Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2.

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.

Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase {alpha} subunit.

The Na(+)/K(+)-ATPase mediates electrogenic transport by exporting three Na(+) ions in exchange for two K(+) ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb(+) ions in the crystal structures of the Na(+)/K(+)-ATPase has defined two "common" cation binding sites, I and II, which accommodate Na(+) or K(+) ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this "unique" Na(+) binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na(+)/K(+)-ATPase alpha(2) subunit decreases the affinity for extracellular and intracellular Na(+), in agreement with previous biochemical studies. Apparently, the DeltaYY deletion changes Na(+) affinity at site III but leaves the common sites unaffected, whereas the more extensive DeltaKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K(+), the DeltaYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na(+) dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na(+)/Na(+) exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na(+) release/binding mechanism. In analogy to the mechanism proposed for the H(+) leak currents of the wild-type Na(+)/K(+)-ATPase, we suggest that the DeltaYY deletion lowers the energy barrier for the intracellular Na(+) occlusion reaction, thus destabilizing the Na(+)-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na(+)/K(+)-ATPase alpha subunit represents a structural and functional relay between Na(+) binding site III and the intracellular cation occlusion gate.

Sodium-dependent potassium channels of a Slack-like subtype contribute to the slow afterhyperpolarization in lamprey spinal neurons.

The slow afterhyperpolarization (sAHP) following the action potential is the main determinant of spike frequency regulation. The sAHP after single action potentials in neurons of the lamprey locomotor network is largely due to calcium-dependent K+channels (80%), activated by calcium entering the cell during the spike. The residual (20%) component becomes prominent during high level activity (50% of the sAHP). It is not Ca2+ dependent, has a reversal potential like that of potassium, and is not affected by chloride injection. It is not due to rapid activation of Na+/K+-ATPase. This non-KCa-sAHP is reduced markedly in amplitude when sodium ions are replaced by lithium ions, and is thus sodium dependent. Quinidine also blocks this sAHP component, further indicating an involvement of sodium-dependent potassium channels (KNa). Modulators tested do not influence the KNa-sAHP amplitude. Immunofluorescence labelling with an anti-Slack antibody revealed distinct immunoreactivity of medium-sized and large neurons in the grey matter of the lamprey spinal cord, suggesting the presence of a Slack-like subtype of KNa channel. The results strongly indicate that a KNa potassium current contributes importantly to the sAHP and thereby to neuronal frequency regulation during high level burst activity as during locomotion. This is, to our knowledge, the first demonstration of a functional role for the Slack gene in contributing to the slow AHP.

Sodium self-inhibition of human epithelial sodium channel: selectivity and affinity of the extracellular sodium sensing site.

The epithelial Na(+) channel (ENaC) is present in the apical membrane of "tight" epithelia in the distal nephron, distal colon, and airways. Its activity controls the rate of transepithelial sodium transport. Among other regulatory factors, ENaC activity is controlled by the concentration of extracellular Na(+), a phenomenon named self-inhibition. The molecular mechanism by which extracellular Na(+) concentration is detected is not known. To investigate the properties of the extracellular Na(+) sensing site, we studied the effects of extracellular cations on steady-state amiloride-sensitive outward currents in Na(+)-loaded oocytes expressing human ENaC and compared them with self-inhibition of inward current after fast solution changes. About half of the inhibition of outward Na(+) currents was due to self-inhibition itself and the rest might be attributed to conduction site saturation. Self-inhibition by extracellular Li(+) was similar to that of Na(+) except for slightly slower kinetics. Ionic selectivity of the inhibition for steady-state outward current was Na(+) > or = Li(+) > K(+). We estimated an apparent inhibitory constant (K(I)) of approximately 40 mM for extracellular Na(+) and Li(+) and found no evidence for a voltage dependence of the K(I). Protease treatment induced the expected increase of the amiloride-sensitive current measured in high-Na(+) concentrations which was due, at least in part, to abolition of self-inhibition. These results demonstrate that both self-inhibition and saturation play a significant role in the inhibition of ENaC by extracellular Na(+) and that Na(+) and Li(+) interact in a similar way with the extracellular cation sensing site.

Functional characterization of high-affinity Na(+)/dicarboxylate cotransporter found in Xenopus laevis kidney and heart.

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na(+)/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is approximately 70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a K(m) for succinate of 4 muM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.

Xoom: a novel oocyte membrane protein maternally expressed and involved in the gastrulation movement of Xenopus embryos.

