Transport of lithium and rectification by frog skin

The isolated frog skin, bathed with Li⁺-Ringer (Na⁺-free) on the outside and Na⁺-Ringer on the inside, can maintain a normal potential difference (PD) and short-circuit current (s.c.c.) for more than 6. h. The s.c.c. correspondended to the Li⁺ influx. The Na⁺ efflux was 4% of the s.c.c. 10⁻⁵ M ouabain depressed Li⁺ influx and s.c.c. 10¹⁰⁻⁵ M amiloride abolished the Li⁺ s.c.c., while 0.1 unit/ml oxytocin stimulated it. When the inside of the skin was bathed with Li⁺-Ringer, PD and s.c.c. fell to zero within 2 h. The oxygen consumption of skin slices bathed in Li⁺-Ringer was 29% lower than controls bathed in Na⁺-Ringer.When the isolated frog skin is bathed in Na₂SO₄-Ringer it shows electrical rectification which has been correlated with the active transport of Na⁺. In skins transporting Li⁺, rectification characteristics are similar to those of skins transporting Na⁺. When the inner face of the skin is bathed with Li⁺-Ringer, rectification, PD and s.c.c. decline in a parallel fashion.It is concluded that: (1) Li⁺ can be transported when Na⁺ is present at the inner face. (2) Amiloride, ouabain and oxytocin affect Li⁺ and Na⁺ transport in a similar manner. (3) Li⁺ transport, like Na⁺ transport, is associated with rectification. (4) Active transport of Na⁺ and Li⁺ seems to depend on two different but associated proceses; one taking place at the external barrier (where rectification occurs) as shown by the effect of amiloride; and the other of an inner site related to energy requirements and affected by ouabain and Li⁺. (5) The cation being transported is not necessarily activating the (Na⁺-K⁺-ATPase.     

Lithium transport across isolated frog skin epithelium

Summary Transepithelial Li⁺ influx was studied in the isolated epithelium from abdominal skin ofRana catesbeiana. With Na⁺-Ringer's as inside medium and Li⁺-Ringer's as outside medium, the Li⁺ influx across the epithelium was 15.6 A/cm². This influx was considerably reduced by removal of either Na⁺ or K⁺ from the inside bath or by the addition of ouabain or amiloride. Epithelial K⁺ or Na⁺ concentration was respectively lower in epithelia bathed in K⁺-free Ringer's or Na⁺-free Ringer's. In conditions of negligible Na⁺ transport, a 20mm Li⁺ gradient (outin) produced across the short-circuited epithelium a Li⁺ influx of 11.8 A/cm² and a mean short-circuit current of 10.2 A/cm². The same Li⁺ gradient in the opposite direction produced a Li⁺ outflux of only 1.9 A/cm². With equal Li⁺ concentration (10.3 and 20.6mm) on both sides of the epithelium, plus Na⁺ in the inside solution only, a stable Li⁺-dependent short-circuit current was observed. Net Li⁺ movement (outin) was also indirectly determined in the presence of an opposing Li⁺ gradient. Although Li⁺ does not substitute for Na⁺ as an activator of the (Na⁺+K⁺)-ATPase from frog skin epithelium, Li⁺ influx appears to be related to Na⁺–K⁺ pump activity. It is proposed that the permeability of the outer barrier to Na⁺ and Li⁺ is regulated by the electrical gradient produced by electrogenic Na⁺–K⁺ pumps located in the membrane of the deeper epithelial cells.     

Role of the inward K rectifier in the repetitive activity at the depolarized level in single ventricular myocytes

The role of the inward K⁺ rectifier in the repetitive activity at depolarized levels was studied in guinea pig single ventricular myocytes by voltage- and current-clamp methods. In action potentials arrested at the plateau by a depolarizing current, small superimposed hyperpolarizing currents caused much larger voltage displacements than at the resting potential and sometimes induced a regenerative repolarization. Around –20 mV, sub- and suprathreshold repetitive inward currents were found. In the same voltage range, small hyperpolarizing currents reversed their polarity. During depolarizing voltage-clamp ramps, around –20 mV there was a sudden decrease in the outward current (I_ns: current underlying the negative slope in the inward K⁺ rectifier steady state I–V relation). During repolarizing ramps, the reincrease in outward current was smaller and slower. During depolarizing and repolarizing current ramps, sudden voltage displacements showed a similar asymmetry. Repetitive I_ns could continue as long as the potential was kept at the level at which they appeared. Depolarizing voltage-clamp steps also caused repetitive I_ns and depolarizing current steps induced repetitive slow responses. Cadmium and verapamil reduced I_ns amplitude during the depolarizing ramp. BRL 34915 (cromakalim), an opener of the ATP-sensitive K⁺ channel, eliminated the negative slope and I_ns, whereas barium increased I_ns frequency (an effect abolished by adding BRL). Depolarization-induced slow responses persisted in an NaCl-Ca-free solution. Thus, the mechanism of repetitive activity at the depolarized level appears to be related to the presence of the negative slope in the inward K⁺ rectifier I–V relation.     

