Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis) either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.

Nitrate, the major nitrogen source for most plants, is widely used as a fertilizer and as a result has become a predominant freshwater pollutant. Plants need nitrate for growth and store most of it in the central vacuole. Some members of the chloride channel (CLC) protein family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels, whereas the bacterial ClC-ec1 and mammalian ClC-4 and ClC-5 have recently been characterized as Cl-/H+ exchangers with unknown cellular functions. Plant members of the CLC family are proposed to be anion channels involved in nitrate homeostasis; however, direct evidence for anion transport mediated by a plant CLC is still lacking. Here we show that Arabidopsis thaliana CLCa (AtCLCa) is localized to an intracellular membrane, the tonoplast of the plant vacuole, which is amenable to electrophysiological studies, and we provide direct evidence for its anion transport ability. We demonstrate that AtCLCa is able to accumulate specifically nitrate in the vacuole and behaves as a NO3-/H+ exchanger. For the first time, to our knowledge, the transport activity of a plant CLC is revealed, the antiporter mechanism of a CLC protein is investigated in a native membrane system, and this property is directly connected with its physiological role.

Anion channels/transporters are key to a wide spectrum of physiological functions in plants, such as osmoregulation, cell signaling, plant nutrition and compartmentalization of metabolites, and metal tolerance. The recent identification of gene families encoding some of these transport systems opened the way for gene expression studies, structure-function analyses of the corresponding proteins, and functional genomics approaches toward further understanding of their integrated roles in planta. This review, based on a few selected examples, illustrates that the members of a given gene family exhibit a diversity of substrate specificity, regulation, and intracellular localization, and are involved in a wide range of physiological functions. It also shows that post-translational modifications of transport proteins play a key role in the regulation of anion transport activity. Key questions arising from the increasing complexity of networks controlling anion transport in plant cells (the existence of redundancy, cross talk, and coordination between various pathways and compartments) are also addressed.

Fundamental plant functions such as control of the membrane potential, osmo-regulation, and turgor-driven growth and movements are based on the availability to gain high cellular potassium concentrations (1). The absorption of this inorganic osmolyte from the soil by the root therefore represents a pivotal process for plant life. Classical experiments by Epstein et al. in 1963 (2) described K ϩ root uptake as a biphasic process mediated by two uptake mechanisms: high affinity potassium transport with apparent affinities of ϳ20 M and a low affinity transport system with K m values in the millimolar range. During the last decades several molecular components of potassium transport systems have been identified and functionally characterized in plants (3, 4). Mutant analyses, heterologous expression, as well as radiotracer uptake experiments characterized the K ϩ channels AKT1⅐AtKC1 and members of the HAK⅐KT⅐KUP family as major components of the Arabidopsis thaliana root-localized potassium transport system (5-9). In this study we focused on AKT1 and AtKC1, members of the Arabidopsis Shaker-like K ϩ channel family. AKT1 is a voltagedependent inward-rectifying K ϩ channel mediating potassium uptake over a wide range of external potassium concentrations (10 -15). Root cells of the akt1-1 loss-of-function mutant completely lack inward rectifying K ϩ currents (12). As a consequence the growth of akt1-1 seedlings is strongly impaired on low potassium medium (100 M and less) (11,12,15). Rescue of yeast growth on 20 M K ϩ and patch clamp experiments (16, 17) directly demonstrated that plant inward rectifying K ϩ channels are capable of serving as high affinity potassium uptake transporters. AtKC1 shares its expression pattern with . AtKC1 ␣-subunits, however, neither form functional channels in akt1-1 knock-out plants nor in heterologous expression systems. In contrast to root cells of akt1-1 loss of function mutants, root protoplasts of AtKC1 null mutants (atkc1-f) still exhibit inward rectifying potassium currents most likely derived from homomeric AKT1 tetramers (20). Inward K ϩ currents in this atkc1-f mutant were characterized by a more positive activation voltage. These data suggested that the AtKC1 ␣-subunits do not form K ϩ channels per se but modulate the properties of the AKT1⅐AtKC1 heterocomplex (20 -22

SUMMARYGas exchange in plants is controlled by guard cells, specialized cells acting as turgor pressure-driven valves. Malate is one of the major anions accumulated inside the vacuole during stomatal opening counteracting the positive charge of potassium. AtALMT6, a member of the aluminum-activated malate transporter family, is expressed in guard cells of leaves and stems as well as in flower organs of Arabidopsis thaliana. An AtALMT6-GFP fusion protein was targeted to the vacuolar membrane both in transient and stable expression systems. Patch-clamp experiments on vacuoles isolated from AtALMT6-GFP over-expressing Arabidopsis plants revealed large inward-rectifying malate currents only in the presence of micromolar cytosolic calcium concentrations. Further analyses showed that vacuolar pH and cytosolic malate regulate the threshold of activation of AtALMT6-mediated currents. The interplay of these two factors determines the AtALMT6 function as a malate influx or efflux channel depending on the tonoplast potential. Guard cell vacuoles isolated from Atalmt6 knock-out plants displayed reduced malate currents compared with wild-type vacuoles. This reduction, however, was not accompanied by phenotypic differences in the stomatal movements in knock-out plants, probably because of functional redundancy of malate transporters in guard cell vacuoles.