During a process of differential display screening for lithium-responsive mRNA in Xenopus embryos, we found a maternal transcript which is remarkably reduced by lithium. The isolated cDNA, designated Xoom, encoded a novel oocyte membrane protein with a signal sequence in the N-terminus and a single transmembrane domain. The extracellular domain contained a cysteine-rich region and a serine/threonine-rich region, which suggests an extracellular association with some proteins. Expression of Xoom maternally occurred in the whole oocyte at the early stage of oogenesis and the transcript was gradually localized in the animal hemisphere of full-grown oocytes. The zygotic expression was detected at first in the dorsoanterior region of the neural fold stage embryo. Thereafter, localized expression of Xoom was observed in neural crest cells of the neural tube stage embryo and in optic and otic vesicles of tadpole. Xoom has been expressed ubiquitously in adult organs, especially with a high level in the eye, heart, liver and kidney. In examining a relation between Xoom and the dorsoventral patterning, lithium-treatment at 32-cell stage embryo decreased Xoom mRNA level within an hour, but coinjection of lithium with myo-inositol reversed the decreasing Xoom mRNA to normal level. UV-irradiation had no effect on the maternal mRNA level of Xoom. Overexpression of Xoom showed no effect on development, but antisense Xoom RNA causes interference with normal gastrulation movement. These results suggest that maternally expressed and membrane-associated Xoom is closely involved in the gastrulation movement through a lithium-inducible signal pathway.

An asparagine residue regulating conductance through P2X2 receptor/channels.

Single channel currents were recorded from Xenopus oocytes expressing wild-type and mutated P2X2 receptors. When 100 mM Na+ was used as the permeant cation, unitary currents of about 80 pS were recorded from the oocyte expressing the wild-type channels. The single channel conductance was roughly halved when Asn333 was replaced by Ile (N333I). A similar decrease in single channel currents was also observed when 100 mM Li+ or Cs+ was used as the permeant cation. With two other mutants, in which Asp315 was replaced by Val (D315V) or Tyr330 was replaced by lie (T333I), single channel conductance was almost the same as that of the wild-type channels. The results suggest that Asn333, which is believed to be involved in the channel pore, plays an essential role in ion transport through P2X2 receptor/channels.

Expression cloning of NaDC-2, an intestinal Na(+)- or Li(+)-dependent dicarboxylate transporter.

A cDNA coding for a Na(+)-dicarboxylate cotransporter from Xenopus laevis intestine, NaDC-2, was isolated by functional expression cloning in Xenopus oocytes. NaDC-2 encodes a 622-residue polypeptide with a predicted mass of 68.6 kDa. The sequence and secondary structure of NaDC-2 are related to the mammalian renal Na(+)-dicarboxylate and Na(+)-sulfate cotransporters. NaDC-2 mRNA is expressed only in the intestine. Oocytes injected with NaDC-2 cRNA exhibit increased transport of succinate, citrate, and glutarate. Transport of succinate by NaDC-2 is stimulated by Na+ or Li+, with Michaelis-Menten constant values for succinate of 0.3 mM (in Na+) and 0.7 mM (in Li+). Na+ and Li+ activation curves show sigmoid kinetics, with Hill coefficients of 1.4 (nNa) and 1.7 (nLi), indicating that multiple cations are involved in the transport of succinate. The transport of succinate by NaDC-2 is insensitive to pH, whereas the transport of citrate is inhibited at high pH. The differences in functional properties between NaDC-2 and the structurally related Na(+)-dicarboxylate cotransporters NaDC-1 and hNaDC-1 will form the basis of detailed structure-function studies.

Monovalent cation conductance of the sustained inward current in rabbit sinoatrial node cells.

Under the whole cell clamp, superfusion of the rabbit sinoatrial node cells with a Na+-free solution suppressed the sustained inward current (Ist), and the L-type Ca2+ current (ICa,L) could be recorded on depolarization less negative than -40 mV from the holding potential of -80 mV. On the other hand, replacement of Ca2+ with Mg2+ in the external solution suppressed inward-going ICa,L and isolated Ist. Under this condition, Ist measured as a nicardipine-sensitive current showed an activation threshold between -60 and -70 mV. The conductance sequence of Ist for monovalent ions was determined as Na+ > Li+ >> K+ approximately = Cs+ by replacing the external Na+ with these alkali metal ions. The contribution of Ist to the diastolic depolarization is discussed.

Confocal microscopic detection of potential-sensitive dyes used to reveal loss of voltage control during patch-clamp experiments.

