Presence Of External Na Research Articles

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

CrossTalk proposal: Apical NKCC1 of choroid plexus epithelial cells works in the net inward flux mode under basal conditions, maintaining intracellular Cl- and cell volume.

Contribution of the Hydroxyl Group and Phenol Ring of Tyrosine 780 of the Alpha-Subunit to NA+ Binding by the Na/K Pump

The Na/K pump is a heterodimeric(αβ) P-type ATPase that exports three Na+ and imports two K+ against their electrochemical gradients using the energy of ATP hydrolysis. Structural studies confirm three ion-binding sites in the catalytic α-subunit. Site-I and Site-II bind either Na+ or K+, while Site-III, the focus here, exclusively binds Na+. To determine the relative energetic contribution to Na+ binding of the hydroxyl group or the π-electrons of Tyr780 in site-III, we introduced the mutation Y780tag into the α-subunit cDNA for the purpose of using nonsense suppression. Y780tag-α- and β3-cRNA were co-injected in Xenopus oocytes with synthetic tRNA (ligated to Tyr, Phe or Phe derivatives) to compare the functional consequences of the chemical modifications at this position using two-electrode voltage clamp. Square voltage pulses in the absence of external K+ (K+o) and presence of external Na+ (Na+o) produce transient currents. The voltage dependence of steady-state charge moved by these transients is described by a Boltzmann distribution with a center (V1/2) related to the relative apparent affinity for Na+o (ΔV1/2= ­25 mV per 2-fold reduction in affinity). The centers of the distribution where V1/2= ­46.9 ±2.5 mV (n=10) for Tyr, V1/2= ­123 ± 9 mV for Phe, and V1/2= −54.6 ±2.1 mV (n=5) for mono-fluorinated at the 3’ position (mono-F)-Tyr. These results indicate ∼8-fold reduction in apparent affinity for Na+o by removal of the hydroxyl group and a smaller reduction (≪ 2-fold) by partial disruption of the cation-π interaction capability by monofluorination. Surprisingly, the apparent affinity for K+o estimated from the [K+]o dependence of pump current activation in the absence of Na+o was reduced by both Phe (∼4-fold) and mono-F-Tyr (∼1.5-fold). Measurements with other derivatives are underway. NSF-MCB 1515434.

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris — functional and kinetic asymmetry revealed in proteoliposomes

The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni2+-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [3H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na+. Internal Na+ slightly stimulated the antiport. Optimal activity was found at pH7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥70%) and by mercurials and methanethiosulfonates (≥80%). Heterologous antiport of [3H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [3H]glutamine/glutamine antiport Km values of 0.097 and 1.8mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na+ on the external side was 32mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.

Erratum to: External K+ Deficiency Inhibits Photosynthetic Activity Through Superoxide Anion Production in Protoplasts Isolated from the Thallus of Ulva pertusa

To investigate the effects of potassium on photosynthetic activity in the green alga Ulva pertusa, we enzymatically isolated protoplasts from thallus samples. Photochemical quenching showed that protoplasts were capable of electron transport by photosystem II (PS II) during illumination. This quenching was dependent on external pH, with a reduced electron transport rate at pH >6.8 and less ability to use HCO 3 − under alkaline conditions. In the presence of external Na+, K+ enhanced PS II quantum yield, indicating a functional role for K+ during photosynthesis. That yield was enhanced in a [K+]-dependent manner, with maximum activity at 100 mM. However, potassium alone did not maintain photochemical activity, and its addition supported photosynthetic O2 evolution only in the presence of Na+. A deficiency of K+ led to the production of superoxide anions. Because of that generation, activities of superoxide dismutase and ascorbate peroxidase, two key enzymes involved in scavenging reactive oxygen species in the water–water cycle, also increased during such stress. These results strongly suggest that a series of ROS-scavenging systems are initiated in Ulva chloroplasts in response to K+ deficiency and that enzyme activities might protect algal cell photosynthesis.

