A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Photophosphorylation in cell envelope vesicles from Halobacteriumhalobium

We have prepared vesicles from cell envelope membranes of $\text{Halobacterium}\text{halobium}$ strains R₁ and ET-15 which are able to synthesize ATP in response to illumination. This photophosphorylation is inhibited by dicyclohexylcarbodiimide (DCCD) and by phloretin. ATP synthesis in L vesicles from the R₁ strain (which contain bacteriorhodopsin) is inhibited by the protonophore 1799 but not by valinomycin. In M vesicles from the R₁ strain and in ET-15 vesicles (both contain halorhodopsin) photophosphorylation is inhibited by both 1799 and valinomycin. These data are consistent with the idea that light-driven ATP synthesis can be coupled to the electrochemical H⁺ gradient generated by bacteriorhodopsin or by halorhodopsin through the membrane potential component of protonmotive force.     

Superoxide dismutase: a photochemical augmentation assay.

Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium, have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na_out⁺ ? Na_in⁺), which arises from Na⁺-efflux coupled to the light-induced H⁺-gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K_T for glutamate is 1.4 × 10^?7m under these conditions, as compared to 1.3 × 10^?7m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na⁺ concentrations on the vesicle exterior and requires low Na⁺ concentrations in the interior. When Na⁺ of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na⁺-gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na⁺-gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.     

The quenching effect of blue light on halorhodopsin

A mutant of Halobacterium halobium which contains halorhodopsin was isolated from strain S₉. An absorbance change at 380 nm caused by steady orange light illumination (λ ?530 nm) was observed. This change depended upon the intensity of the actinic light. The bleached envelope vesicles and vesicles derived from nicotine-grown cells showed a small or no absorbance change at 380 nm, suggesting that the change stemmed from the photochemical intermediate of halorhodopsin (referred to as P-380). When blue light was superimposed on orange background illumination, the membrane potential (Δψ) of the envelope vesicles decreased. Δψ was determined from the tetraphenylphosphonium cation (TPP⁺) distribution by means of a TPP⁺ electrode. When blue light intensity was increased, both Δψ and the amount of P-380 were decreased. An equation was derived which showed that Δψ is proportional to the concentration of P-380 formed by illumination under the assumption that the ionic composition is not significantly changed upon illumination. This equation was checked experimentally from the following three points: The blue light effect, the relationship between Δψ and light intensity, and the effect of gramicidin. The data obtained accorded well with the theoretical relationship.     

The enthalpy changes upon hydrolysis of guanosine triphosphate anhydride and ester bonds

The effects of N,N′-dicyclohexylcarbodiimide (DCCD), triphenyltin chloride (TPT), and 3,5-di-tert-butyl-4-hydroxybenzylidenemalonomtrile (SP6847) were tested on the light-dependent activities of Halobacterium halobium R₁mR which contains a new retinal protein pigment designated as halorhodopsin but no bacteriorhodospin. DCCD inhibited ATP synthesis either in the light- or in the dark-aerobic conditions without affecting the light-induced proton uptake (ΔH⁺). Although DCCD lowered the membrane potential under dark-anaerobic conditions, the potential increased in the light as high as the control (the light-dependent membrane potential increment Δψ became apparently larger in the presence of DCCD). TPT had negligible effect on ATP synthesis both in the dark or in the light but inhibited markedly ΔH⁺ and partly Δψ. After R₁mR was treated with DCCD, TPT abolished ΔH⁺ almost completely but Δψ only partly. The remaining Δψ was collapsed by SF6847 with a concomitant proton incorporation (pH increase). These results led to the following postulations: (i) In R₁mR, ATP is synthesized by a H⁺-ATPase coupled either to respiration and/or light energization by halorhodopsin; (ii) the majority of protons are incorporated in the light by a mechanism which differs from H⁺-ATPase but is driven by the Δψ generated by halorhodopsin; (iii) TPT acts in this system as a chloride/hydroxide exchanger; (iv) the uncoupler SF6847 carries protons into cells in response to Δψ.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Bacterial chimeras and reversible phosphorylation: the work of Walther Stoeckenius

