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

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 Ca2+ and Mg2+ 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

On the role of intracellular concentration of Ca2+ and H+ in thymocyte death after irradiation

AbstractThe role of intracellular Ca2+ and H+ concentrations in radiation-induced interphase death of rat thymocytes has been studied. In response to concanavalin A treatment in the Ca2+-containing medium, or to the CaCl2 treatment in the Ca2+-free medium, the [Ca2+]i rise in irradiated cells was as in the non-treated cells. No changes in the level of [Ca2+]i and pHi were found within l h after irradiation of thymocytes with a dose of 6 Gy. 15 μM 5-(N-ethyl-N-isopropyl)-amiloride. an inhibitor of Na+/H+ exchange, did not affect the DNA fragmentation. The fragmentation was prevented by 2–4 μM (1 -[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)]-2-[(2,4-dichlorophenyl)-methoxy]-ethyl)-1 -H-imidazoliumchloride, an inhibitor of calmodulin. The above data indicate that triggering of interphase death in irradiated thymocytes is not mediated by changes in either [Ca2+]i or pHi. Such changes seem to be involved in intermediate steps of the interphase death process

Homeostatic control of slow vacuolar channels by luminal cations and evaluation of the channel-mediated tonoplast Ca2+ fluxes in situ

Ca2+, Mg2+, and K+ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca2+, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca2+ activity was ∼0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca2+ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca2+) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca2+ within a physiological range of concentrations (0.1–1.0 mM). Aggregation of the physiological vacuolar Na+ (60 mM) and Mg2+ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca2+ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca2+ activates the SV channel in a voltage-independent manner with Kd=0.7–1.5 μM. Comparison of the vacuolar Ca2+ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 μM) cytosolic Ca2+, only 0.5% of SV channels are open. This mediates a Ca2+ release of only a few pA per vacuole (∼0.1 pA per single SV channel). Overall, our data suggest that the release of Ca2+ through SV channels makes little contribution to a global cytosolic Ca2+ signal

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field