Counterflow of L-glutamate in plasma membrane vesicles and reconstituted preparations from rat brain

Membrane vesicles from rat brain exhibit sodium-dependent uptake of L-[3H]glutamate in the absence of any transmembrane ion gradients. The substrate specificity of the process is identical with (Na+ + K+)-coupled L-glutamate accumulation. Although these vesicles are prepared after osmotic shock and are washed repeatedly, they contain about 1.5 nmol/mg of protein endogenous L-glutamate, apparently located inside the vesicles. The affinity of the process (Km approximately 1 microM) is similar to that of (Na+ + K+)-dependent accumulation by the L-glutamate transporter. Membrane vesicles have been disrupted by the detergent cholate, and the solubilized proteins have been subsequently reconstituted into liposomes. The reconstituted proteoliposomes also exhibit the above uptake--with the same characteristics--provided they contain entrapped cold L-glutamate. Counterflow is optimal when sodium is present on both sides of the membrane, but partial activity is still observed when sodium is present either on the inside or on the outside. Increasing the L-glutamate concentration above the Km results in counterflow completely independent of cis sodium. The initial rate of counterflow is 100-200-fold lower than that of net trans potassium dependent flux. The rate of net flux in the presence of trans sodium or lithium is about 10-fold lower than when choline or Tris are used instead. However, the rate of counterflow (no internal potassium present) was not stimulated by replacing internal sodium or lithium by internal choline. Therefore, optimal functioning of the transporter requires internal potassium while internal sodium and lithium are inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dependence of the red blood cell calcium pump on the membrane potential

(1) It is shown that the rate of calcium extrusion from intact human red cells is faster at a membrane potential of approximately +50 mV (inside) than at approximately -50 mV. (2) The positive potential applied was the chloride potential of KCl cells in a K-gluconate medium when the Ca2+ sensitive K+ channel was blocked by 0.3mM quinidine. The negative potential resulted from the high K+ permeability in Ca2+ loaded cells (the cells were loaded to a Ca2+ activity in the cell water of about 50 microM). (3) It is further demonstrated that the Ca2+ affinity of the pump ATPase is decreased both at the internal (high affinity) and external (low affinity) site by increasing the proton concentration. Acidification thus inhibits internally and stimulates externally. (4) An indirect effect of the membrane potential on the pump activity via the accompanying pH shifts on either side of the membrane could be ruled out by choosing Ca2+ concentrations which are fully activating at the internal Ca2+ binding site at pH 6.5 and not yet inhibitory at the external Ca2+ binding site at pH 8. (5) The result is compatible with the assumption that the human red cell Ca-pump is exchanging Ca2+ for protons, yet is electrogenic by virtue of a stoichiometry of 1H+:1Ca2+ for this exchange.     

Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl-/HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization.     

Divalent ion trapping inside potassium channels of human T lymphocytes

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]o or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions approximately 25% of the channels were loaded with Ca2+, whereas under conditions with 10 microM internal Ca2+ the proportion of channels loaded with Ca2+ increased to approximately 50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation.     

Uptake of [3H]serotonin into plasma membrane vesicles from mouse cerebral cortex

Preparations of plasma membrane vesicles were used as a tool to study the properties of the serotonin transporter in the central nervous system. The vesicles were obtained after hypotonic shock of synaptosomes purified from mouse cerebral cortex. Uptake of [3H]serotonin had a Na+-dependent and Na+-independent component. The Na+-dependent uptake was inhibited by classical blockers of serotonin uptake and had a Km of 63-180 nM, and a Vmax of 0.1-0.3 pmol mg-1 s-1 at 77 mM Na+. The uptake required the presence of external Na+ and internal K+. It required a Na+ gradient ([Na+]out greater than [Na+]in) and was stimulated by a gradient of K+ ([K+]in greater than [K+]out). Replacement of Cl- by other anions (NO2-, S2O3-(2-)) reduced uptake appreciably. Gramicidin prevented uptake. Although valinomycin increased uptake somewhat, the membrane potential per se could not drive uptake because no uptake was observed when a membrane potential was generated by the SCN- ion in the absence of internal K+ and with equal [Na+] inside and outside. The increase of uptake as a function of [Na+] indicated a Km for Na+ of 118 mM and a Hill number of 2.0, suggesting a requirement of two sodium ions for serotonin transport. The present results are accommodated very well by the model developed for porcine platelet serotonin transport (Nelson, P. J., and Rudnick, G. (1979) J. Biol. Chem. 254, 10084-10089), except for the number of sodium ions that are required for transport.     