We used a fast, fluorescent, potential-sensitive indicator (Di-8-ANEPPS) in combination with laser-scanning confocal microscopy in the line-scan mode (temporal resolution 500 Hz) to independently determine the transmembrane potential in voltage-clamped cells. While a linear relation between command voltage and Di-8-ANEPPS fluorescence was found in unexcitable Sf9 cells, pronounced nonlinearities were observed in cardiac myocytes. Comparison of the fluorescence records and current traces indicated that most of the observed nonlinearities could be attributed to voltage-escape during flow of membrane current. Voltage-escape during large membrane currents may lead to various experimental difficulties during voltage-clamp experiments. The voltage recording technique based on fluorescence was then used to compare the voltage-escape during flow of Na+ and Li+ ions via voltage-dependent (TTX sensitive) Na+ channels in cardiac myocytes. In these experiments, no significant differences in the degree of voltage-escape was found, suggesting that the two currents were similar in amplitude. In addition to the application presented in this paper, confocal microscopic detection of transmembrane potential with fluorescent dyes may be a useful technique for experiments in preparations that are difficult to impale with microelectrodes because of their small size.

Reappraisal of the role of sodium ions in excitation-contraction coupling in frog twitch muscle.

Tetanic and twitch tension were recorded on isolated frog twitch fibres under experimental conditions modifying the influx of sodium ions. In current clamp conditions replacing Li+ for Na+ did not modify the electrical activity but drastically decreased the plateau of tetanic tension. In voltage clamp conditions replacing Li+ for Na+ did not modify the inward currents but induced a marked decrease of the plateau of the tetanic tension for depolarizations between the activation threshold and the reversal potential of sodium current. Under veratridine treatment, during tetanic depolarization, a slow inward sodium (or lithium) current developed. This induced a parallel increase of the tetanic tension which was much more pronounced in sodium than in lithium containing solution. The twitch tension obtained during short depolarization was increased by greater than 100% during veratridine treatment with a sizeable decrease (40%) of the delay between the end of depolarization and the beginning of tension. All these results could be reproduced in calcium-free solution. Our data confirm that the entry of sodium ions (and to a lesser extent of lithium ions) is able to modulate the release of calcium from the sarcoplasmic reticulum (SR). We discuss these results in terms of a model where sodium ions entering the compartment between the tubular membrane and the SR junctional membrane carry counter charges through the SR K+ channels and help to maintain the SR Ca2+ release. This could occur in particular during a physiological tetanic contraction where the junctional compartment is probably filled with Na+ ions and depleted of K+ ions.

Rhythms and the pharmacology of lithium.

Lithium is the treatment of choice for bipolar affective disorder (manic-depression) and is useful in other recurrent affective and nonaffective illnesses. This review discusses lithium's actions on period, phase, amplitude and coupling of biological rhythms that may relate to its therapeutic effectiveness. Alternatively, lithium might interact with environmental light to influence circadian rhythms by an action on the retina. The mechanisms responsible for lithium's chronopharmacological actions are not known, but cellular cations, phosphoinositide or adenylate cyclase second messenger systems, hormones and neurotransmitters may all be involved.

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)-K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip-V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage-sensitive steps in the cardiac Na(+)-K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Lithium increases the alpha 1-adrenoceptor mediated inotropic effect in rat heart.

The intracellular mechanisms activated by stimulation of myocardial alpha 1-adrenoceptors are not known. As in several other tissues, however, activation of alpha 1-adrenoceptors in heart has been related to breakdown of phosphoinositides resulting in production of putative intracellular messengers: different inositol phosphates and diacylglycerol. Lithium has been shown to inhibit enzymes hydrolyzing inositol phosphates. In the present paper we report studies on the effect of lithium upon the alpha 1-adrenoceptor mediated inotropic response elicited in electrically driven rat papillary muscles. While there was no shift of the horizontal positioning of the dose-response curve to alpha 1-adrenergic stimulation in the presence of lithium, the alpha 1-adrenoceptor mediated inotropic effect was increased in a concentration dependent manner (0.25 to 3.0 mmol/l lithium). For comparison, the effect of lithium upon the beta-adrenoceptor mediated inotropic response was also studied. At 3.0 mmol/l lithium, the horizontal position of the dose-response curve to beta-adrenoceptor stimulation was shifted significantly to the right (to higher agonist concentrations) and the maximal beta-adrenoceptor mediated inotropic response was slightly although not significantly reduced. Thus the augmenting effect of lithium upon the alpha 1-adrenoceptor mediated response was specific for this receptor type. Although the effect of lithium may be complex, the data are compatible with the hypothesis that the inositol phosphates may be of functional importance during stimulation of myocardial alpha 1-adrenoceptors.