In 1971, Walther Stoeckenius discovered that Halobacterium halobium contains a purple pigment that is chemically similar to rhodopsin and works as a light-driven proton pump. This discovery set Stoeckenius on a research path centered on bacteriorhodopsin, which included the creation of a bovine-soybean-halobacteria chimera that was able to produce ATP when exposed to light and the discovery of a class of proteins that are phosphorylated in a light-dependent manner.Reconstitution of Purple Membrane Vesicles Catalyzing Light-driven Proton Uptake and Adenosine Triphosphate Formation (Racker, E., and Stoeckenius, W. (1974) J. Biol. Chem. 249, 662–663)Light-regulated Retinal-dependent Reversible Phosphorylation of Halobacterium Proteins (Spudich, J. L., and Stoeckenius, W. (1980) J. Biol. Chem. 255, 5501–5503)Walther Stoeckenius was born in 1921 in Giessen, Germany. He earned an M.D. degree from the University of Hamburg in 1950, after which he spent 18 months doing clinical work as an intern. In 1952, he began postdoctoral work at the Institute for Tropical Medicine in Hamburg, using electron microscopy to study the development of pox viruses. Two years later, he joined the Department of Pathology at the University of Hamburg as an assistant professor and became Docent for Pathology in 1958. At Hamburg, Stoeckenius continued to use electron microscopy to explore the fine structure of cells and the lipid membrane.In 1959, Stoeckenius left Germany to become a research associate in Keith Porter''s laboratory at Rockefeller University. After a few months, he became an assistant professor at Rockefeller, remaining there for 8 years and eventually becoming an associate professor. He continued to work on membrane structure, studying Halobacterium halobium, until he accepted a professorship at the University of California, San Francisco in 1967.In San Francisco, Stoeckenius focused more on biochemical techniques rather than electron microscopy. In collaboration with Dieter Oesterhelt, he discovered that H. halobium contains a purple pigment (bacteriorhodopsin) that is chemically similar to rhodopsin (1) and plays an important role in light energy storage in halobacteria, working as a light-driven proton pump (2).This discovery led to a collaboration with Journal of Biological Chemistry (JBC) Classic author Efraim Racker (3) in which Stoeckenius and Racker created a thoroughly unnatural vesicle. As reported in the first JBC Classic reprinted here, they used sonication to recombine membrane lipids from soybeans, bacteriorhodopsin from halobacteria, and ATPase from beef mitochondria. The resulting artificial vesicles were able to produce ATP when exposed to light. The chimeric vesicles also formed a simple model system for a biological proton pump capable of generating ATP from ADP and P_i.Stoeckenius continued to study bacteriorhodopsin and its light-driven proton uptake in bacteria. As reported in the second JBC Classic reprinted here, he discovered that phosphorylation is regulated by light absorbed by bacteriorhodopsin (4). Using [³²P]orthophosphate pulse labeling, Stoeckenius and John Spudich identified a class of phosphoproteins in H. halobium. Exposing labeled whole cells to light resulted in rapid dephosphorylation of two of the proteins, which were rapidly rephosphorylated upon darkening of the cells. The light sensitivity of the proteins was responsive to the presence of retinal, indicating that the dephosphorylation depended on rhodopsin-like (retinal-containing) photoreceptors.Stoeckenius currently is Professor Emeritus in the Department of Biochemistry and Biophysics and the Cardiovascular Research Institute at the University of California, San Francisco. He was elected to the National Academy of Sciences in 1978.     

Delta-endotoxin-induced leakage of 86Rb+-K+ and H2O from phospholipid vesicles is catalyzed by reconstituted midgut membrane

Brush border membrane from Heliothis virescens catalyzed delta-endotoxin-induced leakage of ⁸⁶Rb⁺-K⁺ and H₂O from phospholipid vesicles. Activated delta-endotoxin [CrylA(c)-55 kDa] from Bacillus thuringiensis kurstaki strain EG2244 producing a single CrylA(c) toxin, when incorporated into phospholipid vesicles, made these vesicles more leaky to ⁸⁶Rb⁺-K⁺ than phospholipid vesicles without toxin. This effect was assayed by following the movement of ⁸⁶Rb⁺ into the vesicles in response to a KCl gradient. When toxin was added to the outside of phospholipid vesicles, ⁸⁶Rb⁺ uptake was impeded. Vesicles prepared with H. virescens brush border membrane catalyzed the association of the toxin with the vesicle, and stimulated KCl gradient-induced ⁸⁶Rb⁺ uptake. Toxin did not catalyze the leakage of ³⁶Cl⁻, suggesting that the toxin created a cation-selective leak. Toxin enhanced the permeability of phospholipid vesicles to H₂O, demonstrated by the enhanced rate of vesicle shrinking under increased osmotic pressure. This was analyzed spectrophotometrically by following the rate of vesicle shrinking in response to a 10 mM KCl gradient. In the presence of concentrated phosphatidylcholine vesicles, toxin spontaneously associated with the vesicles so as to enhance the rate of vesicle shrinking in an osmotic gradient. The rate of vesicle shrinking the presence of KCl and toxin was catalyzed by the presence of brush border reconstituted into the vesicles, reducing the effective toxin concentration 1000-fold.     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