Probing the Structure of the hSGLT1 Sugar Binding Site

The crystal structure of vSGLT shows that sugar is bound in the center of the protein separated from the extra‐ and intracellular compartments by gates. We have investigated the sugar binding vestibule of the human Na+/glucose cotransporter hSGLT1 by labeling residues in the sugar binding site with tetramethylrhodamine methanethiosulfonate (TMR‐MTS). H83, T287, and Y290 were individually mutated to cysteine, expressed in Xenopus oocytes, and their access to external TMR‐MTS was monitored by simultaneous fluorescence and electrophysiological recordings. Treatment with TMR‐MTS completely inhibited sugar transport but only in the presence of external Na+. Voltage‐jumps elicited changes in rhodamine fluorescence which followed the presteady state charge movements for T287C and Y290C. Labeling blocked the H83C capacitive currents. There was no effect of external sugar on the fluorescence but, in contrast, rhodamine fluorescence changes were dependent on the external Na+. In summary, external TMR‐MTS gains access to the sugar binding site and blocks sugar binding but not sodium‐dependent partial reactions. We conclude that external Na+ triggers a conformational change in hSGLT1 to open the external gate and permit sugar binding. This large conformational change can accommodate molecules with a volume up to 1,700 Å3, i.e. TMR‐MTS or >50 water molecules. Support by NIH grant DK 19567 to E.M.W.

Bridging the gap between structure and kinetics of human SGLT1

The Na(+)-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na(+) electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na(+) cotransporters have shown that Na(+) transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K(0.5)); e.g., mutating sugar binding residues increases the glucose K(0.5) by up to three orders of magnitude. Mutation of the external gate residues increases the Na(+) to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant (K(i)) are proportional to the changes in sugar K(0.5), except in the case of F101C, where phlorizin K(i) increases by orders of magnitude without a change in glucose K(0.5). We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na(+), demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na(+) and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

Sodium instead of potassium and chloride is an important macronutrient to improve leaf succulence and shoot development for halophyte Sesuvium portulacastrum

Soil salinity is contributed largely by NaCl but some halophytes such as Sesuvium portulacastrum have evolved to adapt salinity environment and demonstrate optimal development under moderate salinity. To elucidate the detail mechanisms of the great salt tolerance and determine the respective contributions of Na(+), K(+) and Cl(-) on the development of S. portulacastrum, morphological and physiological analysis were performed using plants supplied with 200 mM of different ions including cations (Na(+), K(+), Li(+)) and anions (Cl(-), NO(3)(-), Ac(-)) respectively. The results revealed that the salt-treated plants accumulated large amounts of sodium in both leaf and stem. There was a greater shoot growth in presence of external Na(+) compared to K(+) and Cl(-). Na(+) was found more effective than K(+) and Cl(-) in cell expansion, leaf succulence, and shoot development. Flame emission and X-Ray microanalysis revealed the relative Na(+) content was much higher than K(+) and Cl(-) in both leaf and stem of well developed S. portulacastrum, leading to a higher Na(+)/K(+) ratio. The effects of different ions on the development of S. portulacastrum were listed as the following: Na(+) > NO(3)(-) > CK > Cl(-) > K(+) > Ac(-) > Li(+). These results demonstrated NaCl toxicity is attributable largely to the effect of Cl(-) but rarely to Na(+), and thus sodium is concluded as a more important macronutrient than potassium and chloride for improving leaf succulence and shoot development of halophyte S. portulacastrum.

Kinetics of hyperosmotically stimulated Na-K-2Cl cotransporter in Xenopus laevis oocytes

A detailed study of hypertonically stimulated Na-K-2Cl cotransport (NKCC1) in Xenopus laevis oocytes was carried out to better understand the 1 K(+):1 Cl(-) stoichiometry of transport that was previously observed. In this study, we derived the velocity equations for K(+) influx under both rapid equilibrium assumptions and combined equilibrium and steady-state assumptions and demonstrate that the behavior of the equations and curves in Lineweaver-Burke plots are consistent with a model where Cl(-) binds first, followed by Na(+), a second Cl(-), and then K(+). We further demonstrate that stimulation of K(+) movement by K(+) on the trans side is an intrinsic property of a carrier that transports multiple substrates. We also demonstrate that K(+) movement through NKCC1 is strictly dependent upon the presence of external Na(+), even though only a fraction of Na(+) is in fact transported. Finally, we propose that the larger transport of K(+), as compared with Na(+), is a result of the return of partially unloaded carriers, which masks the net 1Na(+):1K(+):2Cl(-) stoichiometry of NKCC1. These data have profound implications for the physiology of Na-K-2Cl cotransport, since transport of K-Cl in some conditions seems to be uncoupled from the transport of Na-Cl.

