Insights into structural mechanisms of gating induced regulation of aquaporins

Aquaporin family comprises of transmembrane channels that are specialized in conducting water and certain small, uncharged molecules across cell membranes. Essential roles of aquaporins in various physiological and pathophysiological conditions have attracted great scientific interest. Pioneering structural studies on aquaporins have almost solved the basic question of mechanism of selective water transport through these channels. Another important structural aspect of aquaporins which seeks attention is that how the flow of water through the channel is regulated by the mechanism of gating. Aquaporins are also regulated at the protein level, i.e. by trafficking which includes changes in their expression levels in the membrane. Availability of high resolution structures along with numerous molecular dynamics simulation studies have helped to gain an understanding of the structural mechanisms by which water flux through aquaporins is controlled. This review will summarize the highlights regarding structural features of aquaporins, mechanisms governing water permeation, proton exclusion and substrate specificity, and describe the structural insights into the mechanisms of aquaporin gating whereby water conduction is regulated by post translational modifications, such as phosphorylation.     

Regulation of plant aquaporin activity

Accumulating evidence indicates that aquaporins play a key role in plant water relations. Plant aquaporins are part of a large and highly divergent protein family that can be divided into four subfamilies according to amino acid sequence similarity. As in other organisms, plant aquaporins facilitate the transcellular movement of water, but, in some cases, also the flux of small neutral solutes across a cellular membrane. Plant cell membranes are characterized by a large range of osmotic water permeabilities, and recent data indicate that plant aquaporin activity might be regulated by gating mechanisms. The factors affecting the gating behaviour possibly involve phosphorylation, heteromerization, pH, Ca2+, pressure, solute gradients and temperature. Regulation of aquaporin trafficking may also represent a way to modulate membrane water permeability. The aim of this review is to integrate recent molecular and biophysical data on the mechanisms regulating aquaporin activity in plant membranes and to relate them to putative changes in protein structure.     

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca²⁺ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C^α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.     

On the pH regulation of plant aquaporins

The majority of plants are unable to evade unfavorable conditions such as flooding, salinity, or drought. Therefore, a fine-tuned water homeostasis appears to be of crucial importance for plant survival, and it was assumed that aquaporins play a significant role in these processes. Regulation of plant aquaporin conductivity was suggested to be achieved by a gating mechanism that involves protein phosphorylation under drought stress conditions and protonation after cytosolic acidification during flooding. The effect of protein phosphorylation or protonation of aquaporins was studied on two plasma membrane intrinsic proteins, NtPIP2;1 and NtAQP1 from tobacco, which were heterologously expressed in yeast. Our results on mutated aquaporins with serine-to-alanine exchange indicate that phosphorylation of the two key serine residues did not affect the pH-dependent modification of water permeability. Protonation on a conserved histidine residue decreased water conductivity of NtPIP2;1. Although cells expressing NtPIP2;1 with a replacement of the histidine by an alanine were found to be pH-insensitive with regard to water permeability, these maintain high water transport rates, similar to those obtained under acidic conditions. The data clearly support the role of histidine at 196 as a component of pH-dependent modification of aquaporin-facilitated water transport. The predictions of combined effects from phosphorylation at conserved serines and histidine protonation were not supported by the results of functional analysis. The obtained results challenge the gating model as a general regulation mechanism for plant plasma membrane aquaporins.     

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.     

Plant aquaporins: multifunctional water and solute channels with expanding roles

There is strong evidence that aquaporins are central components in plant water relations. Plant species possess more aquaporin genes than species from other kingdoms. According to sequence similarities, four major groups have been identified, which can be further divided into subgroups that may correspond to localization and transport selectivity. They may be involved in compatible solute distribution, gas-transfer (CO2, NH3) as well as in micronutrient uptake (boric acid). Recent advances in determining the structure of some aquaporins gives further details on the mechanism of selectivity. Gating behaviour of aquaporins is poorly understood but evidence is mounting that phosphorylation, pH, pCa and osmotic gradients can affect water channel activity. Aquaporins are enriched in zones of fast cell division and expansion, or in areas where water flow or solute flux density would be expected to be high. This includes biotrophic interfaces between plants and parasites, between plants and symbiotic bacteria or fungi, and between germinating pollen and stigma. On a cellular level aquaporin clusters have been identified in some membranes. There is also a possibility that aquaporins in the endoplasmic reticulum may function in symplasmic transport if water can flow from cell to cell via the desmotubules in plasmodesmata. Functional characterization of aquaporins in the native membrane has raised doubt about the conclusiveness of expression patterns alone and need to be conducted in parallel. The challenge will be to elucidate gating on a molecular level and cellular level and to tie those findings into plant water relations on a macroscopic scale where various flow pathways need to be considered.     

