Summary A new aquaporin from Nicotiana tabacum (cv. Samsun) was characterized. It shares sequence homology to the Arabidopsis thaliana PIP1 protein family. By two‐phase partitioning and immunoblot analysis, plasma membrane localization could be demonstrated. The corresponding mRNA is highly abundant in roots and flowers, while it is rarely expressed in leaves and stems. Functional expression in Xenopus oocytes revealed that NtAQP1 can mediate glycerol transport in addition to water flow. However, NtAQP1 is impermeable for Na + , K + and Cl – ions. The water permeability and selectivity could not be modulated by addition of mercurials or the activity of cAMP‐dependent protein kinases.

Leaf-moving organs, remarkable for the rhythmic volume changes of their motor cells, served as a model system in which to study the regulation of membrane water fluxes. Two plasma membrane intrinsic protein homolog genes, SsAQP1 and SsAQP2, were cloned from these organs and characterized as aquaporins in Xenopus laevis oocytes. Osmotic water permeability (P(f)) was 10 times higher in SsAQP2-expressing oocytes than in SsAQP1-expressing oocytes. SsAQP1 was found to be glycerol permeable, and SsAQP2 was inhibited by 0.5 mM HgCl(2) and by 1 mM phloretin. The aquaporin mRNA levels differed in their spatial distribution in the leaf and were regulated diurnally in phase with leaflet movements. Additionally, SsAQP2 transcription was under circadian control. The P(f) of motor cell protoplasts was regulated diurnally as well: the morning and/or evening P(f) increases were inhibited by 50 microM HgCl(2), by 2 mM cycloheximide, and by 250 microM phloretin to the noon P(f) level. Our results link SsAQP2 to the physiological function of rhythmic cell volume changes.

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na ؉ , K ؉ , and H ؉ , and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na ؉ /H ؉ /K ؉ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na ؉ -or K ؉ -based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels. Excitatory amino acid transporters (EAATs)1 mediate the removal of glutamate from the synaptic cleft in the central nervous system and the uptake of glutamate in kidney and intestine (1-3). Five structurally distinct subtypes of mammalian glutamate transporters, EAAT1-EAAT5, have been identified in recent years (4 -9). Each of these isoforms exhibits two separate transport processes: a stoichiometrically coupled cotransport of one glutamate with three Na ϩ ions and one H ϩ , in countertransport to one K ϩ ion (10, 11); and an anion conductance that appears to be pore-mediated. The EAAT-associated anion channel is currently thought to function as a glutamategated channel with a tight coupling of channel opening and closing to conformational changes of the corresponding carrier domain. In this model, only certain carrier conformations are associated with conducting anion pores, and the anion channel cycles between conducting and non-conducting states during transitions through various conformational states of the glutamate carrier (8,9,(12)(13)(14)(15).We investigated anion conduction properties of two EAAT isoforms, human EAAT2 and rat EAAT4, using patch clamp recordings of transfected tsA201 cells under conditions that eliminate the Glu/Na ϩ /H ϩ /K ϩ current component. Our results demonstrated that opening and closing of the EAAT-associated anion channels as well as the interaction with the glutamate uptake process are more complex than previously assumed. External glutamate modifies intrinsic properties of EAAT2-and EAAT4-associated anion channels such as anion selectivity and the rate constants of anion permeation. Moreover, EAAT4 anion channels exhibit voltage-dependent opening and closing transitions that are modified by glutamate. These results provide novel insights into the function of neurotransmitter transporters and illustrate similarities as well as differences between transporter-associated pores and ion channe...

The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-␤-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxylterminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.ClC channels are found in almost all prokaryotic and in eukaryotic cells. Nine isoforms (ClC-1 to ClC-7, ClC-Ka and ClC-Kb) were shown to be expressed in human tissues and to fulfil a variety of functional roles. ClC-1 is the major muscle chloride channel responsible for the regulation of muscle excitability (1, 2), and ClC-2 is crucial for neuronal chloride homeostasis (3). ClC-Kb is involved in transepithelial NaCl movement in the thick ascending limb of Henle (4), and ClC-3, -5, and -7 are necessary for the pH adjustment of several cell compartments (5-7).Eukaryotic and prokaryotic ClC channels exhibit 18 transmembrane domains (8) followed by cytoplasmic carboxyl termini of variable sequences. The carboxyl-terminal tails of mammalian isoforms contain between 146 and 404 amino acids and exhibit two structurally defined domains, so-called CBS domains (9, 10). The functional importance of the carboxyl terminus is illustrated by disease-causing mutations in ClC-1, -2, -5 and ClC- Kb (3,6,11,12). Moreover, truncations removing parts of the distal cystathionine-␤-synthase (CBS) 1 domain of ClC channels were shown to abolish functional expression in heterologous systems (6,(13)(14)(15)(16). Although co-expression of the complemen...