Ion Translocation Process Research Articles

Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase

Three Na+ sites are defined in the Na+-bound crystal structure of Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Remarkably, Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane – in fact, mutations Q925A and Q925G increased the apparent Na+ affinity – but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na+-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side.

Energy coupling in ABC exporters

Multidrug transporters are important and interesting molecular machines that extrude a wide variety of cytotoxic drugs from target cells. This review summarizes novel insights in the energetics and mechanisms of bacterial ATP-binding cassette multidrug transporters as well as recent advances connecting multidrug transport to ion and lipid translocation processes in other membrane proteins.

Multi-frequency electrical impedance tomography as a non-invasive tool to characterize and monitor crop root systems

Abstract. A better understanding of root–soil interactions and associated processes is essential in achieving progress in crop breeding and management, prompting the need for high-resolution and non-destructive characterization methods. To date, such methods are still lacking or restricted by technical constraints, in particular the charactization and monitoring of root growth and function in the field. A promising technique in this respect is electrical impedance tomography (EIT), which utilizes low-frequency (< 1 kHz)- electrical conduction- and polarization properties in an imaging framework. It is well established that cells and cell clusters exhibit an electrical polarization response in alternating electric-current fields due to electrical double layers which form at cell membranes. This double layer is directly related to the electrical surface properties of the membrane, which in turn are influenced by nutrient dynamics (fluxes and concentrations on both sides of the membranes). Therefore, it can be assumed that the electrical polarization properties of roots are inherently related to ion uptake and translocation processes in the root systems. We hereby propose broadband (mHz to hundreds of Hz) multi-frequency EIT as a non-invasive methodological approach for the monitoring and physiological, i.e., functional, characterization of crop root systems. The approach combines the spatial-resolution capability of an imaging method with the diagnostic potential of electrical-impedance spectroscopy. The capability of multi-frequency EIT to characterize and monitor crop root systems was investigated in a rhizotron laboratory experiment, in which the root system of oilseed plants was monitored in a water–filled rhizotron, that is, in a nutrient-deprived environment. We found a low-frequency polarization response of the root system, which enabled the successful delineation of its spatial extension. The magnitude of the overall polarization response decreased along with the physiological decay of the root system due to the stress situation. Spectral polarization parameters, as derived from a pixel-based Debye decomposition analysis of the multi-frequency imaging results, reveal systematic changes in the spatial and spectral electrical response of the root system. In particular, quantified mean relaxation times (of the order of 10 ms) indicate changes in the length scales on which the polarization processes took place in the root system, as a response to the prolonged induced stress situation. Our results demonstrate that broadband EIT is a capable, non-invasive method to image root system extension as well as to monitor changes associated with the root physiological processes. Given its applicability on both laboratory and field scales, our results suggest an enormous potential of the method for the structural and functional imaging of root systems for various applications. This particularly holds for the field scale, where corresponding methods are highly desired but to date are lacking.

Development of Functional Artificial Ion Channels using Peptide Nanostructures

Ion channel proteins are of fundamental importance in numerous biological processes and constitute a key target in pharmaceutical research. However, many aspects of their mechanism of action still remain poorly understood. In order to shed light on these complex membrane transport systems, we have developed a family of peptide nanostructures that create channels for ions by aligning crown ethers on top of each other when adopting an α-helical conformation. We will present different analogs of such artificial ion channels and how they were used to study ion translocation processes in membranes. Also, we will report on the engineering of their ion-selectivity and their incorporation into new biodetection systems.

Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters

The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu2+- and Ni2+-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co2+. Indeed, the structure of TtNikM2 containing a bound Co2+ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.

