We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane. Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Λ ϭ 145pS. Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient. Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening. In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75. The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (d pore ≅ 8-9 Å). Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.

The channel properties of Toc75 (the protein import pore of the outer chloroplastic membrane) were further characterized by electrophysiological measurements in planar lipid bilayers. After improvement of the Toc75 reconstitution procedure the voltage dependence of the channel open probability resembled those observed for other beta-barrel pores. Studies concerning the pore size of the reconstituted Toc75 indicate the presence of a narrow restriction zone corresponding to the selectivity filter and a wider pore vestibule with diameters of approximately 14 A and 26 A, respectively. Interactions between Toc75 and different peptides (a genuine chloroplastic transit peptide, a synthetic peptide resembling a transit peptide, and a mitochondrial presequence) show that Toc75 itself is able to differentiate between these peptides and the recognition is based on both conformational and electrostatic interactions.

The reconstituted pea chloroplastic outer envelope protein of 16 kDa (OEP16) forms a slightly cationselective, high-conductance channel with a conductance of ⌳ ‫؍‬ 1,2 nS (in 1 M KCl). The open probability of OEP16 channel is highest at 0 mV (P open ‫؍‬ 0.8), decreasing exponentially with higher potentials. Transport studies using reconstituted recombinant OEP16 protein show that the OEP16 channel is selective for amino acids but excludes triosephosphates or uncharged sugars. Crosslinking indicates that OEP16 forms a homodimer in the membrane. According to its primary sequence and predicted secondary structure, OEP16 shows neither sequence nor structural homologies to classical porins. The results indicate that the intermembrane space between the two envelope membranes might not be as freely accessible as previously thought.

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares Ϸ22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltagegated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.

Isolated chloroplast envelope membranes were fused with azolectin liposomes. Ion transport across the membrane of these liposomes was investigated by the patch-clamp technique and in planar bilayers. Our results show that the chloroplast envelope contains voltage-dependent anion- and cation-selective channels as well as anion- and cation-selective pores with high conductances. At least one of the high-conductance pores could be located in the chloroplast outer envelope membrane. The low-conductance chloride channel and the potassium channel showed complex gating behavior with subconductant states. Potassium channel gating was affected by monovalent and divalent cations as well as by millimolar concentrations of ATP. Low concentrations of Cs+ induced a flickering block. Voltage dependence of the open probability reveals that macroscopic currents of potassium channels are rectified with preferential potassium uptake into the chloroplast. Flux measurements and determinations of the stroma pH of intact chloroplasts confirm the presence of a potassium channel that is regulated by divalent cations (Mg2+) and by ATP. The fully open potassium channel revealed a conductance of lambda approximately equal to 100 pS in asymmetric KCl (250/20 mM KCl), and the fully open chloride channel revealed a conductance of lambda approximately equal to 60 ps in 100 mM Tris/HCl. One high-conductance channel, mainly active at holding potentials > 60 mV, was slightly selective for glutamate anions (PK+/PGlu- approximately equal to 2) and revealed fast voltage-dependent gating. This high-conductance channel had a conductance of lambda approximately equal to 540 pS (in 250/20 mM potassium glutamate) and was closed most of the time. A second type of high-conductance channel, mainly open and active at holding potentials below 30 mV, was slightly selective for cations (PGlu-/PK+ approximately equal to 2) with a conductance of lambda approximately equal to 1.14 nS (in 250/20 mM potassium glutamate).