Plant aquaporins: Roles in plant physiology

Background

Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules across biological membranes of most living organisms.

Scope of review

Here, we present comprehensive insights made on plant aquaporins in recent years, pointing to their molecular and physiological specificities with respect to animal or microbial counterparts.

Major conclusions

In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular localizations and various physiological substrates in addition to water. Of particular relevance for plants is the transport by aquaporins of dissolved gases such as carbon dioxide or metalloids such as boric or silicic acid. The mechanisms that determine the gating and subcellular localization of plant aquaporins are extensively studied. They allow aquaporin regulation in response to multiple environmental and hormonal stimuli. Thus, aquaporins play key roles in hydraulic regulation and nutrient transport in roots and leaves. They contribute to several plant growth and developmental processes such as seed germination or emergence of lateral roots.

General significance

Plants with genetically altered aquaporin functions are now tested for their ability to improve plant resistance to stresses. This article is part of a Special Issue entitled Aquaporins.     

A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.     

Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype

The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant–water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular mechanisms governing aquaporin relocalisation

The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.     

Plant aquaporins: novel functions and regulation properties

Aquaporins are water channel proteins of intracellular and plasma membranes that play a crucial role in plant water relations. The present review focuses on the most recent findings concerning the molecular and cellular properties of plant aquaporins. The mechanisms of transport selectivity and gating (i.e. pore opening and closing) have recently been described, based on aquaporin structures at atomic resolution. Novel dynamic aspects of aquaporin subcellular localisation have been uncovered. Also, some aquaporin isoforms can transport, besides water, physiologically important molecules such as CO(2), H(2)O(2), boron or silicon. Thus, aquaporins are involved in many great functions of plants, including nutrient acquisition, carbon fixation, cell signalling and stress responses.     

Yeast water channels: an overview of orthodox aquaporins

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N-terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.     

Arabidopsis SNAREs SYP61 and SYP121 Coordinate the Trafficking of Plasma Membrane Aquaporin PIP2;7 to Modulate the Cell Membrane Water Permeability

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.     

The role of aquaporins in cellular and whole plant water balance

Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants. In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents. Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes. A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level. Plants contain a large number of aquaporin isoforms with distinct cell type- and tissue-specific expression patterns. Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity. At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins.     

水通道蛋白研究动态

水通道蛋白是对水专一的通道蛋白,它普遍存在于动、植物及微生物中,不同水通道蛋白之间具有类似特征.哺乳动物中水通道蛋白主要分为六类,分布于水分代谢活跃的器官中;植物除了质膜上水通道蛋白外,液泡膜也存在着水通道蛋白,它们在植物生长,发育及胁迫适应中起着重要作用.目前有关水通道蛋白的详细的结构和功能信息主要来自对红细胞膜上水通道蛋白的研究,它由同源的四聚体组成,每个单体具有独立的水通道功能,四聚体在膜上分布具有不对称性,在膜内侧四聚体呈伸展状态,在膜外侧形成大的中心空腔.     