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na + dependent [ 3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K +. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [ 3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na +and internal K +. The transporter showed low if there is any tolerance towards the substitution of Na + or K + for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na + concentration cooperativity index close to 1 was derived, indicating that 1 Na + should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na + transport. Optimal rate of 0.1 mM [ 3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5–7.0. [ 3H]glutamine (and [ 3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl 2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na + dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.

Phosphorylation of Phospholemman (FXYD1) by Protein Kinases A and C Modulates Distinct Na,K-ATPase Isozymes

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K(+) and Na(+) affinity of Na,K-ATPase ( Crambert, G., Fuzesi, M., Garty, H., Karlish, S., and Geering, K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11476-11481 ). In this study, we use the Xenopus oocyte expression system to study the role of phospholemman phosphorylation by protein kinases A and C in the modulation of different Na,K-ATPase isozymes present in the heart. Phosphorylation of phospholemman by protein kinase A has no effect on the maximal transport activity or on the apparent K(+) affinity of Na,K-ATPase alpha1/beta1 and alpha2/beta1 isozymes but increases their apparent Na(+) affinity, dependent on phospholemman phosphorylation at Ser(68). Phosphorylation of phospholemman by protein kinase C affects neither the maximal transport activity of alpha1/beta1 isozymes nor the K(+) affinity of alpha1/beta1 and alpha2/beta1 isozymes. However, protein kinase C phosphorylation of phospholemman increases the maximal Na,K-pump current of alpha2/beta1 isozymes by an increase in their turnover number. Thus, our results indicate that protein kinase A phosphorylation of phospholemman has similar functional effects on Na,K-ATPase alpha1/beta and alpha2/beta isozymes and increases their apparent Na(+) affinity, whereas protein kinase C phosphorylation of phospholemman modulates the transport activity of Na,K-ATPase alpha2/beta but not of alpha1/beta isozymes. The complex and distinct regulation of Na,K-ATPase isozymes by phosphorylation of phospholemman may be important for the efficient control of heart contractility and excitability.

Heterologous Expression of the Glutamine Transporter SNAT3 in Xenopus Oocytes Is Associated with Four Modes of Uncoupled Transport

The glutamine transporter SNAT3 (SLC38A3, former SN1) plays a major role in glutamine release from brain astrocytes and in glutamine uptake into hepatocytes and kidney epithelial cells. Here we expressed rat SNAT3 in oocytes of Xenopus laevis and reinvestigated its transport modes using two-electrode voltage clamp and pH-sensitive microelectrodes. In addition to the established coupled Na+-glutamine-cotransport/H+ antiport, we found that there are three conductances associated with SNAT3, two dependent and one independent of the amino acid substrate. The glutamine-dependent conductance is carried by cations at pH 7.4, whereas at pH 8.4 the inward currents are still dependent on the presence of external Na+ but are carried by H+. Mutation of threonine 380 to alanine abolishes the cation conductance but leaves the proton conductance intact. Under Na+-free conditions, where the substrate-dependent conductance is suppressed, a substrate-independent, outwardly rectifying current becomes apparent at pH 8.4 that is carried by K+ and H+. In addition, we identified a glutamine-dependent uncoupled Na+/H+ exchange activity that becomes apparent upon removal of Na+ in the presence of glutamine. In conclusion, our results suggest that, in addition to coupled transport, SNAT3 mediates four modes of uncoupled ion movement across the membrane.

Kinetic evidence that the Na-PO4 cotransporter is the molecular mechanism for Na/Li exchange in human red blood cells.