Light-driven proton translocations in Halobacterium halobium

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating an electrochemical proton gradient across the cell membrane. However, the typical response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light is turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium.The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow.The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N′-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor.Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.     

Extracellularly truncated desmoglein 1 compromises desmosomes in MDCK cells

Bacteriorhodopsin and Halorhodopsin present in Halobacterium halobium strains have been investigated in relation to Na⁺/H⁺ exchange in isolated cell envelope vesicles. Upon illumination, these retinal proteins result in extrusion of sodium ions by either an electrogenic Na⁺/H⁺ antiporter and/or a direct sodium pump. Since a molecular characterization of these mechanism(s) of sodium extrusion has not yet been realized, it was of interest to measure directly the light- and sodium-dependent changes in ΔpH and membrane potential under nearly identical conditions in S₉ and R¹mR cell membrane vesicles to gain information on the relation of these retinal proteins to sodium extrusion. These activities were evaluated in terms of their dependence on light intensity, and on the inhibitory effect of chemical modifiers of carboxyl groups (carbodiimides); electroneutral exchanges (monensin and triphenyltin); digitoxin and some analogues; and phloretin. Under most of the conditions and treatments employed, light- and sodium-dependent ΔpH led to similar effects in both membrane vesicle types. Hence, it is concluded that the ΔpH and Δξ which arise from sodium transport occur by either a single mechanism or by one which shares common features.     

Why 11-cis-Retinal?

The C₂₀ diterpenoid compound retinal is the chromophore of thevisual pigments the rhodopsins, and the pigments present inHalobacterium halobium, namely, bacteriorhodopsin (proton pump),halorhodopsin (chloride pump), and the sensory rhodopsins (phototaxisreceptor). In all cases, they are bound covalently to the receptorprotein by a protonated Schiff base. However, in rhodopsins,the retinal is the 11-cis isomer, whereas in H. halobium pigmentsit is the all-trans isomer. Why did Nature choose retinal asthe chromophore, and why 11-cis in some cases and all-transin other cases? Also why is the chromophore a protonated Schiffbase? These points are addressed after giving an outline ofthe current status of the various photoreceptor pigments     

In?Vitro Demonstration of Dual Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A subfamily of rhodopsin pigments was recently discovered in bacteria and proposed to function as dual-function light-driven H⁺/Na⁺ pumps, ejecting sodium ions from cells in the presence of sodium and protons in its absence. This proposal was based primarily on light-induced proton flux measurements in suspensions of Escherichia coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the putative Na⁺ and H⁺ transport definitively. We expressed IAR, an uncharacterized member from Indibacter alkaliphilus in E. coli cell suspensions, and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na⁺ pumping activity in vitro, namely, light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na⁺-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore. These results meet the criteria for electrogenic Na⁺ transport and electrogenic H⁺ transport, respectively, in the presence and absence of Na⁺. The direction of proton fluxes indicated that IAR was inserted inside-out into our sealed LUV system, which we confirmed by site-directed spin-label electron paramagnetic resonance spectroscopy. We further demonstrate that Na⁺ transport by IAR requires Na⁺ only on the cytoplasmic side of the protein. The in vitro LUV system proves that the dual light-driven H⁺/Na⁺ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo.     

Direct measurement of k channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers

Light-driven electron transfer reactions cause the active accumulation of protons inside thylakoids, yet at steady state the electrical potential difference across the thylakoid membrane is very small; therefore, there must be a flux of other ions to balance the charge that would otherwise be built up by the net movement of H⁺. This paper presents direct measurements of ion movements through channels in the thylakoid membrane. These were made possible by fusing thylakoid vesicles from spinach (Spinacia oleracea L.) into planar lipid bilayers, using techniques developed originally to study sarcoplasmic reticulum. No Mg²⁺ current was found, but voltage-dependent channels have been characterized, these being somewhat selective for K⁺ over Cl⁻. The data are consistent with a role for these channels in charge balance during light-driven H⁺ movements.     