Diffusion and Electric Mobility of Ions within Isolated Cuticles of Citrus aurantium: Steady-State and Equilibrium Values

We report a new method for measuring cation and anion permeability across cuticles of sour orange, Citrus aurantium, leaves. The method requires the measurement of two electrical parameters: the diffusion potential arising when the two sides of the cuticle are bathed in unequal concentrations of a Cl⁻ salt; and the electrical conductance of the cuticle measured at a salt concentration equal to the average of that used in the diffusion-potential measurement. The permeabilities of H⁺, Li⁺, Na⁺, K⁺, and Cs⁺ ranged from 2 × 10⁻⁸ to 0.6 × 10⁻⁸ meters per second when cuticles were bathed in 2 moles per cubic meter Cl⁻ salts. The permeability of Cl⁻ was 3 × 10⁻⁹ meters per second. The permeability of Li⁺, Na⁺, and K⁺ was about five times less when measured in 500 moles per cubic meter Cl⁻ salts. We also report an asymmetry in cuticle-conductance values depending on the magnitude and the direction of current flow. The asymmetry disappears at low current-pulse magnitude and increases linearly with the magnitude of the current pulse. This phenomenon is explained in terms of transport-number effects in a bilayer model of the cuticle. Conductance is not augmented by current carried by exchangeable cations in cuticles; conductance is rate limited by the outer waxy layer of the cuticle.     

Intracellular Electrical Potentials in Frog Skin

The influence of changes in ionic composition of the bathing solutions on intracellular electrical potentials in frog skin has been examined. When the skin bathed in SO₄ Ringer''s solution is penetrated with a microelectrode two approximately equal potential jumps were frequently observed and most experiments were carried out with the electrode located between these steps. Substitution of Cl for SO₄ in the bathing solutions caused a decrease in PD across both the "outer" and "inner" barriers. When the skin was short-circuited an average intracellular potential of -18 mv was found with both Cl and SO₄ Ringer''s. With the skin in SO4 Ringer''s, decrease in Na concentration of the outside solution caused a decrease in PD between the microelectrode and the outside solution which was approximately the same as the decrease in total skin PD. With SO₄ Ringer''s, an increase in K concentration in the inside solution caused a marked decrease in total skin PD. However, only 50 per cent of this change occurred at the inner barrier, between the microelectrode and the inside solution. The remainder of the change occurred at the outer barrier. This observation does not appear to be consistent with the model of the skin proposed by Koefoed-Johnson and Ussing (Acta Physiol. Scand., 1958, 42, 298).     

A tracer method to study unidirectional fluxes of lithium: Application to frog skin

We describe a new tracer method to measure unidirectional fluxes of Li⁺, despite the lack of any utilizable radioisotope of lithium. This method uses the purified stable isotopes, ⁶Li and ⁷Li, detected with an ion-probe microanalyser. The accuracy is comparable to that obtained for other ions (e.g., Na⁺) with radiotracers.The method has been applied to frog skin with both faces bathed in a 20% lithium/80% sodium medium. Sodium and lithium unidirectional fluxes have been measured simultaneously. The results are consistent with lithium being actively pumped, the outflux of lithium being, however, much larger than that of sodium.     

Structure of aggregates of water and Li+, Na+, or K+ counterions with nucleic acid in solution

The position and orientation of water molecules hydrating fragments of DNA in the B and Z conformations are analyzed with the help of computer simulations. Monte Carlo studies are carried out at room temperature, high relative humidity (500 water molecules per pitch) and in the presence of counterions such as Li⁺, Na⁺, and K⁺. Differences in hydration patterns and in the counterionic structures were found by compairing B-DNA with Z-DNA double helices and B-DNA helices with different base-pair distributions. The present extension of our similations to Z-DNA and to Li⁺ and K⁺ counterions permits some general conclusions concerning nucleic acids in solution.     