Molecular dynamics of ion transport through the open conformation of a bacterial voltage-gated sodium channel

The crystal structure of the open conformation of a bacterial voltage-gated sodium channel pore from Magnetococcus sp. (NaVMs) has provided the basis for a molecular dynamics study defining the channel's full ion translocation pathway and conductance process, selectivity, electrophysiological characteristics, and ion-binding sites. Microsecond molecular dynamics simulations permitted a complete time-course characterization of the protein in a membrane system, capturing the plethora of conductance events and revealing a complex mixture of single and multi-ion phenomena with decoupled rapid bidirectional water transport. The simulations suggest specific localization sites for the sodium ions, which correspond with experimentally determined electron density found in the selectivity filter of the crystal structure. These studies have also allowed us to identify the ion conductance mechanism and its relation to water movement for the NavMs channel pore and to make realistic predictions of its conductance properties. The calculated single-channel conductance and selectivity ratio correspond closely with the electrophysiology measurements of the NavMs channel expressed in HEK 293 cells. The ion translocation process seen in this voltage-gated sodium channel is clearly different from that exhibited by members of the closely related family of voltage-gated potassium channels and also differs considerably from existing proposals for the conductance process in sodium channels. These studies simulate sodium channel conductance based on an experimentally determined structure of a sodium channel pore that has a completely open transmembrane pathway and activation gate.

E. Coli F1Fo ATP Synthase Subunit C: Solution NMR Structure by CS-Rosetta

The ATP synthase is the main source of ATP in cells, whose function is to inter-convert chemical energy and transmembrane electrochemical potential difference. The enzyme contains two domains: a soluble F1 domain where ATP is synthesized/hydrolyzed and a transmembrane FO domain, which functions as an ion pump. Multiple (9-15) copies of subunit c form an oligomeric ring in the FO domain. The ion translocation activity takes place at the interface of c-ring and subunit a where an essential acidic residue in middle of the outer helix in subunit c undergoes a protonation/deprotonation process, which couples with the relative rotation of c-ring to subunit a. Although high-resolution X-ray structures of the c-rings from several organisms have been solved, the mechanism of ion translocation still needs to be clarified at atomic resolution.We aim to solve the structures of E. coli subunit c at both protonation states of the essential Asp61 and elucidate any conformational changes at the active site during the ion translocation process. Due to the difficulty of obtaining unambiguous long-range NOE constraints in membrane proteins, traditional solution NMR methods were unsuccessful in this case. We employed CS-Rosetta, which utilizes minimal NMR constraints, in our study.Our study of the subunit c in the protonated state shows that the choice of proper scoring weights is essential in the application of CS-Rosetta to membrane proteins. Using limited NMR restraints this method converges on a structure for E. coli subunit c that is similar to the X-ray structures of subunit c from other organisms. Further improving the quality of the structure may be achieved by optimizing the weights in the calculation. We are also using this approach on deprotonated subunit c and hoping to model the c-ring from our results.

Structure of the rotor ring modified with N , N ′ -dicyclohexylcarbodiimide of the Na + -transporting vacuolar ATPase

The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na(+), which can be detected by radioisotope (22Na(+)) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for 22Na(+) decreased approximately 30-fold by reaction with N,N(')-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na(+)-bound and Na(+)-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na(+) from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an "open" conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na(+). This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na(+) decreases due to the neutralization of E139, thus releasing bound Na(+), and that the structures of Na(+)-bound and Na(+)-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na(+) during rotational catalysis of V-ATPase, respectively.

Concentration Gradient Effects of Sodium and Lithium Ions and Deuterium Isotope Effects on the Activities of H +-ATP Synthase from Chloroplasts

We explored the concentration gradient effects of the sodium and lithium ions and the deuterium isotope's effects on the activities of H +-ATP synthase from chloroplasts (CF 0F 1). We found that the sodium concentration gradient can drive the ATP synthesis reaction of CF 0F 1. In contrast, the lithium ion can be an efficient enzyme-inhibitor by blocking the entrance channel of the ion translocation pathway in CF 0. In the presence of sodium or lithium ions and with the application of a membrane potential, unexpected enzyme behaviors of CF 0F 1 were evident. To account for these observations, we propose that both of the sodium and lithium ions could undergo localized hydrolysis reactions in the chemical environment of the ion channel of CF 0. The protons generated locally could proceed to complete the ion translocation process in the ATP synthesis reaction of CF 0F 1. Experimental and theoretical deuterium isotope effects of the localized hydrolysis on the activities of CF 0F 1, and the energetics of these related reactions, support this proposed mechanism. Our experimental observations could be understood in the framework of the well-established ion translocation models for the H +-ATP synthase from Escherichia coli, and the Na +-ATP synthase from Propionigenium modestum and Ilyobacter tartaricus.