The molecular basis for Na/Li exchange is unknown. Li can be transported by the Na pump, anion exchanger (AE1), a background leak, and the Na/Li exchanger. In vivo the intraerythrocyte concentration of Li results from the balance of passive entry, mostly on AE1, and the active extrusion on the Na/Li exchanger. Here we show that erythrocytes have Li-activated PO4 transport that behaves as if it is mediated by the Na-PO4 cotransporter (hBNP1) and provide evidence that this Na/Li-PO4 cotransporter is also the mechanism for Na/Li exchange. First, external Li (>20 mM) activated PO4 influx severalfold. Li activation of PO4 influx was potentiated by the presence of external Na. Second, the ouabain-insensitive 22Na efflux was stimulated by external Li and then inhibited by external PO4. Third, phloretin inhibited Na- and Li-activated PO4 flux with the same Ki, 0.25 mM. Fourth, external PO4 (0.1-1.0 mM) inhibited ouabain-insensitive Li efflux only if external Na was present. Fifth, arsenate, a phosphate congener, inhibited both Na-PO4 cotransport and Li-activated PO4 flux with similar kinetics when Na or Li concentration was high but did not inhibit Liout/Nain exchange when Liout concentration was low. The collective results suggest that both Na and Li are substrates for at least two sites on the same PO4 cotransporter and that Na/Li exchange behaves as if it is mediated by this Na/Li-PO4 cotransporter when only one cation is bound. Plasma and intracellular PO4 concentrations may be important regulators of Li transport and its therapeutic effects.

Role of Na+/Ca2+ exchange in [Ca2+]i clearance in rat culture Purkinje neurons requires reevaluation.

Previous studies have shown that in contrast to other neuronal cells, Na(+)/Ca(2+) exchange contributes little to Ca(i)(2+) homeostasis in rat cerebellar Purkinje neurons under intracellular perfused conditions and at room temperature [Fierro et al.: J Physiol (Lond) 510: 499-512, 1998]. The purpose of this study was to clarify the role of this transporter in cerebellar Purkinje neurons by using intact cells at nearly physiological body temperature. Using Fluo-3 microfluorometry, we have examined the role of the Na(+)/Ca(2+) exchange in the buffering of calcium loads in cultured rat Purkinje neurons at two temperatures: 20 and 34 degrees C. At 20 degrees C, the recovery of the K(+)-induced [Ca(2+)](i) signal was little affected by the presence of external Na(+) (tau(e) = 35.5 +/- 1.2 s [n = 49]), or by its absence (tau(e) = 36.6 +/- 2.2 s [n = 29]), i.e. in a Li(+)-containing medium. In contrast, at 34 degrees C, the recovery of the [Ca(2+)](i) signal was highly dependent on external Na, i.e. tau(e) = 19.9 +/- 1.2 s (n = 119) and tau(e) = 41.7 +/- 2.6 s (n = 39), in Li(+)-containing media, respectively. A comparison of the rate of clearance of [Ca(2+)](i) in Na(+) or Li(+) media, shows that at a room temperature of 20 degrees C, the Na(+)/Ca(2+) exchange contributes at most to 15-20% of the total [Ca(2+)](i) clearance, compared to 55-65% at 34-36 degrees C. We also demonstrate that under normal physiological conditions forward and reverse Na(+)/Ca(2+) exchanges operate in the same neuron. We conclude that the Na(+)/Ca(2+) exchange is strongly suppressed at room temperature and therefore its role should be reevaluated among different neuronal preparations.

The ionic composition of the contractile vacuole fluid of Paramecium mirrors ion transport across the plasma membrane

In vivo K+, Na+, Ca2+, Cl- and H+ activities in the cytosol and the contractile vacuole fluid, the overall cytosolic osmolarity, the fluid segregation rate per contractile vacuole and the membrane potential of the contractile vacuole complex of Paramecium multimicronucleatum were determined in cells adapted to 24 or 124 mosm l(-1) solutions containing as the monovalent cation(s): 1) 2 mmol l(-1) K+; 2) 2 mmol l(-1) Na+; 3) 1 mmol l(-1) K+ plus 1 mmol l(-1) Na+; or 4) 2 mmol l(-1) choline. In cells adapted to a given external osmolarity i) the fluid segregation rate was the same if adapted to either K+ or Na+, twice as high when adapted to solutions containing both K+ and Na+, and reduced by 50% or more in solutions containing only choline, ii) the fluid of the contractile vacuole was always hypertonic to the cytosol while the sum of the ionic activities measured in the fluid of the contractile vacuole was the same in cells adapted to either K+ or Na+, at least 25% higher in cells adapted to solutions containing both K+ and Na+, and was reduced by 55% or more in solutions containing only choline, iii) the cytosolic osmolarity was the same in cells adapted to K+ alone, to Na+ alone or to both K+ and Na+, whereas it was significantly lower in cells adapted to choline. At a given external osmolarity, a positive relationship between the osmotic gradient across the membrane of the contractile vacuole complex and the fluid segregation rate was observed. We conclude that both the plasma membrane and the membrane of the contractile vacuole complex play roles in fluid segregation. The presence of external Na+ moderated K+ uptake and caused the Ca2+ activity in the contractile vacuole fluid to rise dramatically. Thus, Ca2+ can be eliminated through the contractile vacuole complex when Na+ is present externally. The membrane potential of the contractile vacuole complex remained essentially the same regardless of the external ionic conditions and the ionic composition of the fluid of the contractile vacuole. Notwithstanding the large number of V-ATPases in the membrane of the decorated spongiome, the fluid of the contractile vacuole was found to be only mildly acidic, pH 6.4.