Na transport across frog skin at low external Na concentrations

Isolated frog skin was bathed with a dilute solution containing 1 mm NaCl on the outside and with normal Ringer’s solution on the inner surface. Net Na flux was determined by simultaneous measurement of unidirectional fluxes with Na²² and Na²⁴ and intracellular electrical potentials were examined with microelectrodes. There was a net inward transport of Na under both open-circuit and short-circuit conditions. The short-circuit current was approximately 15% greater than the net Na flux; the discrepancy could be accounted for by a small outward flux of Cl. The electrical potential profile did not differ greatly from that observed in skins bathed on the outside with normal Ringer’s solution. Under open-circuit conditions, there were usually several potential steps and under short-circuit conditions the cells were negative relative to the bathing solutions. Estimates of epithelial Na concentrations utilizing radioactive Na suggested that if all epithelial Na were in a single compartment, an active entry step would be necessary to allow a net inward Na transport. The results could also be explained by a series arrangement of Na compartments without necessarily postulating an active Na entry. The behavior of the potential profile suggested that this latter alternative was more likely.     

Presence of a Na+/H+ antiporter in Methanobacterium thermoautotrophicum and its role in Na+ dependent methanogenesis

Methane formation from H₂/CO₂ by methanogenic bacteria is dependent on Na⁺ ions. In this communication it is shown with Methanobacterium thermoautotrophicum that a Na⁺/H⁺ antiporter plays a role in methane formation from H₂ and CO₂ and in the regulation of the ΔpH. This is based on the following findings:

1. Li⁺ ions, an alternative substrate of Na⁺/H⁺ antiporters, could replace Na⁺ in stimulating methanogenesis from H₂ and CO₂.
2. Harmaline, amiloride, and NH ₄ ⁺ , which are inhibitors of Na⁺/H⁺ antiporters, inhibited methanogenesis; inhibition was competitive to Na⁺ or Li⁺.
3. Addition of Na⁺ or Li⁺ rather than of other cations to cell suspensions resulted in an acidification of the suspension medium. The rate and extent of acidification was affected by those inhibitors, which inhibited methanogenesis competitively to Na⁺ or Li.
4. During methane formation from H₂ and CO₂ the generation of a ΔpH (inside alkaline) was dependent on the presence of Na⁺ or Li⁺. However, methanogenesis was also dependent on Na⁺ or Li⁺ under conditions where ΔpH was zero.
5. ATP synthesis driven by an electrogenic potassium efflux was significantly enhanced in the presence of Na⁺ or Li⁺. Na⁺ or Li⁺ were shown to prevent acidification of the cytoplasm under these conditions.

     

Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more potent than adenosine triphosphate in eliciting an increase in cytosolic Ca²⁺ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca²⁺ were monitored by using single channel recordings of the high conductance Ca²⁺-activated K⁺ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na⁺-saline, UTP (>30 nm) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 m. Cells bathed in K⁺ saline (150 m) were less sensitive to UTP (5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed in cells previously depolarized with K⁺ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca²⁺ entry blockers or by removal of extracellular Ca²⁺. However, in the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca²⁺ ionophores (<5 m) to that of UTP indicate that both agents elevate cytosolic Ca²⁺ by mobilization of this ion from intracellular pools. However, the Ca²⁺ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca²⁺ from intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues. PKC does not appear to be directly involved, even though the UTP response is blocked by low nm levels of PMA. While the latter data implicate PKC in diminishing the UTP response, agents that inhibit either PKC or phosphatase activity did not prevent abolition of UTP responses by PMA, nor did they modify basal channel activity.     

Graded and All-or-None Electrogenesis in Arthropod Muscle : I. The effects of alkali-earth cations on the neuromuscular system of Romalea microptera

Graded electrically excited responsiveness of Romalea muscle fibers is converted to all-or-none activity by Ba⁺⁺, Sr⁺⁺, or Ca⁺⁺, the two former being much the more effective in this action. The change occurs with as little as 7 to 10 per cent of Na⁺ substituted by Ba⁺⁺. The spikes now produced have overshoots and may be extremely prolonged, lasting many seconds. During the spike the membrane resistance is lower than in the resting fiber, but the resting resistance and time constant are considerably increased by the alkali-earth ions. The excitability is also increased, spikes arising neurogenically from spontaneous repetitive discharges in the axon as well as myogenically from spontaneous activity in the muscle fibers. Repetitive responses frequently occur on intracellular stimulation with a brief pulse. The data indicate that the alkali-earth ions exert a complex of effects on the different action components of electrically excitable membrane. They may be described in terms of the ionic theory as follows: The resting K⁺ conductance is diminished. The sodium inactivation process is also diminished, and sodium activation may be increased. Together these changes can act to convert graded responsiveness to the all-or-none variety. The alkali-earth ions can also to some degree carry inward positive charge during activity, since spikes are produced when Na⁺ is fully replaced with the divalent ions.     