Effect of R56865 on cardiac sarcoplasmic reticulum function and its role as an antagonist of digoxin at the sarcoplasmic reticulum calcium release channel.

1. The effect of R56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl]-N-methyl-2- benzothiazolamine) on cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel function was investigated. The effect of R56865 on [3H]-ryanodine and [3H]-digoxin binding to SR vesicles and its effect on the ATP-stimulated 45Ca2+ uptake into SR vesicles was also studied. 2. R56865 (0.5-50 microM) had no effect on single-channel open probability (Po) when added to native cardiac SR Ca(2+)-release channels, incorporated into planar phospholipid bilayers, that had previously been activated by 10 microM Ca2+. The single-channel conductance (93 pS) and the Ca2+/Tris+ permeability ratio (12.5) were also unaffected by R56865. 3. R56865 failed to affect the rapid Ca(2+)-induced efflux of 45Ca2+ from cardiac SR vesicles. The initial efflux rate at an extravesicular [Ca2+] of 0.1 microM was 176 +/- 33 nmol 45Ca2+ mg-1 protein s-1 (n = 5). Addition of 0.5-50 microM R56865 to the efflux solution did not affect the initial efflux rate or the total amount of 45Ca2+ released from the vesicles. 4. The specific binding of [3H]-ryanodine to SR vesicles can be viewed as a marker for SR Ca(2+)-release channel activation. R56865 (0.05-50 microM) did not change the amount of specific [3H]-ryanodine bound at 10 microM activating Ca2+. Taken together these data (points 2, 3 and 4) suggest that R56865 does not affect the Ca2+ activation of the cardiac SR Ca(2+)-release channel. 5. R56865 (0.5-50 microM) decreased the ATP-stimulated uptake of 45Ca2+ into cardiac SR vesicles. The total amount of 45Ca2+ taken up into the vesicles was decreased by 26-37% and the initial rate of uptake was also reduced.6. R56865 decreased the binding of [3H]-digoxin to cardiac SR vesicles. It inhibited binding of[3H]-digoxin to both sites on SR membranes. However, it acted as a mixed type of non-competitive antagonist, as it increased the Kd and reduced the Bmax for [3H]-digoxin. Additionally, when SRCa2+-release channels incorporated into bilayers were activated by 1 nM digoxin at 10 micro M Ca2+, 5 microMR56865 decreased single-channel Po to that seen prior to activation by digoxin, confirming that R56865is an antagonist at the cardiac glycoside receptor site on the cardiac SR Ca2+-release channel.7. The results presented here show that R56865 decreases 45Ca2+ uptake into SR vesicles and also non-competitively decreases the binding of cardiac glycosides to their activation sites on the SRCa2+-release channel. Since this compound fails to affect other aspects of the functioning of the SR, we conclude that inhibition of Ca2+-uptake into the SR and/or antagonism of digoxin binding to its high affinity sites on the cardiac SR Ca2+-release channel may contribute to the protection against glycoside induced toxicity seen with R56865. Indeed the reduction of Ca2+ uptake into the SR during Ca2+overload may contribute to the protective action of R56865 against arrhythmias induced by veratridine or by ischaemia. Notwithstanding this, it is possible that the multitude of other actions on specific ion translocation processes, as well as the possible non-specific membrane effects of R56865, may be as responsible for the protection against arrhythmias as the effects on SR function we have documented here. However, the simultaneous interference with more than one of these processes by R56865 may be required for its anti-arrhythmic action.

