Ion Channels in Native Chloroplast Membranes: Challenges and Potential for Direct Patch-Clamp Studies

Abstract

Photosynthesis without any doubt depends on the activity of the chloroplast ion channels. The thylakoid ion channels participate in the fine partitioning of the light-generated proton-motive force (p.m.f.). By regulating, therefore, luminal pH, they affect the linear electron flow and non-photochemical quenching. Stromal ion homeostasis and signaling, on the other hand, depend on the activity of both thylakoid and envelope ion channels. Experimentally, intact chloroplasts and swollen thylakoids were proven to be suitable for direct measurements of the ion channels activity via conventional patch-clamp technique; yet, such studies became infrequent, although their potential is far from being exhausted. In this paper we wish to summarize existing challenges for direct patch-clamping of native chloroplast membranes as well as present available results on the activity of thylakoid Cl(-) (ClC?) and divalent cation-permeable channels, along with their tentative roles in the p.m.f. partitioning, volume regulation, and stromal Ca(2+) and Mg(2+) dynamics. Patch-clamping of the intact envelope revealed both large-conductance porin-like channels, likely located in the outer envelope membrane and smaller conductance channels, more compatible with the inner envelope location. Possible equivalent model for the sandwich-like arrangement of the two envelope membranes within the patch electrode will be discussed, along with peculiar properties of the fast-activated cation channel in the context of the stromal pH control.

Keywords: ClC channel; cation channel; chloroplast envelope; magnesium; patch-clamp; porin; proton-motive force; thylakoid.

Figure 1

Chloroplast ion transport under the light. Light-driven export of H⁺ into the thylakoid lumen by photosynthetic electron transfer chain (PS) causes a hyperpolarization of the thylakoid ΔΨ. At steady state, this voltage difference is partly dissipated by channel-mediated fluxes of anions, K⁺, and Mg²⁺. Light-driven H⁺ and parallel Cl⁻ fluxes to the thylakoid lumen cause the depletion of these ions in stroma, which is compensated by their uptake across the envelope. For maintenance of alkaline stromal pH, H⁺ could be actively extruded to cytosol by the IE H⁺ pump, which requires a counter influx of monovalent cations across the envelope for electrical balance. K⁺/H⁺ exchange across the envelope is essential for control of the chloroplast volume and stromal pH. Abbreviations: TM, IE, and OE are thylakoid, inner envelope, and outer envelope membranes, F0F1 is thylakoid ATP-synthetase (F-type H⁺-ATPAse), TPK3 (tandem-pore K⁺ 3 channel, functionally characterized in recombinant system). In situ functionally (by patch-clamp) detected channels were: ClC (anion-selective channel from a ClC family), ICTCC (intermediate-conductance thylakoid cation channel), FACC (fast activating chloroplast cation channel), PIRAC (protein import related anion channel), and outer envelope porins (most possibly, active OEP24 or OEP21). Other: GLR3.4 (glutamate receptor type 3.4 channel) and KEA1/2 (cation/proton antiporters from family 2, CPA2). Another member of the CPA2 family, the thylakoid-localized KEA3, accelerates dissipation of the transthylakoid ΔpH upon the light offset.

Figure 2

Hypothetical equivalent electrical circuit for recording on a sandwich-like envelope patch. It is assumed that the intermembrane space was tightly sealed (${\text{R}}_{i/m}^{\#}\text{ >>}$ R_o) and that inner envelope electrical resistance was much higher than of the outer envelope, R_i >> R_o (due to the presence of multiple porins in the latter), so that command voltage (V_comm) dropped almost entirely across the inner envelope membrane patch (V_comm ~V_i). R_o represents the access resistance, which was ~1 GOhm for measurements on pea chloroplasts, equivalent to a presence of few (2–3) open porins in the outer patch membrane. Average patch capacitance was about 0.35 pF; on the basis of specific capacitance for biological membranes (~1 μF/cm²) this transforms to 35 μm² or 70% of the pea chloroplast surface, additional argument for the presence of a double membrane vesicle in the patch (Pottosin et al., 2005).