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.     

The first water channel protein (later called aquaporin 1) was first discovered in Cluj-Napoca, Romania.

This invited review briefly outlines the importance of membrane water permeability, highlights the landmarks leading to the discovery of water channels. After a decade of systematic studies on water channels in human RBC Benga's group discovered in 1985 the presence and location of the water channel protein among the polypeptides migrating in the region of 35-60 kDa on the electrophoretogram of RBC membrane proteins. The work was extended and reviewed in several articles. In 1988, Agre and coworkers isolated a new protein from the RBC membrane, nick-named CHIP28 (channel-forming integral membrane protein of 28 kDa). However, in addition to the 28 kDa component, this protein had a 35-60 kDa glycosylated component, the one detected by the Benga's group. Only in 1992 Agre's group suggested that "it is likely that CHIP28 is a functional unit of membrane water channels". Half of the 2003 Nobel Prize in Chemistry was awarded to Peter Agre (Johns Hopkins University, Baltimore, USA) "for the discovery of water channels", actually the first water channel protein from the human red blood cell (RBC) membrane, known today as aquaporin 1 (AQP1). The seminal contributions from 1986 of the Benga's group were grossly overlooked by Peter Agre and by the Nobel Prize Committee. Thousands of science-related professionals from hundreds of academic and research units, as well as participants in several international scientific events, have signed as supporters of Benga; his priority is also mentioned in several comments on the 2003 Nobel Prize.     

Concerted action of two cation filters in the aquaporin water channel

Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H⁺ ≫K⁺ >Rb⁺ >Na⁺ >Cs⁺ >Li⁺. Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.     

Identification and characterization of functional aquaporin water channel protein from alimentary tract of whitefly, Bemisia tabaci

Some hemipteran xylem and phloem-feeding insects have evolved specialized alimentary structures or filter chambers that rapidly transport water for excretion or osmoregulation. In the whitefly, Bemisia tabaci, mass movement of water through opposing alimentary tract tissues within the filter chamber is likely facilitated by an aquaporin protein. B. tabaci aquaporin-1 (BtAQP1) possesses characteristic aquaporin topology and conserved pore-forming residues found in water-specific aquaporins. As predicted for an integral transmembrane protein, recombinant BtAQP1 expressed in cultured insect cells localized within the plasma membrane. BtAQP1 is primarily expressed in early instar nymphs and adults, where in adults it is localized in the filter chamber and hindgut. Xenopus oocytes expressing BtAQP1 were water permeable and mercury-sensitive, both characteristics of classical water-specific aquaporins. These data support the hypothesis that BtAQP1 is a water transport protein within the specialized filter chamber of the alimentary tract and functions to translocate water across tissues for maintenance of osmotic pressure and/or excretion of excess dietary fluid.     

The grapevine tonoplast aquaporin TIP2;1 is a pressure gated water channel

In plants, the vacuole is a multifunctional organelle with an important role in the maintenance of the intracellular space. Tonoplast membranes are highly permeable to water due to their content in aquaporins TIPs (Tonoplast Intrinsic Proteins) that allow the rapid water influx creating an internal turgor pressure responsible for cell expansion, elongation and shape.     

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3?.     

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and functional characterization of a vasotocin-sensitive aquaporin water channel in quail kidney

Both mammals and birds can concentrate urine hyperosmotic to plasma via a countercurrent multiplier mechanism, although evolutionary lines leading to mammals and birds diverged at an early stage of tetrapod evolution. We reported earlier (Nishimura H, Koseki C, and Patel TB. Am J Physiol Regul Integr Comp Physiol 271: R1535-R1543, 1996) that arginine vasotocin (AVT; avian antidiuretic hormone) increases diffusional water permeability in the isolated, perfused medullary collecting duct (CD) of the quail kidney. In the present study, we have identified an aquaporin (AQP) 2 homolog water channel in the medullary cones of Japanese quail, Coturnix coturnix (qAQP2), by RT-PCR-based cloning techniques. A full-length cDNA contains an 822-bp open reading frame that encodes a 274-amino acid sequence with 75.5% identity to rat AQP2. The qAQP2 has six transmembrane domains, two asparagine-proline-alanine (NPA) sequences, and putative N-glycosylation (asparagine-124) and phosphorylation sites (serine-257) for cAMP-dependent protein kinase. qAQP2 is expressed in the membrane of Xenopus laevis oocytes and significantly increased its osmotic water permeability (P(f)), inhibitable (P < 0.01) by mercury chloride. qAQP2 mRNA (RT-PCR) was detected in the kidney; medullary mRNA levels were higher than cortical levels. qAQP2 protein that binds to rabbit anti-rat AQP2 antibody is present in the apical/subapical regions of both cortical and medullary CDs from normally hydrated quail, and the intensity of staining increased only in the medullary CDs after water deprivation or AVT treatment. The relative density of the approximately 29-kDa protein band detected by immunoblot from the medullary cones was modestly higher in water-deprived/AVT-treated quail. The results suggest that 1) medullary CDs of quail kidneys express a mercury-sensitive functioning qAQP2 water channel, and 2) qAQP2 is at least partly regulated by an AVT-dependent mechanism. This is the first clear identification of AQP2 homolog in nonmammalian vertebrates.     

Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris

The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C-terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation-mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix-assisted laser desorption ionization-mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea- and alkali-stripped Pichia membranes by octyl-beta-D-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.     

Mercury inhibits the L170C mutant of aquaporin Z by making waters clog the water channel

We conduct in silico experiments of the L170C mutant of the Escherichia coli aquaporin Z (AQPZ) with and without mercury bonded to residue Cys 170. We find that bonding mercury to Cys 170 does not induce consequential structural changes to the protein. We further find that mercury does not stick in the middle of the water channel to simply occlude water permeation, but resides on the wall of the water pore. However, we observe that the water permeation coefficient of L170C-Hg⁺ (with one mercury ion bonded to Cys 170) is approximately half of that of the mercury-free L170C. We examine the interactions between the mercury ion and the waters in its vicinity and find that five to six waters are strongly attracted by the mercury ion, occluding the space of the water channel. Therefore we conclude that mercury, at low concentration, inhibits AQPZ-L170C mutant by making water molecules clog the water channel.     

A model linking water and protein structures

This communication presents a new picture of protein and solvent in which they are much more closely related both structurally and functionally than hitherto described. The picture is based on the recently published model of liquid structure, which proposes that clusters in water have long-term rather than flickering existence and are as large as proteins. Their spacial dimensions ensure that the two structures have a mutual tendency to pack together and cooperate in their vibrational motion.     

Immunohistochemical localization of a water channel, aquaporin 7 (AQP7), in the rat testis

Cell volume reduction is one of the most distinct morphological changes during spermiogenesis and may be largely attributable to water efflux from the cell. A strong candidate for a water efflux route, aquaporin 7 (AQP7), which is a water channel, was studied immunohistochemically in the rat testis. Immunoreactivity was restricted within the elongated spermatids, testicular spermatozoa, and residual bodies remaining in the seminiferous epithelium. Weak but distinct immunoreactivity was first observed in the cytoplasmic mass of the spermatid at step 8 of spermiogenesis. The Golgi-like apparatus became steadily immunoreactive at step 10. The plasma membrane covering the cytoplasmic mass showed strong immunoreactivity after step 16. At this step, the middle piece of the tail also showed immunoreactivity at the portion protruding into the lumen. The whole head and distal tail, where the elongated spermatid had only a limited amount of cytoplasm, showed no immunoreactivity throughout spermiogenesis. After spermiation, the immunoreactivity of AQP7 remained at the middle piece and in the cytoplasmic droplet in the testicular spermatozoon. The present observations suggest that AQP7 contributes to the volume reduction of spermatids, since this water channel protein is localized on the plasma membrane covering the condensing cytoplasmic mass of the elongated spermatid, and since the seminiferous tubule fluid is hypertonic.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Role of nonacidic endosomes in recycling of ADH-sensitive water channel structures.

Toad urinary bladder epithelial cells respond to the hormone ADH by increasing the water permeability of their luminal membrane. This action is mediated by insertion into the apical membrane of specific water channels. In the absence of ADH these channels appear to be present in tubular cytoplasmic vesicles as morphologically distinctive intramembrane structures called particle aggregates. ADH induces these vesicles to fuse with the apical membrane, transferring their aggregate-water channels into the apical membrane. When ADH stimulation is removed (ADH reversal), aggregates and fluid-phase markers from the mucosal bath appear in water-permeable vesicles in the cytoplasm. We have examined the fate of fluid-phase markers and aggregates with time after ADH reversal. Although the fluid-phase markers horseradish peroxidase and colloidal gold are initially found predominantly in tubular vesicles near the apical surface, by 30 min the markers were found in perinuclear multivesicular bodies (MVBs) of heterogeneous size and shape. These MVBs appear to be nonacidic since they fail to accumulate DAMP. Acid phosphatase (AcPase) was undetectable in these structures. After 60 min, labeled MVBs tended to be smaller, and some of these structures displayed DAMP accumulation and AcPase activity. By evaluation of uncleaned replicas it was possible to localize recycled aggregate-water channels with respect to internalized fluid-phase markers. Thirty minutes after retrieval from the apical surface in tubular vesicles, aggregates could be localized to both the central body and tubular projections of labeled MVBs. At 60 min following reversal, most MVBs had a reduced number of aggregates compared with 30 min, and compact structures could be identified that contained markers but no detectable aggregates. These observations show that aggregates and fluid-phase markers enter a nonacidic endosomal compartment with an MVB morphology following ADH reversal. At extended times following reversal, labeled MVBS having lysosomal characteristics and labeled MVBs having no detectable aggregates can be found, suggesting that aggregates are sorted or degraded prior to this stage.     

Potassium channel structures

The molecular basis of K+ channel function is universally conserved. K+ channels allow K+ flux and are essential for the generation of electric current across excitable membranes. K+ channels are also the targets of various intracellular control mechanisms, such that the suboptimal regulation of channel function might be related to pathological conditions. Because of the fundamental role of K+ channels in controlling membrane excitability, a structural understanding of their function and regulation will provide a useful framework for understanding neuronal physiology. Many recent physiological and crystallographic studies have led to new insights into the workings of K+ channels.