Characterization of the ion transport activity of the budding yeast Na/H antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H⁺ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na⁺/H⁺ but not K⁺/H⁺ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na⁺ and K⁺ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb⁺ and Li⁺. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na⁺/H⁺ antiporters, whereas other known eukaryotic Na⁺/H⁺ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na⁺/H⁺ antiporters.     

Large Deformation of Helix F during the Photoreaction Cycle of Pharaonis Halorhodopsin in Complex with Azide

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl⁻ and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.     

Volume regulation by human lymphocytes: Characterization of the ionic basis for regulatory volume decrease

The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5–0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K⁺ decreased during RVD, while Na⁺ content remained unchanged. Similarly, the efflux of ⁸⁶Rb (used as a K⁺ analog) increased upon dilution, whereas ²²Na efflux was not altered. ⁸⁶Rb uptake was enhanced by hypotonic stress and both ouabain-sensitive and -insensitive components were affected. A ouabain-sensitive stimulation was also seen in Na⁺- free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K⁺-free media, and also in Na⁺-free media when Li⁺, choline⁺, or Tris⁺ were the substitutes. In high K⁺ or Rb⁺ hypotonic media shrinking was absent and a second swelling phase was observed. Cs⁺ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K⁺ concentration was varied and, with 50 mM K⁺, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K⁺, resulting from a selective increase in permeability to this ion. In addition, the (Na-K) pump appears to be activated upon cell swelling by a mechanism other than Na⁺ entry into the cell, but this activation is not essential for RVD.     

Light-induced generation of a protonmotive force and Ca2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The light-driven primary proton pump bacteriorhodopsin has been incorporated in the cytoplasmic membrane of Streptococcus cremoris, in order to generate a protonmotive force across these membranes. This has been achieved by fusion of S. cremoris membrane vesicles with bacteriorhodopsin proteoliposomes. This fusion occurred when both preparations were mixed at low pH (less than 6.0), as shown by sucrose density gradient centrifugation and by dilution of fluorescent phospholipids incorporated into the bacteriorhodopsin proteoliposomes. Fusion was strongly enhanced by the presence of negatively charged phospholipids in the liposomal bilayer. When proteoliposomes were used that showed light-dependent proton uptake, the orientation of bacteriorhodopsin in the fused membranes was inside-out with respect to the in vivo orientation in Halobacterium halobium. Consequently, in the light a ΔΨ, interior positive and a ΔpH, interior acid were generated. This protonmotive force could drive calcium uptake in the fused membranes. The uptake increased hyperbolically with increasing light intensity and was abolished by bleaching of bacteriorhodopsin. Addition of the ionophore valinomycin stimulated calcium uptake and led to an increase of the ΔpH. Calcium uptake was strongly decreased in the dark and in the light in the presence of uncouplers, nigericin or both valinomycin and nigericin.     

Potassium Channels in Motor Cells of Samanea saman: A Patch-Clamp Study

Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K⁺, Cl⁻ and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K⁺ currents that flow during leaflet movement. Recordings in the “whole-cell” mode reveal depolarization-activated K⁺ currents in extensor and flexor cells that increase slowly (t_½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K⁺ currents is adequate to account quantitatively for K⁺ loss, previously measured in vivo during cell shrinkage. The K⁺ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.     

The uptake and extrusion of monovalent cations by isolated heart mitochondria

Summary The factors involved in the movement of monovalent cations across the inner membrane of the isolated heart mitochondrion are reviewed. The evidence suggests that the energy-dependent uptake of K⁺ and Na⁺ which results in swelling of the matrix is an electrophoretic response to a negative internal potential. There are no clear cut indications that this electrophoretic cation movement is carrier-mediated and possible modes of entry which do not require a carrier are examined. The evidence also suggests that the monovalent cation for proton exchanger (Na⁺ > K⁺) present in the membrane may participate in the energy-dependent extrusion of accumulated ions. The two processes, electrophoretic cation uptake (swelling) and exchange-dependent cation extrusion (contraction) may represent a means of controlling the volume of the mitochondrion within the functioning cell. A number of indications point to the possibility that the volume control process may be mediated by the divalent cations Ca⁺² and Mg⁺². Studies with mercurial reagents also implicate certain membrane thiol groups in the postulated volume control process.An invited article.