60 mV, the distribution of channel closed times could be fit by the sum of four exponentials with time constants of 0.20, 1.46, 8.92 and 69.9 msec, respectively. The three longer time constants decreased, while the shortest time constant did not vary with the concentration of Na⁺. Membrane depolarization decreased all four closed time constants. Burst duration was unaffected by the concentration of Na⁺, but was increased by membrane depolarization. Permeability for monovalent cations relative to that of Na⁺ (P _X /P _Na ), calculated from the reversal potential, was: Li⁺ (1.11) > Na⁺ (1.0) > K⁺ (0.54) > Rb⁺ (0.36) > Cs⁺ (0.20). Extracellular divalent cations (10 mm) blocked the inward Na⁺ current at −60 mV according to the following sequence: Mn²⁺ > Ca²⁺ > Sr²⁺ > Mg²⁺ > Ba²⁺. Relative permeabilities for divalent cations (P _Y /P _Na ) were Ca²⁺ (39.0) > Mg²⁺ (34.1) > Mn²⁺ (15.5) > Ba²⁺ (13.8) > Na⁺ (1.0). Both the reversal potential and the conductance determined in divalent cation-free mixtures of Na⁺ and Cs⁺ or Li⁺ were monotonic functions of the mole fraction, suggesting that the channel is a single-ion pore that behaves as a multi-ion pore when the current is carried exclusively by divalent cations. The properties of the channel are consistent with the channel playing a role in odor activation of these primary receptor neurons. Received: 17 September 1996/Revised: 15 November 1996     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Sequential Amino Acid Exchange across b0,+-like System in Chicken Brush Border Jejunum

In the small intestine, cationic amino acids are transported by y⁺-like and b^0,+-like systems present in the luminal side of the epithelium. Here, we report the characterization of a b^0,+-like system in the apical membrane of the chicken jejunum, and its properties as an amino acid exchanger. Analysis of the brush border membrane by Western blot points out the presence of rBAT (protein related to b^0,+ amino acid transport system) in these membranes. A functional mechanism for amino acid exchange across this system was established by kinetic analysis measuring fluxes at varying substrate concentrations both in internal (in) and external (out) vesicle compartments. This intestinal b^0,+-like system functions for l-arginine as an obligatory exchanger since its transport capacity increases 100–200 fold in exchange conditions, thus suggesting an important role in the intestinal absorption of cationic amino acids. The kinetic analysis of Arg_in efflux velocities is compatible with the formation of a ternary complex and excludes a model involving a ping-pong mechanism. The binding affinity of Arg_out is higher than that of Arg_in, suggesting a possible order of binding (Arg_out first) for the formation of the ternary complex during the exchange cycle. A model of double translocation pathways with alternating access is discussed. Received: 23 March 2000/Revised: 29 December 2000     

Sodium Blocking Induced by a Point Mutation at the C-Terminal End of the Pore Helix of the KAT1 Channel

A plant hyperpolarization-activating K⁺ channel, KAT1, is highly selective for K⁺ over Na⁺ and is little affected by external Na⁺, which is crucial to take up K⁺ effectively in a Na⁺-containing environment. It has been shown that a mutation at the location (Thr256) preceding the selectivity signature sequence dramatically enhanced the sensitivity of the KAT1 channel to external Na⁺. We report here electrophysiological experiments for the mechanism of action of external Na⁺ on KAT1 channels. The Thr256 residue was substituted with either glutamine (Q) or glutamate (E). The wild-type channel was insensitive to external Na⁺. However, the activity of both mutant channels was significantly depressed by Na⁺ with apparent dissociation constants of 6.7 mm and 11.3 mm for T256Q and T256E, respectively. The instantaneous current-voltage relationships revealed distinct blocking mechanisms for these mutants. For T256Q a typical voltage-dependent fast blocking was shown. On the other hand, the blocking for the T256E mutant was voltage-independent at low Na⁺ concentrations and became voltage-dependent at higher concentrations. At extreme hyperpolarization the blocking was relieved significantly. These data strongly suggest that the mutation at the end of the pore helix rearranged the selectivity filter and allows Na⁺ to penetrate into the pore. Received: 16 October 2000/Revised: 20 February 2001     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October