ABC transporters: how small machines do a big job

Transporters from the ATP-binding cassette (ABC) superfamily operate in all organisms, from bacteria to humans, to pump substances across biological membranes. Recent high-resolution views of ABC transporters in different conformational states provide clues as to how ATP might be used to drive the structural reorganizations that accompany membrane transport. Importantly, it now appears that a putative translocation pathway running through the center of the transporter might be gated alternately, either at the inside or the outside of the cytoplasmic membrane, coupling substrate translocation to a cycle of ATP-dependent conformational changes. ATP binding and ATP hydrolysis have distinct roles in this cycle: binding favors the outward-facing orientation, whereas hydrolysis returns the transporter to an inward-facing conformation.     

High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.

Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.     

Inhibition of Ca2+-activated K+ channels in pig pancreatic acinar cells by Ba2+, Ca2+, quinine and quinidine

Patch-clamp whole-cell and single-channel current recordings were made from pig pancreatic acinar cells to test the effects of quinine, quinidine, Ba2+ and Ca2+. Voltage-clamp current recordings from single isolated cells showed that high external concentrations of Ba2+ or Ca2+ (88 mM) abolished the outward K+ currents normally associated with depolarizing voltage steps. Lower concentrations of Ca2+ only had small inhibitory effects whereas 11 mM Ba2+ almost blocked the K+ current. 5.5 mM Ba2+ reduced the outward K+ current to less than 30% of the control value. Both external quinine and quinidine (200-500 microM) markedly reduced whole-cell outward K+ currents. In single-channel current studies it was shown that external Ba2+ (1-5 mM) markedly reduced the probability of opening of high-conductance Ca2+ and voltage-activated K+ channels whereas internal Ba2+ (6 X 10(-6) to 3 X 10(-5) M) caused activation at negative membrane potentials and inhibition at positive potentials. Quinidine (200-400 microM) evoked rapid chopping of single K+ channel openings acting both from the outside and inside of the membrane and in this way markedly reduced the total current passing through the channels.     

Kinetics of Cl-dependent K fluxes in hyposmotically swollen low K sheep erythrocytes

A detailed kinetic study of K:Cl cotransport in hyposmotically swollen low K sheep red blood cells was carried out to characterize the nature of the outwardly poised carrier. The kinetic parameters were determined from the rate of K efflux and influx under zero-K-trans conditions in red cells with cellular K altered by the nystatin method and with different extracellular K or Rb concentrations. Although apparent affinities for efflux and influx were quite similar, the maximal velocity for K efflux was approximately two times greater than for influx. Furthermore, at thermodynamic equilibrium (i.e., when the ion product of K and Cl within the cell was equal to that outside) a temperature-dependent net K efflux was observed, approaching zero only when the external product reached approximately two times the internal product. The binding order of the ions to the transporter was asymmetric, being ordered outside (Cl binding first, followed by K) and random inside. K efflux but not influx was trans-inhibited by KCl. Trans inhibition of K efflux was used to verify the order of binding outside: trans inhibition by external Cl occurred in the absence of external K, but not vice versa. Thus K:Cl cotransport is kinetically asymmetric in hyposmotically swollen low K sheep red cells.     

Ionic currents in the human serotonin transporter reveal inconsistencies in the alternating access hypothesis

We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore.     

Potassium blocks barium permeation through a calcium-activated potassium channel

Single high-conductance Ca2+-activated K+ channels from rat skeletal muscle were inserted into planar lipid bilayers, and discrete blocking by the Ba2+ ion was studied. Specifically, the ability of external K+ to reduce the Ba2+ dissociation rate was investigated. In the presence of 150 mM internal K+, 1-5 microM internal Ba2+, and 150 mM external Na+, Ba2+ dissociation is rapid (5 s-1) in external solutions that are kept rigorously K+ free. The addition of external K+ in the low millimolar range reduces the Ba2+ off-rate 20-fold. Other permeant ions, such as Tl+, Rb+, and NH4+ show a similar effect. The half-inhibition constants rise in the order: Tl+ (0.08 mM) less than Rb+ (0.1 mM) less than K+ (0.3 mM) less than Cs+ (0.5 mM) less than NH4+ (3 mM). When external Na+ is replaced by 150 mM N-methyl glucamine, the Ba2+ off-rate is even higher, 20 s-1. External K+ and other permeant ions reduce this rate by approximately 100-fold in the micromolar range of concentrations. Na+ also reduces the Ba2+ off-rate, but at much higher concentrations. The half-inhibition concentrations rise in the order: Rb+ (4 microM) less than K+ (19 microM) much less than Na+ (27 mM) less than Li+ (greater than 50 mM). The results require that the conduction pore of this channel contains at least three sites that may all be occupied simultaneously by conducting ions.     

