水通道蛋白的生理功能 ——水通道基因敲除小鼠表型研究进展

水通道蛋白 (aquaporin, AQP) 是一族细胞膜上选择性高效转运水分子的特异孔道. 自从 Agre 等于 1992 年从红细胞膜发现第一个水通道蛋白 AQP1以来,有关水通道蛋白结构与功能的研究取得了迅速的、系列性的进展 . 已报道的哺乳动物 AQP 家族已有 11 个在蛋白质序列上有同源性成员 (AQP0~AQP10). AQP 在体内各系统组织中广泛表达,除了在与体液分泌和吸收密切相关的多种上皮和内皮细胞高表达外,在一些与体液转运无明显关系的组织细胞如红细胞、白细胞、脂肪细胞和骨骼肌细胞等处也有表达,提示 AQP 可能在多种器官生理和病理中发挥重要作用. 基因打靶技术是研究特定基因在体内生理功能的有力手段. 目前 AQP1、3、4、5 基因敲除和 AQP2 基因点突变的基因敲入小鼠模型 ( 模拟人类常染色体隐性遗传尿崩症 ) 已成功建立并广泛用于表型研究,在 AQP 水通道蛋白生理功能方面获得许多重要进展.     

The structural basis of water permeation and proton exclusion in aquaporins

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.     

The structural basis of water permeation and proton exclusion in aquaporins (Review)

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.     

Aquaporin water channels and lung physiology

Fluid transport across epithelial and endothelial barriers occurs in the neonatal and adult lungs. Biophysical measurements in the intact lung and cell isolates have indicated that osmotic water permeability is exceptionally high across alveolar epithelia and endothelia and moderately high across airway epithelia. This review is focused on the role of membrane water-transporting proteins, the aquaporins (AQPs), in high lung water permeability and lung physiology. The lung expresses several AQPs: AQP1 in microvascular endothelia, AQP3 in large airways, AQP4 in large- and small-airway epithelia, and AQP5 in type I alveolar epithelial cells. Lung phenotype analysis of transgenic mice lacking each of these AQPs has been informative. Osmotically driven water permeability between the air space and capillary compartments is reduced approximately 10-fold by deletion of AQP1 or AQP5 and reduced even more by deletion of AQP1 and AQP4 or AQP1 and AQP5 together. AQP1 deletion greatly reduces osmotically driven water transport across alveolar capillaries but has only a minor effect on hydrostatic lung filtration, which primarily involves paracellular water movement. However, despite the major role of AQPs in lung osmotic water permeabilities, AQP deletion has little or no effect on physiologically important lung functions, such as alveolar fluid clearance in adult and neonatal lung, and edema accumulation after lung injury. Although AQPs play a major role in renal and central nervous system physiology, the data to date on AQP knockout mice do not support an important role of high lung water permeabilities or AQPs in lung physiology. However, there remain unresolved questions about possible non-water-transporting roles of AQPs and about the role of AQPs in airway physiology, pleural fluid dynamics, and edema after lung infection.     

Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.     

Modulating the expression of aquaporin genes in planta: A key to understand their physiological functions?

Aquaporins (AQPs) are believed to act as "cellular plumbers", allowing plants to rapidly alter their membrane water permeability in response to environmental cues. This study of AQP regulation at both the RNA and protein levels has revealed a large number of possible mechanisms. Currently, modulation of AQP expression in planta is considered the strategy of choice for elucidating the role of AQPs in plant physiology. This review highlights the fact that this strategy is complicated by many factors, such as the incomplete characterization of transport selectivity of the targeted AQP, the fact that AQPs might act as multifunctional channels with multiple physiological roles, and the number of post-translational regulation mechanisms. The classification of AQPs as constitutive or stress-responsive isoforms is also proposed.     

Aquaporins are multifunctional water and solute transporters highly divergent in living organisms

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agre's team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.     

