Oep23 forms an ion channel in the chloroplast outer envelope

Abstract

Background: Metabolite, ion and protein translocation into chloroplasts occurs across two membranes, the inner and the outer envelope. Solute and metabolite channels fulfill very important functions in integrating the organelles into the metabolic network of the cell. However so far only a few have been identified. Here we describe the identification and the characterization of the outer envelope protein of 23 kDa, Oep23 from garden pea.

Results: Oep23 is found in the entire plant lineage from green algae to flowering plants. It is expressed in all organs and developmental states tested so far. The reconstituted recombinant protein Oep23 from pea forms a high conductance ion channel with a maximal conductance in the fully open state of 466 ± 14pS at a holding potential of +100 mV (in 250 mM KCl). The Oep23 channel is cation selective (PK+ : PCl- = 15 : 1) with a voltage dependent open probability of maximal Vmem = 0 mV.

Conclusion: The data indicate that the Oep23 activity represents a single channel unit and does not assemble into a multiple pore complex like bacterial type porins or mitochondrial voltage dependent anion channel. Thus, Oep23 represents a new member of ion channels in the outer envelope of chloroplasts involved in solute exchange.

Figure 1

PsOep23 shows channel activity. Representative current traces of a bilayer containing a single active PsOep23 channel. Holding potential is set to +100 and −100 mV, respectively. The channel activity is characterized by short gating events from the open (o-main) to the sub-conductance state (o-sub). Some events are faster than the time resolution of the measurement which becomes apparent in the current histogram and a zoom into the trace. The electrolyte solution contained 250 mM KCl, 50 mM Mops/Tris pH 7.0 (symmetrical cis/trans).

Figure 2

PsOep23 temporarily fully opens. Current trace of a bilayer containing a single active Oep23 channel at a holding potential of +100 mV. Apart from the main open (o-main) and the sub-conductance state (o-sub) the channel sometimes switched to its fully open state (o-full) and especially at holding potentials above +/−100 mV to the closed state (c). Electrolyte solution 250 mM KCl, 50 mM Mops/Tris pH 7.0 (symmetrical cis/trans).

Figure 3

Verification of PsOep23 orientation in a lipid bilayer. The I/V relationship revealed a slightly rectifying current of all open states. This rectification helps to identify the orientation of the channel after fusion with the bilayer, which was randomly distributed. For clarity the large conductance was always allocated to positive holding potentials. The data points represent the average of 16 independent experiments. Electrolyte solution 250 mM KCl, 50 mM Mops/Tris pH 7.0 (symmetrical cis/trans).

Figure 4

PsOep23 is cation selective. Current response of a voltage ramp from −50 to +100 mV (voltage slope: 7.5 mV/s) in asymmetric electrolyte conditions (cis/trans 20/250 mM KCl). Oep23 has a pronounced cation selectivity. The reversal potential (E_rev) of +46 mV translates into a permeability ratio of K⁺: Cl⁻ of 15 : 1 by using the GHK equation and activities instead of concentrations.

Figure 5

Channel behavior of PsOep23 in presence of spermine. Current trace of a single active Oep23 channel at a holding potential of (A) +100 and (B) -100 mV in control conditions and after addition of 5 and 10 mM of spermine, respectively. The presence of spermine induced a concentration dependent reduction of the open channel conductance (o - > o’) and the open probability, apparent by a more frequent and prolonged likelihood to remain in a closed or blocked state. This block is completely reversible by washing out spermine (data not shown).