Single channels of 9, 11, 13, 15-destryptophyl-phenylalanyl-gramicidin A

Analysis of the single-channel behavior of an analogue of gramicidin A in which all four tryptophyl residues are substituted by phenylalanyl suggests that the nature of the side chains may play an important role in the ion translocation process. Indeed, while infrared spectroscopy indicates that both peptides have very similar backbone conformations, they have different single-channel characteristics. The unit conductance of the analogue is much smaller than that of the natural product. Moreover, contrary to gramicidin A, it is voltage dependent.

Ionic channels of some glycine-rich synthetic polypeptides

The ion transport properties of glycine-rich synthetic polypeptides (Ser-Leu-Gly)n, (Leu-Ser-Leu-Gly)n and their O-benzyl derivatives have been examined. When incorporated into lipid bilayers membranes, all these model molecules give rise to ion permeation through single channel events. Their behavior, peculiarly that of the O-protected derivatives rules out the channeled model previously proposed for (Leu-Ser-Leu-Gly)n in which the hydroxyl groups of the seryl residues were supposed to play a role in the ion translocation process. This latter polypeptide also permeates membranes to calcium. Preliminary investigations suggest an α-like helical conformation rather than a β helix with an intramolecular channel.

Mechanism of inactivation (desensitization) of acetylcholine receptor. Investigations by fast reaction techniques with membrane vesicles

Exposure of the acetylcholine receptor to acetylcholine, or its stable analogue carbamylcholine, inactivates (desensitizes) the receptor. Inactivation of receptor-controlled ion (86Rb+) flux in the presence of different concentrations of carbamylcholine (12.5 microM to 28 mM) was measured in the millisecond to minute time region, using a quench flow technique and membrane vesicles prepared from the electric organ of Electrophorus electricus. Three different kinetic measurements were made to establish the relationship between carbamylcholine concentration and the ion translocation process: (i) the rate of inactivation of the ion translocation process; (ii) the rate of recovery of the inactivated receptor upon removal of carbamylcholine; and (iii) the rate of the ion flux mediated by equilibrium mixtures of active and inactive receptor forms. The kinetics of these three processes follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence could be used. Therefore, it was possible to determine the value of the rate constants in a scheme relating the ligand binding steps to ion translocation, and to predict the dependence of these rate constants on carbamylcholine concentration over the 200-fold range investigated.

Structure of the squid axon membrane as derived from charge-pulse relaxation studies in the presence of absorbed lipophilic ions.

Charge-pulse relaxation studies were performed on squid giant axons in the presence of membrane absorbed lipophilic anions, dipicrylamine (DPA) and tetraphenylborate (TPhB), and of specific blockers of sodium and potassium active currents. With the instrumentation used in this work a time resolution of 5 to 10 μsec was easily obtained without any averaging, although the voltage relaxations were always smaller than 5 mV in amplitude in order to keep the membrane voltage in a range where the used theory cyn be linearized. Two well distinguishable linear relaxations were invariably observed in the presence of the lipophilic anions. With DPA the fast relaxation (time constants between 8 and 70 μsec) was attributed to the redistribution of the lipophilic ions within the membrane following the change in membrane potential. The long relaxation process (time constant in the millisecond range) corresponds to the normal voltage relaxation of the passive squid axon membrane slightly modified by the process of redistribution of the extrinsic ions. The results support the same model for the translocation of lipophilic ions within the nerve membrane proposed earlier for artificial lipid bilayers. The fit of the data with a single barrier model yields the translocation rate constant,K, and the total concentration,N t , of membrane absorbed ions, from which the membrane-solution partition coefficient, β, can be derived. Both for DPA and TPhB,K had values close to those measured for solvent-free artificial lipid bilayers. The axon membrane appears as fluid mosaic membrane with a thickness of about 2.5 nm for the lipid bilayer part. In axons treated with DPA the dependence of relaxation data upon the holding membrane potential, $$\bar E_m$$ , provided information on the asymmetry of the membrane structure. The data were best fitted by assuming that nearly 100% of the membrane potential drops between the two free energy minima where the extrinsic ions are located, indicating that these minima lie very close to the membrane-solution interfaces, in the region of the phospholipid polar heads. The asymmetry voltage,E o, at which the extrinsic ions are expected to be equally distributed between the two sides of the membrane was found to range between −35 and −65 mV (inside negative), depending on the assumed shape of the free energy barrier describing the ion translocation process. This voltage is of the same sign and of the same order of magnitude as the equilibrium voltages for the open-close transitions of the gates of sodium and potassium channels, suggesting that all these voltages result from the same membrane asymmetry. A similar analogy was found between the asymmetry of the free energy barrier which best fitted DPA relaxation data and the asymmetrical voltage dependence of the gating of ionic channels. Our data were best fitted by assuming that about 70% of the potential drop occurs between the free energy minimum on the intracellular membrane face and the top of the barrier.