Amphibian aquaporins and adaptation to terrestrial environments: a review

In many anurans, the pelvic patch of the ventral skin and the urinary bladder are important osmoregulatory organs. Since the discovery of water channel protein, aquaporin (AQP), in mammalian erythrocytes, 17 distinct full sequences of AQP mRNAs have been identified in anurans. Phylogenetic tree of AQP proteins from amphibians and mammals suggested that anuran AQPs can be divided into six types: i.e. types 1, 2, 3, and 5, and anuran-specific types a1 and a2. Among them, two types of anuran AQPs (types 1 and a2) are localized in the skin and urinary bladder by immunohistochemistry. Tree frog type-a2 AQPs, AQP-h2 and AQP-h3, are vasotocin-regulated water channels predominant in the osmoregulatory organs. Both the AQP-h2 and AQP-h3 are expressed at the granular cells underneath the keratinized layer in the pelvic patch, whereas only AQP-h2 is detected at the granular cells in the urinary bladder. In response to vasotocin, both the molecules seem to be translocated from the cytoplasmic pool to the apical plasma membrane of the granular cells. On the other hand, type-1 AQPs, Rana FA-CHIP and Hyla AQP-h1, are detected at the endothelial cells of blood capillaries in frog osmoregulatory organs. These findings suggest that AQP-h2 and AQP-h3 are key players for transepithelial water movement, and that FA-CHIP and AQP-h1 might be important for the transport of absorbed water into the blood flow. Comparative investigation of type-a2 AQPs in anurans further revealed that AQP-h2 and -h3-like molecules might exist at the urinary bladder and the pelvic skin, respectively, in various anurans from aquatic species to arboreal dwellers. AQP-h2-like protein is also detected in the pelvic skin of terrestrial and arboreal species. It is possible that this molecule might have occurred in the pelvic skin as anurans penetrated into drier environments.     

Cellular and subcellular localization of aquaporins 1, 3, 8, and 9 in amniotic membranes during pregnancy in mice

The amniotic membrane encloses the amniotic fluid and plays roles in the regulation of amniotic fluid flux through the intramembranous pathway during pregnancy. Aquaporins (AQPs) 1, 3, 8, and 9 are expressed in amniotic membranes. AQPs are water channel proteins that facilitate the rapid flux of water or small molecules across the plasma membrane. Recently, additional roles of AQPs in facilitating cell migration, proliferation, and apoptosis have been suggested, with AQPs being distributed in the appropriate subcellular regions for their functions. The cellular and subcellular distributions of AQPs in the amniotic membrane however remain unclear. We have examined the cellular and subcellular localization of AQPs in amniotic membranes during pregnancy in mice. After embryonic day 12 (E12), AQP1 was distributed in the plasma membrane of finely branched cell processes in the amniotic fibroblasts. AQP3 was present in both epithelial cells and fibroblasts between E10 and E12. The distribution of AQP3 in the epithelial cells dynamically changed as follows: at E14 in the lateral membrane and apical junction; at E16 in the lateral membrane alone; at E17 in the lateral membrane and cytoplasm. AQP8 was expressed in the epithelial cells and complementarily localized in the apical junction and the lateral membrane. AQP9 was detected only in the apoptotic cells of the epithelium. These cellular and subcellular localizations of amniotic AQPs indicate that each AQP plays distinct functional roles, such as in water and urea transport, cell migration, cell proliferation, and apoptosis, for amniotic fluid homeostasis or tissue remodeling of amniotic membranes.     

Modulating the expression of aquaporin genes in planta: A key to understand their physiological functions?

Aquaporins (AQPs) are believed to act as “cellular plumbers”, allowing plants to rapidly alter their membrane water permeability in response to environmental cues. This study of AQP regulation at both the RNA and protein levels has revealed a large number of possible mechanisms. Currently, modulation of AQP expression in planta is considered the strategy of choice for elucidating the role of AQPs in plant physiology. This review highlights the fact that this strategy is complicated by many factors, such as the incomplete characterization of transport selectivity of the targeted AQP, the fact that AQPs might act as multifunctional channels with multiple physiological roles, and the number of post-translational regulation mechanisms. The classification of AQPs as constitutive or stress-responsive isoforms is also proposed.     

Aquaporin-3 functions as a glycerol transporter in mammalian skin

The AQPs (aquaporins) are a family of homologous water transporting proteins expressed in many mammalian epithelial, endothelial and other cell types. Phenotype analysis of mice lacking individual AQPs has been informative in elucidating their role in mammalian physiology. For example, phenotype analysis has indicated an important role of AQPs in the renal urinary concentrating mechanism (AQP1-AQP4), brain water balance and neural signal transduction (AQP4), exocrine gland secretion (AQP5) and ocular fluid balance (AQP1, AQP5). In skin, the aquaglyceroporin AQP3 is expressed in the basal layer of epidermal keratinocytes. Mice deficient in AQP3 have dry skin with reduced SC (stratum corneum) hydration, decreased elasticity and impaired biosynthesis. Mechanistic analysis of the altered skin phenotype in AQP3 deficiency suggested that the glycerol rather than the water transporting function of AQP3 is important in skin physiology. The glycerol content of SC and epidermis of AQP3 deficient mice is reduced, whereas that of dermis and serum is normal. The dry, relatively inelastic skin in AQP3 null mice is probably related to the humectant properties of glycerol, and the impaired SC repair to impaired epidermal biosynthetic function. The key role of AQP3 in epidermal physiology might be exploited in the development of improved cosmetics and new therapies for skin diseases associated with altered skin water content.     

Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways.

Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion. Many of the tissues involved in these processes are highly water permeable and express aquaporin (AQP) water channels. AQP1 is expressed in microvascular endothelia throughout the lung and airways, AQP3 in epithelia in large airways, AQP4 in epithelia throughout the airways, and AQP5 in type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these AQPs increases near the time of birth and is regulated by growth factors, inflammation, and osmotic stress. Transgenic mouse models of AQP deletion have provided information about their physiological role. In lung, AQP1 and AQP5 provide the principal route for osmotically driven water transport; however, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion nor is lung CO(2) transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, regulation of the airway surface liquid layer, and isosmolar fluid absorption are not impaired by AQP deletion. In contrast to these negative findings, AQP5 deletion in submucosal glands in upper airways reduced fluid secretion and increased protein content by greater than twofold. Thus, although AQPs play a major physiological role outside of the airways and lung, AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of fluid transport in airways, pleura, and lung compared with renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress and/or injury not yet tested or in functions unrelated to transepithelial fluid transport.     

Upregulation and protein trafficking of aquaporin-2 attenuate cold-induced osmotic damage during cryopreservation

Aquaporins (AQPs) are a recently discovered family of proteins that function as transmembrane water channels. These proteins regulate the delicate osmotic balance across the cell plasma membrane. Given that osmotic damage is the major contributing factor to cell death during freezing, we hypothesized that regulation of AQPs may have an unrealized role in protecting cells from osmotic damage during cryopreservation. Rat kidney inner medullar collecting duct (IMCD) cells were treated with arginine vasopressin (AVP) to increase the amount of AQP2 in the external plasma membrane before freezing in University of Wisconsin solution at -4 degrees C for 24 h. This resulted in a significant increase in cell viability on warming. Conversely, treatment of IMCD cells with AVP and W7 (which inhibits AQP2 protein trafficking to the plasma membrane) before freezing resulted in a 55% decrease in cell viability. These preliminary data indicate that regulation of AQP2 can attenuate cold-induced osmotic damage in rat kidney IMCD cells.     

Roles of aquaporins in the brain

It is now over 10 years ago that aquaporin 1 (AQP1) was discovered and cloned from the red blood cells, and in 2003 the Nobel price in Chemistry was awarded to Pr. Peter Agre for his work on AQPs, highlighting the importance of these proteins in life sciences. AQPs are water channels. To date this protein family is composed of 11 sub-types in mammalians. Three main AQPs described in the mammalian brain are AQP1, AQP4 and AQP9. Several recent studies have shown that these channels are implicated in numerous physiological functions. AQP1 has a role in cerebrospinal fluid formation, whereas AQP4 is involved in water homeostasis and extracellular osmotic pressure in brain parenchyma. AQP4 seems also to have an important function in oedema formation after brain trauma or brain ischemia. AQP9 is implicated in brain energy metabolism. The level of expression of each AQP is highly regulated. After a trauma or an ischemia perturbation of the central nervous system, the level of expression of each AQP is differentially modified, resulting in facilitating oedema formation. At present, the exact role of each AQP is not yet determined. A better understanding of the mechanisms of AQP regulation should permit the development of new pharmacological strategies to prevent oedema formation. AQP9 has been recently specifically detected in the catecholaminergic neurons of the brain. This new result strengthens the hypothesis that the AQPs are not only water channels, but that some AQPs may play a role in energy metabolism as metabolite channels.     

Water channel proteins in the inner ear and their link to hearing impairment and deafness

The inner ear is a fluid-filled sensory organ that transforms mechanical stimuli into the senses of hearing and balance. These neurosensory functions depend on the strict regulation of the volume of the two major extracellular fluid domains of the inner ear, the perilymph and the endolymph. Water channel proteins, or aquaporins (AQPs), are molecular candidates for the precise regulation of perilymph and endolymph volume. Eight AQP subtypes have been identified in the membranous labyrinth of the inner ear. Similar AQP subtypes are also expressed in the kidney, where they function in whole-body water regulation. In the inner ear, AQP subtypes are ubiquitously expressed in distinct cell types, suggesting that AQPs have an important physiological role in the volume regulation of perilymph and endolymph. Furthermore, disturbed AQP function may have pathophysiological relevance and may turn AQPs into therapeutic targets for the treatment of inner ear diseases. In this review, we present the currently available knowledge regarding the expression and function of AQPs in the inner ear. We give special consideration to AQP subtypes AQP2, AQP4 and AQP5, which have been studied most extensively. The potential functions of AQP2 and AQP5 in the resorption and secretion of endolymph and of AQP4 in the equilibration of cell volume are described. The pathophysiological implications of these AQP subtypes for inner ear diseases, that appear to involve impaired fluid regulation, such as Menière's disease and Sj?gren's syndrome, are discussed.