A QM/QTAIM detailed look at the Watson–Crick↔wobble tautomeric transformations of the 2-aminopurine·pyrimidine mispairs

This work is devoted to the careful QM/QTAIM analysis of the evolution of the basic physico-chemical parameters along the intrinsic reaction coordinate (IRC) of the biologically important 2AP·T(WC)?2AP·T*(w) and 2AP·C*(WC)?2AP·C(w) Watson–Crick(WC)?wobble(w) tautomeric transformations obtained at each point of the IRC using original authors’ methodology. Established profiles reflect the high similarity between the courses of these processes. Basing on the scrupulous analysis of the profiles of their geometric and electron-topological parameters, it was established that the dipole-active WC?w tautomerizations of the Watson–Crick-like 2AP·T(WC)/2AP·C*(WC) mispairs, stabilized by the two classical N3H?N1, N2H?O2 and one weak C6H?O4/N4 H-bonds, into the wobble 2AP·T*(w)/2AP·C(w) base pairs, respectively, joined by the two classical N2H?N3 and O4/N4H?N1 H-bonds, proceed via the concerted stepwise mechanism through the sequential intrapair proton transfer and subsequent large-scale shifting of the bases relative each other, through the planar, highly stable, zwitterionic transition states stabilized by the participation of the four H-bonds – N1⁺H?O4^–/N4^–, N1⁺H?N3^–, N2⁺H?N3^–, and N2⁺H?O2^–. Moreover, it was found out that the 2AP·T(WC)?2AP·T*(w)/2AP·C*(WC)?2AP·C(w) tautomerization reactions occur non-dissociatively and are accompanied by the consequent replacement of the 10 unique patterns of the specific intermolecular interactions along the IRC. Obtained data are of paramount importance in view of their possible application for the control and management of the proton transfer, e.g. by external electric or laser fields.     

Molecular simulation of Tyr105 phosphorylated pyruvate kinase M2 to understand its structure and dynamics

Microstructure of dibenzo-18-crown-6 (DB18C6) and DB18C6/Li⁺ complex in different solvents (water, methanol, chloroform, and nitrobenzene) have been analyzed using radial distribution function (RDF), coordination number (CN), and orientation profiles, in order to identify the role of solvents on complexation of DB18C6 with Li⁺, using molecular dynamics (MD) simulations. In contrast to aqueous solution of LiCl, no clear solvation pattern is found around Li⁺ in the presence of DB18C6. The effect of DB18C6 has been visualized in terms of reduction in peak height and shift in peak positions of g_Li-Ow. The appearance of damped oscillations in velocity autocorrelation function (VACF) of complexed Li⁺ described the high frequency motion to a “rattling” of the ion in the cage of DB18C6. The solvent-complex interaction is found to be higher for water and methanol due to hydrogen bond (HB) interactions with DB18C6. However, the stability of DB18C6/Li⁺ complex is found to be almost similar for each solvent due to weak complex-solvent interactions. Further, Li⁺ complex of DB18C6 at the liquid/liquid interface of two immiscible solvents confirm the high interfacial activity of DB18C6 and DB18C6/Li⁺ complex. The complexed Li⁺ shows higher affinity for water than organic solvents; still they remain at the interface rather than migrating toward water due to higher surface tension of water as compared to organic solvents. These simulation results shed light on the role of counter-ions and spatial orientation of species in pure and hybrid solvents in the complexation of DB18C6 with Li⁺. Graphical Abstract

DB18C6/Li⁺ complex in pure solvents (water, methanol, chloroform, and nitrobenzene) and water/nitrobenzene interface     

Membrane potential genesis in<Emphasis Type="Italic"> Nitella</Emphasis> cells,mitochondria, and thylakoids