The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu 73 to Val 91 ) did not change the properties of the OEP16 channel. The only exception was a Cys 71 3 Ser mutation, which led to a loss of the CuCl 2 sensitivity of the channel. Analysis of N-and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.The plastid organelle family conducts vital biosynthetic functions in every plant cell. Chloroplasts carry out photosynthesis, which converts atmospheric carbon dioxide to carbohydrates like triosephosphate, starch, and others. These and other biosynthetic pathway products and intermediates are steadily exchanged with the surrounding cell by the assistance of specific carrier proteins, localized in the plastidic inner envelope and solute channels located in the chloroplast outer envelope. Although the inner envelope transport proteins, such as the triosephosphate/phosphate translocator, the dicarboxylate translocator, and the hexose phosphate translocator, show distinct substrate selectivity and specificity (1), it is not known to what extent transport through the outer membrane channels is selective and regulated. In particular, the number of different channels present in the outer membrane has not been determined.In mitochondria, a single anion selective channel with high conductance (VDAC) 1 has been discovered and extensively characterized at a molecular level (2). A second protein with high homology to the VDAC channel has also been identified (3). The VDAC channel formed by a 30-kDa protein with presumed ␤-sheet topology (4) is thought to be responsible for most of the metabolite flux across the outer mitochondrial membrane (5).In Gram-negative bacteria, however, several different types of high conductance channels exist in the outer membrane (6): (i) So-called porins, forming water-filled pores that allow the downhill diffusion of solutes, provided that the size of the solutes does not exceed the exclusion limit (ϳ600 Da) of the chann...

Ion channels in the thylakoid membrane were investigated by direct patch clamping on swollen thylakoids. A preparation method has been developed in order to release osmotically swollen intact thylakoids from pea protoplasts derived from cotyledons of young Pisum sativum plants. The swollen thylakoids with typical diameters between 10 µm and 20 µm formed reproducibly high-resistance seals with patch pipettes. We observed a potassium channel with a main conductant state of Λ Ϸ 40 pS and a conductance of Λ Ϸ 90 pS (in asymmetric 20/100 mM KCl) for the fully open channel. Surprisingly, the thylakoid membranes also contained a high-conductance channel with a main conductant state of Λ Ϸ 620 pS (in asymmetric 20/100 mM KCl), revealing also higher and lower conductant states. With a different experimental approach we showed that thylakoids are able to accumulate transiently the membrane impermeant fluorescent dye Lucifer Yellow which likewise suggests the presence of a pore-like channel with a diameter large enough to allow permeation of Lucifer Yellow.Keywords : chloroplast; thylakoid; ion channel; patch clamp; fluorescence.Light-induced electron flow in the photosynthetic mem-electrophysiologically, a voltage-dependent Cl Ϫ channel has branes of chloroplasts causes proton pumping across these mem-been identified in patch-clamp studies on swollen chloroplasts branes. The proton motive force generated by the light-driven of Peperomia metallica revealing a unit conductance of Λ ϭ redox pumps is used for ATP synthesis by the H ϩ -ATPase 110 pS (Ω Ϫ1 ) (Schoenknecht et al., 1988). Besides, voltage-de-(Mitchell, 1966). In thylakoids a proton gradient of pH 3Ϫ4 is pendent potassium channels with unit conductances of Λ ϭ established under saturating steady-state illumination (Rumberg 14 pS and Λ ϭ 20 pS have been observed upon incorporation of and Siggel, 1969 ;Pick et al., 1974), whereas at the same time vesicles derived from thylakoid membranes into planar bilayers the transmembrane electric potential between the two aqueous (Tester and Blatt, 1991). phases appears to be small (Ϸ10 mV, positive in the lumen; RotIn addition, a cation-selective channel with a conductance of tenberg and Grunwald, 1972;Junge and Jackson, 1982). By way 60 pS in symmetrical 105 mM KCl was characterized in isolated of contrast, a large membrane potential is observed across the patches of osmotically swollen spinach thylakoids (Pottosin and inner mitochondrial membrane (Holian and Wilson, 1980). In Schoenknecht, 1996). thylakoids, the membrane potential has to be electrically balUsing the patch-clamp technique we have previously idenanced by the countercurrent of K ϩ and Mg 2ϩ (Hind et al., 1974; tified three types of voltage-dependent conductances in the Chow et al., 1976) and uptake of Cl Ϫ (Vambutas and Schechter, membrane of giant thylakoid/liposome vesicles (Enz et al., 1983; Vambutas, 1984) during the onset of steady-state accumu-1993). A chloride conductance of Λ Ϸ 220 pS, a potassium conlation of protons in the lumen. Ion channels would ...