RNA from heart of young and old rats leads to the expression of protein(s) in Xenopus oocytes that alter the transport activity of rat Na+,K+-ATPases differently.

To address the question of whether the function of Na+,K+-ATPases differs in the heart of young and old rats, enzymes formed from the alpha1 or alpha2 isoform with the beta1 subunit of rat were expressed in Xenopus oocytes. In addition to injections of the cRNA coding for the respective subunits, oocytes were co-injected with total RNA from the left ventricle of young or old rats. To assess alterations in transport activity due to the co-injections, ouabain-sensitive 86Rb+ uptake was measured. Co-injection of the RNA from young rats led to 31% inhibition of 86Rb+ uptake into oocytes with the alpha1/beta1 pumps while uptake into oocytes with the alpha2/beta1 pumps was hardly affected. Co-injection of the RNA from old rats, on the other hand, reduced 86Rb+ uptake only in cells with the alpha2/beta1 isoform (to 85%). The steady-state current generated in the absence of external Na+ by the alpha1/beta1 ATPase was significantly reduced by co-injection of RNA only from young rats to 70%, and this inhibition was hardly affected by membrane potential. For the alpha2/beta1 ATPase co-injection of RNA only from old rats also led to a significant reduction of pump-mediated current at potentials more negative than -70 mV to 70-80%. In the presence of Na+, inhibition of the alpha1 isoform by co-injection of RNA from young rats is voltage-dependent, increasing with more negative potentials. For the alpha2/beta1 pump, co-injection of RNA from old rats was no longer effective, but voltage-dependent inhibition by co-injection of RNA from young rats became apparent. The data indicate that changes in protein expression occurring in young and old rat hearts may modulate transport activity of the Na+,K+-ATPase and this modulation depends on membrane potential and the presence of external Na+. We propose that the described mechanisms may play a functional role in working myocardium, and may form a basis for processes involved in heart aging.

Na+-Ca2+ exchange and its implications for calcium homeostasis in primary cultured rat brain microvascular endothelial cells.

1. The role of Na+-Ca2+ exchange in the regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was studied in primary cultured rat brain capillary endothelial cells. [Ca2+]i was measured by digital fluorescence imaging in cells loaded with fura-2. 2. ATP (100 microM) applied for a short period of time (6 s) caused a rise in [Ca2+]i from 127 +/- 3 (n = 290) to 797 +/- 25 nM, which then declined to the resting level, with a t time required for [Ca2+]i to decline to half of peak [Ca2+]i) of 5.4 +/- 0.09 s. This effect was independent of external Ca2+ and could be abolished by previously discharging the Ca2+ pool of the endoplasmic reticulum with thapsigargin (1 microM). 3. Application of thapsigargin (1 microM) or cyclopiazonic acid (10 microM) to inhibit the Ca2+-ATPase of the endoplasmic reticulum 6 s prior to ATP application did not influence the peak [Ca2+]i but greatly reduced the rate of decline of [Ca2+]i, with t values of 15 +/- 1.6 and 23 +/- 3 s, respectively. 4. In the absence of external Na+ (Na+ replaced by Li+ or N-methylglucamine) the basal [Ca2+]i was slightly elevated (152 +/- 6 nM) and the restoration of [Ca2+]i after the ATP stimulation was significantly slower (t , 7.3 +/- 0.46 s in Li+ medium, 8.12 +/- 0.4 s in N-methylglucamine medium). 5. The external Na+-dependent component of the [Ca2+]i sequestration was also demonstrated in cells stimulated by ATP subsequent to addition of cyclopiazonic acid; in a Na+-free medium [Ca2+]i remained at the peak level in 88 % of the cells after stimulation with ATP. 6. Addition of monensin (10 microM) in the presence of external Na+ increased the resting [Ca2+]i to 222 +/- 9 nM over approximately 1 min and subsequent removal of extracellular sodium resulted in a further increase in [Ca2+]i to a peak of 328 +/- 11 nM, which was entirely dependent on external Ca2+. 7. These findings indicate that a functional Na+-Ca2+ exchanger is present at the blood-brain barrier, which plays a significant role in shaping the stimulation-evoked [Ca2+]i signal and is able to work in reverse mode under pharmacological conditions.