The resting membrane potential of Nitella cells shifts in parallel with the change in H⁺ equilibrium potential, but is not equal to the H⁺ equilibrium potential. The deviation of the membrane potential from the H⁺ equilibrium potential depends on the extrusion rate of H⁺ by the electrogenic H⁺-pump. The activity of the electrogenic H⁺-pump was formulated in terms of the change in the free energy of ATP hydrolysis. The deviation of membrane potential from the H⁺ equilibrium potential induces a passive H⁺ flow. The passive inward H⁺ current may be coupled with Cl^– uptake. The coupling rate of H⁺,Cl^– co-transport was discussed. The membrane potential of mitochondria was electrochemically formulated in terms of oxidation–reduction H₂/H⁺ half-cells spontaneously formed at the inner and outer boundaries of each trans-membrane electron-conducting pathway. The membrane potential formed by a pair of H₂/H⁺ redox cells is pH-sensitive in its nature, but deviates from the H⁺ equilibrium potential to an extent that depends on the logarithm of the ratio of H₂ concentrations at the inner and outer boundaries. The membrane potential of thylakoids is considered to be primarily due to the electromotive force of photocells embedded in the thylakoid membrane, as far as the anode and cathode of each photocell are in contact with the inner and outer solutions, respectively. The light-induced electronic current yields oxygen at the inner boundary and causes an increase in the H₂ pool at the outer boundary of the electron-conducting pathway, which has no shunting plastoquinone chain between these two boundaries.     

Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K+ reduction and acidification

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K _o ⁺ ] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K⁺ concentration. As well as changes in serum [K _o ⁺ ], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K _o ⁺ -dependent, and the suggestion has been made that pH and K _o ⁺ may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K _o ⁺ . It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K⁺ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K⁺ channels in the open vs inactivated states of the channels by physiological H⁺ and K⁺ concentrations represents an important pathway to control Kv channel function in health and disease.     