Binding order of substrates to the sodium and potassium ion coupled L-glutamic acid transporter from rat brain

Efflux of L-glutamic acid from synaptic plasma membrane vesicles requires external potassium. This requirement is saturated by concentrations of about 15 mequiv/L potassium. In the absence of potassium, L-glutamic acid can be released from the vesicles in the presence of external L-glutamic acid. This stimulation does not require external sodium but is dependent on the external concentration of L-glutamic acid. Half-maximal effects are obtained by concentrations of about 1 microM which are very similar to the apparent Km for L-glutamic acid influx. Efflux of labeled glutamate driven by external sodium plus glutamate requires internal sodium. These findings suggest that the transporter displays an asymmetric behavior toward sodium. This ion dissociates much more slowly than L-glutamic acid on the external surface of the membrane but not on the internal surface. Furthermore, it appears that the transporter translocates potassium in a step distinct from the L-glutamic acid translocation step. The simplest explanation is that upon translocation of sodium and L-glutamic acid and their release to the inside, potassium binds to the transporter, enabling it to return to the outside to allow initiation of a new transport cycle.     

External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter

Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving approximately 37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located approximately 13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

Potassium-dependent changes in the conformation of the Kv2.1 potassium channel pore.

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = approximately 265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30-100 microM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.     

The hFbpABC transporter from Haemophilus influenzae functions as a binding-protein-dependent ABC transporter with high specificity and affinity for ferric iron

Pathogenic Haemophilus influenzae, Neisseria spp. (Neisseria gonorrhoeae and N. meningitidis), Serratia marcescens, and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe3+ transport assay, we established an apparent Km=0.9 microM and Vmax=1.8 pmol/10(7)cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m-chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe3+ and selectivity for trivalent metals, including Ga3+ and Al3+, over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli. Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity Kd=5.2 x 10(-4) M(-1), approximately 14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, approximately 35% of wild-type transport, with Km=1.2 microM and Vmax=0.5 pmol/10(7)cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe3+) binding and membrane transport.     

Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels

Nitric oxide (NO) is an inhibitor of hemoproteins including cytochrome P-450 enzymes. This study tested the hypothesis that NO inhibits cytochrome P-450 epoxygenase-dependent vascular responses in kidneys. In rat renal pressurized microvessels, arachidonic acid (AA, 0.03-1 microM) or bradykinin (BK, 0.1-3 microM) elicited NO- and prostanoid-independent vasodilation. Miconazole (1.5 microM) or 6-(2-propargyloxyphenyl)hexanoic acid (30 microM), both of which are inhibitors of epoxygenase enzymes, or the fixing of epoxide levels with 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 3 microM) inhibited these responses. Apamin (1 microM), which is a large-conductance Ca2+-activated K+ (BKCa) channel inhibitor, or 18alpha-glycyrrhetinic acid (30 microM), which is an inhibitor of myoendothelial gap junctional electromechanical coupling, also inhibited these responses. NO donors spermine NONOate (1 and 3 microM) or sodium nitroprusside (0.3 and 3 microM) but not 8-bromo-cGMP (100 microM), which is an analog of cGMP (the second messenger of NO), blunted the dilation produced by AA or BK in a reversible manner without affecting that produced by hydralazine. However, the non-NO donor hydralazine did not affect the dilatory effect of AA or BK. Spermine NONOate did not affect the dilation produced by 11,12-EET, NS-1619 (a BKCa channel opener), or cromakalim (an ATP-sensitive K+ channel opener). AA and BK stimulated EET production, whereas hydralazine had no effect. On the other hand, spermine NONOate (3 microM) attenuated basal (19 +/- 7%; P < 0.05) and AA stimulation (1 microM, 29 +/- 9%; P < 0.05) of renal preglomerular vascular production of all regioisomeric EETs: 5,6-; 8,9-; 11,12-; and 14,15-EET. These results suggest that NO directly and reversibly inhibits epoxygenase-dependent dilation of rat renal microvessels without affecting the actions of epoxides on K+ channels.     