Characterization of the electrogenic Na +–K + pump in horizontal cells isolated from the carp retina

The electrogenic Na +–K + pump current in horizontal cells acutely dissociated from the carp retina was investigated using a nystatin-perforated patch recording configuration under voltage-clamp conditions. In the presence of suitable blockers for known voltage-dependent Na +, K + and Ca 2+ conductances, the pump current was activated in a concentration-dependent manner by adding K + ions to external solution. The EC 50 value and Hill coefficient for the external K + concentration were 0.66 mM and 1.39, respectively. The pump current did not show any significant voltage dependency at the physiological potential range between −90 and 20 mV either with or without external Na + ions. In the presence of 120 mM external Na + concentration, the addition of 3 mM K + to the external solution induced a steady outward pump current even when the patch-pipette (internal) solution did not contain Na +. A large outward shift of the holding current was observed by removing external Na +. The result thus suggests that continuous Na + influxes exist across the plasma membrane in the presence of external Na +. When Na + was removed from both external and internal solutions, a transient outward pump current was observed by adding K + to the external solution, thus indicating that the transient pump current was activated by the residual intracellular Na + ions. The pump current was suppressed by ouabain in a concentration-dependent manner, and the ouabain-sensitive inhibition curve was fitted by two components. The IC 50 values of high- and low-sensitive pump currents for ouabain were 20 nM and 10.4 μM, respectively, indicating the existence of at least two isoforms of the pump in the horizontal cells.

Verapamil block of the delayed rectifier K current in chick embryo dorsal root ganglion neurons.

We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.

Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore.

To explore the role of pore-lining amino acids in Na+ channel ion-selectivity, pore residues were replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+ > Li+ > NH4+ > > Cs+ and were impermeable to divalent cations. Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4+ and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4+ > K+ > Na+ > or = Li+ approximately Cs+), D1532C, and G1533C (Na+ > Li+ > or = NH4+ > K+ > Cs+). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH4+ > K+ > Na+ > or = Li+ approximately Cs+) and was permeable to divalent cations having the selectivity sequence Ca2+ > or = Sr2+ > Mg2+ > Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na+ channel.

Voltage-dependent Na(+)-HCO3- cotransporter and Na+/H+ exchanger are involved in intracellular pH regulation of cultured mature rat cerebellar oligodendrocytes.

Intracellular pH (pHi) was measured at 37 degrees C in mature rat cerebellar oligodendrocytes dissociated in culture by using the pH-sensitive probe BCECF. Cells were identified by anti-galactocerebroside antibody. The mean steady-state pHi was 7.02 in the absence of CO2/bicarbonate (Hepes-buffered solution) at an external pH of 7.40 and 7.04 in 5% CO2/25 mM bicarbonate-buffered solution at the same external pH; this value was modified neither by the removal of external chloride nor by the addition of the chloride-coupled transport blocker DIDS. In both external solutions steady-state pHi values were strongly dependent on external pH. In Hepes-buffered solution pHi recovery following an acid load required external Na+ and was completely inhibited by amiloride, indicating the presence of a Na+/H+ exchanger. In CO2/bicarbonate-buffered solution amiloride partially reduced the pHi recovery rate, indicating the presence of a bicarbonate-dependent pHi regulating mechanism. Membrane depolarization induced by increasing external K+ concentration elicited an alkalinization only in the presence of external Na+ and bicarbonate. Analysis of the calculated HCO3- fluxes with respect to membrane potential indicated that these fluxes were mediated by a Na(+)-HCO3- cotransport with a stoichiometry of 1:3. These results demonstrate that a Na+/H+ exchanger and a Na(+)-HCO3- cotransporter are involved in pHi regulation of mature oligodendrocytes.