Elucidating Conformational Changes in the ��-Aminobutyric Acid Transporter-1

The GABA transporter-1 (GAT-1) has three current-generating modes: GABA-coupled current, Li⁺-induced leak current, and Na⁺-dependent transient currents. We earlier hypothesized that Li⁺ is able to substitute for the first Na⁺ in the transport cycle and thereby induce a distinct conformation in GAT-1 and that the onset of the Li⁺-induced leak current at membrane potentials more negative than −50 mV was due to a voltage-dependent conformational change of the Li⁺-bound transporter. In this study, we set out to verify this hypothesis and seek insight into the structural dynamics underlying the leak current, as well as the sodium-dependent transient currents, by applying voltage clamp fluorometry to tetramethylrhodamine 6-maleimide-labeled GAT-1 expressed in Xenopus laevis oocytes. MTSET accessibility studies demonstrated the presence of two distinct conformations of GAT-1 in the presence of Na⁺ or Li⁺. The voltage-dependent fluorescence intensity changes obtained in Li⁺ buffer correlated with the Li⁺-induced leak currents, i.e. both were highly voltage-dependent and only present at hyperpolarized potentials (<−50 mV). The transient currents correlated directly with the voltage-dependent fluorescence data obtained in sodium buffer and the associated conformational changes were distinct from those associated with the Li⁺-induced leak current. The inhibitor potency of SKF89976A of the Li⁺- versus Na⁺-bound transporter confirmed the cationic dependence of the conformational occupancy. Our observations suggest that the microdomain situated at the external end of transmembrane I is involved in different conformational changes taking place either during the binding and release of sodium or during the initiation of the Li⁺-induced leak current.γ-Aminobutyric acid (GABA)² is the major inhibitory neurotransmitter in the mammalian central nervous system. Continuous GABAergic neurotransmission is efficiently prevented by a GABA re-uptake system that transports GABA back into the synaptic processes via the GABA transporters (GAT). Four isoforms of the mammalian GAT have been found: GAT-1, GAT-2, GAT-3, and BGT-1 (betaine transporter-1) (1). These membrane proteins couple the transport of one GABA molecule to the transport of two Na⁺ and one Cl⁻ (2, 3). Accordingly, the transport process is electrogenic and the transport activity can therefore be monitored by electrophysiological methods. GAT-1 has also been shown to generate: (i) an inwardly rectifying leak current in the presence of Li⁺ (and in complete absence of Na⁺) when the membrane potential is more negative than −50 mV (4–6) and (ii) a presteady-state transient current in the presence of Na⁺ but in the absence of GABA in response to step jumps in membrane voltage (4, 7).GAT-1 is strictly dependent on external Na⁺ to drive the transport of GABA (7) and external GABA does not affect the Li⁺-induced leak current (8). Taken together, this suggests that Li⁺ cannot induce the same conformation in GAT-1 as Na⁺ is capable of inducing: namely the conformation that is required for the binding and translocation of GABA. This is in contrast to, e.g. the related serotonin transporter and dopamine transporter in which substrate inhibits the Li⁺-induced leak current (9, 10) and the Na⁺/glucose cotransporter (SGLT1) where Li⁺ is able to sustain substrate transport (11). We have in an earlier study shown that Li⁺ is able to bind to the first low apparent affinity cation-binding site in the transport cycle (and replace Na⁺) but not to the second high apparent affinity cation-binding site (8). This finding was later confirmed by Kanner and co-workers (12), who were able to identify the cation-binding site with which Li⁺ interacts and is able to replace Na⁺. According to our model, the transporter in the presence of Li⁺ is “stalled” in the conformation in which only the first cation-binding site is occupied, in contrast to the presence of Na⁺, where both cation-binding sites are occupied.An unresolved question on the Li⁺-induced leak current for GAT-1 is the mechanism of the prominent inward rectification. The electrochemical driving force for Li⁺ predicts a Li⁺-induced inward current originating at much more positive membrane potentials, but this current is not detected unless the membrane potential is more negative than −50 mV. We previously hypothesized that the Li⁺-induced leak mode would commence only at the hyperpolarized membrane potentials due to a voltage-dependent conformational change in the Li⁺-bound GABA transporter (8). In the present study, we set out to test this hypothesis by introduction of a fluorescent probe in GAT-1 to monitor voltage-dependent local conformational changes and relate these to the different current-generating modes of GAT-1.We expressed GAT-1 in Xenopus laevis oocytes and used simultaneous electrical and optical measurements (voltage clamp fluorometry) (13) to monitor the currents and conformational changes of the transporter in the presence of Li⁺ and Na⁺ in response to step changes in membrane potential. By labeling Cys⁷⁴ at the external end of transmembrane helix (TM) I of rat GAT-1 (see Fig. 1) with the cysteine-reactive fluorescent probe, tetramethylrhodamine 6-maleimide (TMR6M), we were able to correlate the voltage dependence of the Li⁺-induced leak current and the Li⁺- and voltage-dependent changes in conformations observed by fluorescence intensity changes. The voltage dependence of the Li⁺-induced conformational changes appeared distinct from the Na⁺-induced conformational changes associated with the Na⁺-dependent transient currents. We also explored differences in the inhibitor potency of the GAT-1-specific inhibitor SKF89976A (14) as well as the differential inhibition of the GABA transport by the cysteine-reactive methanethiosulfonate ethyltrimethylammonium (MTSET) in the presence of either Na⁺ or Li⁺. Finally, we prepared a homology model of GAT-1 (Fig. 1) by using the bacterial leucine transporter, LeuT_Aa (15), as a template and dock the TMR6M into the model to provide a framework for interpreting the putative conformational rearrangements that may explain the observed changes in fluorescence intensity.Open in a separate windowFIGURE 1.Three-dimensional model of GAT-1 with the fluorophore, TMR6M, covalently attached to Cys⁷⁴. The model was made by homology modeling with the bacterial LeuT_Aa transporter as template. A, side view of the GAT-1 model. The 12 transmembrane helices are shown in different colors; TM1 being blue and TM12 being red. The two sodium ions are purple spheres, chloride is a green sphere, and GABA is shown next to the sodium ions as red, light and dark blue spheres. TMR6M is located in the external surface of the model (shown as green, blue, and red spheres) and is attached to Cys⁷⁴ (shown as blue, red, and yellow spheres). B, magnified view of the local environment of TMR6M embedded in a hydrophobic cleft between EL3 (green), the beginning of EL4 (yellow), and the outer part of TM1 (blue). Below TMR6M, sodium, chloride, and GABA can be seen as spheres.Altogether, the present data support that local conformational changes taking place at the external surface of TM1 to mirror the global conformational changes taking place during the current-generating modes of the GABA transporter. Moreover, our data demonstrate that voltage dependence of the conformational changes associated with the Li⁺-induced leak current is different from the Na⁺-dependent conformational changes required for GABA transport.     