Pharmacological characterization of the serotonin-sensitive potassium channel of Aplysia sensory neurons

The effects of a variety of K+ channel blockers on current flow through single serotonin-sensitive K+ channels (the S channels) of Aplysia sensory neurons were studied using the patch-clamp technique. Tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Co2+ and Ba2+ were first applied to the external membrane surface using cell-free outside-out patches. At concentrations up to 10 mM, these agents had little or no effect on single S-channel currents. At higher concentrations, external TEA acted as a fast open-channel blocker, reducing the single-channel current amplitude according to a simple one-to-one binding scheme with an apparent Kd of 90 mM. Blockage by external TEA is voltage independent. Internal TEA also acts as an open-channel blocker, with an apparent Kd of approximately 40 mM and a relatively weak voltage dependence, corresponding to an apparent electrical distance to the internal TEA-binding site of 0.1. Both internal and external TEA block the open channel selectively, with an affinity that is 10-100-fold greater than the affinity for the closed channel. Internal Ba2+ acts as a slow channel blocker, producing long closures of the channel, and binding with an apparent Kd of approximately 25-30 microM. These results show that single S-channel currents share a similar pharmacological profile with the macroscopic S current previously characterized with voltage clamp. On the basis of these results, a structural model for S-channel opening is proposed.     

Two conformational changes are associated with glutamate translocation by the glutamate transporter EAAC1

Glutamate is transported across membranes by means of a carrier mechanism that is thought to require conformational changes of the transport protein. In this work, we have determined the thermodynamic parameters of glutamate and the Na+ binding steps to their extracellular binding sites along with the activation parameters of rapid, glutamate-induced processes in the transport cycle by analyzing the temperature dependence of glutamate transport at steady state and pre-steady state. Our results suggest that glutamate binding to the transporter is driven by a negative reaction enthalpy (DeltaH0 = -33 kJ/mol), whereas the tighter binding of the non-transportable inhibitor TBOA is caused by an additional increase in entropy. Processes linked to the binding of glutamate and Na+ to the transporter are associated with low activation barriers, indicative of diffusion-controlled reactions. The activation enthalpies of two processes in the glutamate translocation branch of the transport cycle were DeltaH++ = 95 kJ/mol and DeltaH++ = 120 kJ/mol, respectively. Such large values of DeltaH++ suggest that these processes are rate-limited by conformational changes of the transporter. We also found a large activation barrier for steady-state glutamate transport, which is rate-limited by the K+-dependent relocation of the empty transporter. Together, these results suggest that two conformational changes accompany glutamate translocation and at least one conformational change accompanies the relocation of the empty transporter. We interpret the data with an alternating access model that includes the closing and opening of an extracellular and an intracellular gate, respectively, in analogy to a hypothetical model proposed previously on the basis of the crystal structure of the bacterial glutamate transporter GltPh.     

Calcium transport in brain synaptosomes during depolarization. The role of potential-dependent channels and Na+/Ca2+ metabolism

The contribution of Ca2+ channels and Na+/Ca2+ exchange to Ca2+ uptake in rat brain synaptosomes upon long- (t greater than or equal to 30 s) and short-term (t less than 30 s) depolarization by high K+ was studied by measuring the 45Ca content and free Ca2+ concentration (from Quin-2 fluorescence). At 37 degrees C, the system responsible for the K+-stimulated uptake of 45Ca (t greater than or equal to 30 s) and the Na+/Ca+ exchanger are characterized by a similar concentration dependence of external Ca2+ (Ca0(2+] and K0+ as well as by an equal sensitivity to verapamil (Ki = approximately 20-40 microM) and La2+ (Ki = approximately 50 microM). These data and the results from predepolarization suggest that the 45Ca entry into synaptosomes at t greater than or equal to 30 s is due to the activation of Na+/Ca+ exchange caused by its electrogenic component, while the insignificant contribution of Ca2+ channels can be accounted for by their inactivation. At low temperatures (2-4 degrees C) which decelerate the inactivation, the initial phase of 45Ca uptake is fully provided for by Ca2+ channels, showing a lower (as compared to the exchanger) affinity for Ca0(2+) (K0.5 greater than 1 mM)m a greater sensitivity to La3+ (Ki = approximately 0.2-0.3 microM) and verapamil (Ki = approximately 2-3 microM); these channels are fully inactivated by predepolarization with K0+, ouabain and batrachotoxin. The Ca2+ channels can be related to T-type channels, since they are not blocked by nicardipine and niphedipine.