The role of aquaporins in root water uptake

The capacity of roots to take up water is determined in part by the resistance of living tissues to radial water flow. Both the apoplastic and cell-to-cell paths mediate water transport in these tissues but the contribution of cell membranes to the latter path has long been difficult to estimate. Aquaporins are water channel proteins that are expressed in various membrane compartments of plant cells, including the plasma and vacuolar membranes. Plant aquaporins are encoded by a large multigene family, with 35 members in Arabidopsis thaliana, and many of these aquaporins show a cell-specific expression pattern in the root. Mercury acts as an efficient blocker of most aquaporins and has been used to demonstrate the significant contribution of water channels to overall root water transport. Aquaporin-rich membranes may be needed to facilitate intense water flow across root tissues and may represent critical points where an efficient and spatially restricted control of water uptake can be exerted. Roots, in particular, show a remarkable capacity to alter their water permeability over the short term (i.e. in a few hours to less than 2-3 d) in response to many stimuli, such as day/night cycles, nutrient deficiency or stress. Recent data suggest that these rapid changes can be mostly accounted for by changes in cell membrane permeability and are mediated by aquaporins. Although the processes that allow perception of environmental changes by root cells and subsequent aquaporin regulation are nearly unknown, the study of root aquaporins provides an interesting model to understand the regulation of water transport in plants and sheds light on the basic mechanisms of water uptake by roots.     

A cohesion/tension mechanism explains the gating of water channels (aquaporins) in Chara internodes by high concentration

Isolated internodes of Chara corallina have been used to study the gating of aquaporins (water channels) in the presence of high concentrations of osmotic solutes of different size (molecular weight). Osmolytes were acetone and three glycol ethers: ethylene glycol monomethyl ether (EGMME), diethylene glycol monomethyl ether (DEGMME), and triethylene glycol monoethyl ether (TEGMEE). The 'osmotic efficiency' of osmolytes was quite different. Their reflection coefficients ranged between 0.15 (acetone), 0.59 (EGMME), 0.78 (DEGMME), and 0.80 (TEGMEE). Bulk water permeability (Lp) and diffusive permeabilities (Ps) of heavy water (HDO), hydrogen peroxide (H2O2), acetone, and glycol ethers (EGMME, DEGMME, and TEGMEE) were measured using a cell pressure probe. Cells were treated with different concentrations of osmotic solutes of up to 800 mM ( approximately 2.0 MPa of osmotic pressure). Inhibition of aquaporin activity increased with both increasing concentration and size of solutes (reflection coefficients). As cell Lp decreased, Ps increased, indicating that water and solutes used different passages across the plasma membrane. Similar to earlier findings of an osmotic gating of ion channels, a cohesion/tension model of the gating of water channels in Chara internodes by high concentration is proposed. According to the model, tensions (negative pressures) within water channels affected the open/closed state by changing the free energy between states and favoured a distorted/collapsed rather than the open state. They should have differed depending on the concentration and size of solutes that are more or less excluded from aquaporins. The bigger the solute, the lower was the concentration required to induce a reversible closure of aquaporins, as predicted by the model.     

Membrane trafficking and osmotically induced volume changes in guard cells

Guard cells rapidly adjust their plasma membrane surface area while responding to osmotically induced volume changes. Previous studies have shown that this process is associated with membrane internalization and remobilization. To investigate how guard cells maintain membrane integrity during rapid volume changes, the effects of two membrane trafficking inhibitors on the response of intact guard cells of Vicia faba to osmotic treatments were studied. Using confocal microscopy and epidermal peels, the relationship between the area of a medial paradermal guard-cell section and guard-cell volume was determined. This allowed estimates of guard-cell volume to be made from single paradermal confocal images, and therefore allowed rapid determination of volume as cells responded to osmotic treatments. Volume changes in control cells showed exponential kinetics, and it was possible to calculate an apparent value for guard-cell hydraulic conductivity from these kinetics. Wortmannin and cytochalasin D inhibited the rate of volume loss following a 0-1.5 MPa osmotic treatment. Cytochalasin D also inhibited volume increases following a change from 1.5 MPa to 0 MPa, but wortmannin had no effect. Previous studies showing that treatment with arabinanase inhibits changes in guard-cell volume in response to osmotic treatments were confirmed. However, pressure volume curves show that the effects of arabinanase and the cytochalasin D were not due to changes in cell wall elasticity. It is suggested that arabinanase, cytochalasin D, and wortmannin cause reductions in the hydraulic conductivity of the plasma membrane, possibly via gating of aquaporins. A possible role for aquaporins in co-ordinating volume changes with membrane trafficking is discussed.     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.