Electrophysiology of the marine diatom Coscinodiscus wailesii. II. Potassium currents

The dominating mechanism of K⁺ uniport through the plasmalemma of Coscinodiscus wailesii has been studied in some detail as part of a general study of ionic relations in marine diatoms. Electrical measurements with double-barrelled glass-microelectrodes have been made in intact cells (diameter 100 m) bathed in artificial sea-water in which [K⁺] has been changed from 3 mM to 100 mM. Using a modified Goldman equation, these results provide an estimate of [K⁺]i of about 400 mM and a selectivity for K⁺ over Na⁺ and CI^-, which could spontaneously vary by orders of magnitude and reach values of about 1000. Voltage-clamp experiments have been carried out in these states of high K⁺ selectivity using bipolar staircase command voltages over a range from -180 to +60 mV. The resulting steady-state current-voltage relationships have inward rectifying sigmoid characteristics with a negative saturation current around -30 nA, and a slope conductance of the order or 1 S at free running voltages <-60 mV. Temporal responses of the clamp currents upon rectangular voltage steps were basically rectangular, i.e. they did not show the familiar relaxation kinetics of voltage-induced activation/inactivation. The sigmoid steady-state current-voltage relationships could not be described by a usual model of constant-field currents through a voltage-gated pore, where the positive current of an inward rectifier would not saturate but vanish. Alternatively, the observed steady-state inward rectifying current-voltage relationship and its changes upon changes in [K⁺]o, are well described by a three-state reaction cycle for catalysis of K⁺ translocation with a steady activity.     

Microsomal (Na + K)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors

A method is described for the extraction of microsomal ouabain-sensitive (Na⁺ + K⁺)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na⁺ + K⁺)-stimulated activity in the range of 30–40 nmol · mg⁻¹ · min⁻¹ at 26 °C. This portion, which is also ouabain sensitive, is about half of the total activity in media containing Mg²⁺, Na⁺ and K⁺. These preparations also contain Mg²⁺-dependent or Ca²⁺-dependent activities which are not additive and which are not significantly affected by ouabain, Na⁺, K⁺ or Li⁺.The activations of the ouabain-sensitive ATPase activity by Mg²⁺, Na⁺, and K⁺ are similar to those described in other tissues. It is found that Li⁺ does not substitute for Na⁺ as an activator but in high concentrations does produce partial activation in the presence of Na⁺ with no K⁺. These results are pertinent to the reported observations of ouabain-sensitive Li⁺ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li⁺ on the sodium pump.     

Coupled transepithelial sodium and potassium transport across isolated frog skin: Effect of ouabain,amiloride and the polyene antibiotic filipin

Summary Addition of the polyene antibiotic filipin (50 m) to the outside bathing solution (OBS) of the isolated frog skin resulted in a highly significant active outward transport of K⁺ because filipinper se increases the nonspecific Na⁺ and K⁺ permeability of the outward facing membrane. The K⁺ transport was calculated from the chemically determined changes in K⁺ concentrations in the solution bathing the two sides of the skin. The active transepithelial K⁺ transport required the presence of Na⁺ in the OBS, but not in the inside bathing solution (IBS), and it was inhibited by the Na⁺, K⁺-ATPase inhibitor ouabain. The addition of Ba⁺⁺ to the IBS in the presence of filipin in the OBS resulted in an activation of the transepithelial K⁺ transport and in an inhibition of the active Na⁺ transport. This is in agreement with the notion that Ba⁺⁺ decreases the passive K⁺ permeability of the inward facing membrane. In the presence of amiloride (which blocks the specific Na permeability of the outward facing membrane) and Ba⁺⁺ there was a good correlation between the active Na⁺ and K⁺ transport. It is concluded that the active transepithelial K⁺ transport is carried out by a coupled electrogenic Na–K pump, and it is suggested that the pump ratio (Na/K) is 1.5.     

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon

Summary In this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na⁺ or K⁺ Ringer's solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na⁺ Ringer's solution (the mucosal solution contained 50 m amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K⁺ Ringer's (containing 50 m amiloride), a Lorentzian component appeared with an average corner frequency,f _c=15.6±0.91 Hz and a mean plateau valueS _o=(7.04±2.94)×10^–20 A² sec/cm². The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K⁺ entry across the apical membrane. Elimination of the K⁺ gradient by bathing the colon on both sides with K⁺ Ringer's solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs⁺ or tetraethylammonium (TEA) and by serosal addition of Ba²⁺. The one-sided action of these K⁺ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K⁺ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na⁺ transport pathway.