Kinetic mechanism of acetylcholine receptor-controlled ion flux: Flow quench kinetic measurements of acetylcholine-induced flux in membrane vesicles

The dependence of acetylcholine receptor-controlled transmembrane ion flux on acetylcholine concentration was measured in the msec time region using membrane vesicles and a quench flow technique. Four measurements were made: (1) transmembrane ion flux, (2) rate of inactivation of the receptor, (3) rate of recovery, and (4) ion flux mediated by “inactivated” receptor. A minimum model which relates the ligand binding and ion translocation processes, which has been previously proposed to account for carbamylcholine-induced ion flux, can also account for acetylcholine-induced flux. The integrated rate equation, based on the model, predicts the time dependence of the ion flux over the 160-fold concentration range of acetylcholine investigated. The receptor-controlled ion flux exhibits simple kinetics, and this has permitted the use of simple analytical expressions for the dependence on acetylcholine concentration of the various constants of the minimum mechanism. The evaluation of the constants, and methods for the separation of vesicles which contain functional receptors from those which do not, have led to the determination of the specific reaction rate, J , of the acetylcholine receptor-controlled translocation of inorganic ions. J = 3 × 10 7 M −1 sec −1. The value for J allows one to calculate the number of ions translocated per receptor per msec, ∼6 × 10 3. A value of 1 × 10 7 ions translocated per receptor site per unit time has also been determined by analysis of acetylcholine-induced noise in cells. Therefore it becomes possible to integrate the results obtained in two types of measurement of receptor function: chemical kinetics, which establishes the relationship between the ligand binding and ion translocation processes, and noise analysis, which measures elementary steps in the formation of receptor-formed ion channels.

The influence of surface charge on the kinetics of ion-translocation across biological membranes

Rate equations have been developed which describe the concentration dependence for ion-translocation across charged membranes for those cases in which the translocation process can be considered to be formally equivalent with an enzymic process of a Michaelis-Menten type. We have limited ourselves to those cases in which the ion-translocational step through the membrane is electroneutral. In addition it is assumed that the sites on the membrane involved in the ion-translocation process can not move through the membrane when these sites are not occupied by ions. It is shown that in general deviations from Michaelis-Menten kinetics may be expected. In case of monovalent ion-translocation across oppositely charged membranes apparent negative homotrope cooperative effects may occur, whereas for ion-translocation across equally charged membranes apparent positive homotrope cooperative effects may be found. When the bulk aqueous phase also contains polyvalent ions both types of effects may occur both in the case of ion-translocation across oppositely charged membranes as well as with ion-translocation across a membrane of which the sign of the surface charge is the same as that of the ion translocated. Under limited conditions, also apparent single Michaelis-Menten kinetics may be observed. In these cases, however, the apparent K m generally is no linear function of the concentration of a competing ion. It is shown that even when an ion does not bind to the translocation sites the K m is affected by increasing concentrations of this ion, a phenomenon which is not expected when the membrane is not charged. The effects of divalent ions, added to the bulk aqueous phase as 1-1-electrolytes, upon the K m are discussed in connection with in literature reported effects of Ca ++ upon the rate of uptake of several monovalent ions into plant cells.

