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

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.

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).

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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...