An electron microscopic study of the formation of amorphous calcium phosphate and its transformation to crystalline apatite.

Dried amorphous calcium phosphate (ACP) can exist in discoidal and spheroidal forms. The disk-shaped particles are most prominent in dried ACP specimens isolated immediately following precipitation. The spherical forms become dominant in dried specimens taken from older suspensions. The disk-like morphology is a result of sample drying. Spherules can also arise during the drying step but are present in the native suspension as well. Both the disks and spherules appear to have a common solution progenitor in the form of a highly hydrated ACP aggregate of unknown morphology. The formation of crystalline apatite in solution is intimately connected with ACP. The first crystals appear on the surface of the amorphous spherules and in contact with the amorphous disks. In the case of the spherules, the initial crystals increase in size and generate additional crystals by secondary nucleation until the spherules become enveloped by apatite. However, when conversion is completed, the apatite does not fill appreciably the space previously occupied by the dissolved amorphous spherules. These observations do not support the concept of anin situ solid state conversion in this case, but indicate a heterophase transition supported by a solution-mediated ion translocation process. The occurrence of crystals in the amorphous disks suggest that apatite can also form directly from the solution progenitor.

Light-Dependent Ion Translocation in Spinach Chloroplasts

The absorption of light by chloroplasts is the first step for several energy-requiring systems such as ATP synthesis and CO2 fixation. Recently, it has been found that light can alter the structure and volume of the chloroplast (7, 15, 17, 26). Therefore the question was raised whether light could affect ion movements in isolated chloroplasts under conditions where structural changes occur. This action of light on ion uptake by chloroplasts has received little attention, even though ion movements in algae and higher plants have been known for some time. Hoagland and Davis in 1923 were among the earliest in demonstrating a role of light in the movement of ions in plant cells by showing light-induced increases of chloride and bromide in Nitella cells (10). These observations were extended in 1934 by Jacques and Osterhout, who found that light increased the rate of K entry into Valonia macrophysa (16). Later, Arens reported Ca movements through Elodea leaves by a process dependent on photosynthesis (1), and Ingold found that chloride , K and phosphate were accumulated by such leaves in the light (14). Although subsequent investigations showed that lightdependent uptake of ions is a general process in plants (4, 19), still little is known about the mechanism of this process in chlorophyll-containing tissues. Certain features of the problem outlined above for chloroplasts are relevant to the properties of mitochondria. Mitochondria are known to regulate their structure and also to translocate ions by an energy-dependent mechanism. In 1961, Ca uptake by mitochondria was reported by Vasington and Murphy (32) and by DeLuca and Engstrom (6). Ca uptake in rat kidney mitochondria was dependent on respiration and required ATP, Mg, and phosphate (33). Ca binding was prevented by uncouplers of oxidative phosphorylation and electron-transport inhibitors. Brierley, M.urer, and Green found that ADP inhibited divalent cation uptake, possibly by competing with the ion for a high-energy intermediate (3). The close association of ion uptake with phosphorylation in mitochondria was shown by Rossi and Lehninger, who reported that for each pair of electrons traversing a phosphorvlating site in the respiratory chain, 1.0 molecules of phosphate and 1.67 of Ca were accumulated (29). During Ca and phosphate accumulation, other ions also undergo concentration changes; e.g., the intramitochondrial K content falls and Na rises, although only 2 to 3 % as much monovalent ion is moved as Ca (5). It now appears that ion uptake by plant mitochondria occurs under similar conditions as ion uptake by animal mitochondria (12, 22). By an approach initially based on mitochondrial investigations for the uptake of Ca and phosphate, it has now been discovered that the basic conditions of electron flow, Mg and ATP support the uptake of these ions by isolated spinach chloroplasts. [Preliminary reports of this research have recently appeared (24, 25)]. This ion translocation process is associated with the light-triggered Mg-activated hydrolysis of ATP in chloroplasts (11, 21, 28). The ability of spinach chloroplasts to take up other ions has been surveyed and it has been found that Na is also taken up by an energy-dependent process.