Molecular Characterization of a Broad Selectivity Neutral Solute Channel

Abstract

In all living cells, coordination of solute and water movement across cell membranes is of critical importance for osmotic balance. The current concept is that these processes are of distinct biophysical nature. Here we report the expression cloning of a liver cDNA encoding a unique promiscuous solute channel (AQP9) that confers high permeability for both solutes and water. AQP9 mediates passage of a wide variety of non-charged solutes including carbamides, polyols, purines, and pyrimidines in a phloretin-and mercury-sensitive manner, whereas amino acids, cyclic sugars, Na ؉ , K ؉ , Cl ؊ , and deprotonated monocarboxylates are excluded. The properties of AQP9 define a new evolutionary branch of the major intrinsic protein family of aquaporin proteins and describe a previously unknown mechanism by which a large variety of solutes and water can pass through a single pore, enabling rapid cellular uptake or exit of metabolites with minimal osmotic perturbation.Transport of solutes such as ions, nutrients, neurotransmitters, and metabolic waste products across cell membranes is of fundamental importance to all mammalian cells. Despite the identification of many selective solute transporters and water channels (1-4), it has remained unclear how transport of large amounts of solutes is coordinated with water movement in metabolically highly active cells such as hepatocytes, spermatocytes, neurons, and glia. The liver is a major site of production and elimination of metabolites such as urea, nucleotides, and ketone bodies, and substantial amounts of these solutes must rapidly cross the hepatocyte plasma membrane with minimal osmotic perturbation (5). In testis, a solute transport mechanism is presumably required to supply nutrients to rapidly growing spermatocytes and to provide an exit pathway for metabolites. In brain, regulation of solute transport is critical because osmolality changes in extracellular fluids can affect neuronal cell function (6).Among metabolically active tissues, liver was selected as a target for expression cloning of a new solute-transporting protein because a phloretin-sensitive urea exit mechanism had been described (7-9). MATERIALS AND METHODSExpression Cloning-Total RNA was extracted from rats fed a high protein diet (50%, w/w) for 2 weeks. Poly(A) ϩ RNA purified by oligo(dT) chromatography was size-fractionated by preparative agarose gel electrophoresis (30). Specific fractions were screened for 1 mM [14 C]urea uptake activity in RNA-injected Xenopus oocytes (4, 30). A directional cDNA library was constructed from the positive fraction by using the SuperScript Plasmid System (Life Technologies, Inc.), and cDNA clones were screened for urea uptake (4). Northern Analysis and in Situ Hybridization-Poly(A)ϩ RNA (3 g) from rat tissues was electrophoresed in a formaldehyde-agarose gel and transferred to a nylon membrane. The filter was probed with 32 Plabeled full-length AQP9 1 cDNA, hybridized at 42°C, and washed with 0.1% SDS, 0.1ϫ SSC, at 65°C. Autoradiography was performed at Ϫ80°C for 5 days...

Aquaporins in spermatozoa and testicular germ cells: identification and potential role

“…In situ hybridization has revealed mRNA in immature germ cells [64] and cDNA microarrays have shown high expression in pachytene spermatocytes [53,54] with upregulation at the onset of spermatid elongation [52]. In the human testis, the entire ORF of the mRNA has been detected and the protein was occasionally observed in a npg few spermatocytes and Sertoli cells [58].…”

Members of the aquaporin family in the developing pea seed coat include representatives of the PIP, TIP, and NIP subfamilies

“…Some mammalian MIPs transport larger molecules or ions. AQP9 from the liver allows the passage of water and a variety of neutral molecules including carbamides, polyols, purines and pyrimidines (Tsukaguchi et al, 1998), whereas AQP6, an endomembrane aquaporin in renal epithelia, is permeable to anions, particularly nitrate (Ikeda et al, 2002).…”

Cytoprotective effects of growth factors: BDNF more potent than GDNF in an organotypic culture model of Parkinson's disease

“…When expressed in Xenopus oocytes, it is shown to be permeable to water, polyols (glycerol, mannitol, sorbitol) carmabides (urea, thiourea) purines (adenine) and purimidines (uracil and the chemotherapeutic agent 5 fluorouracil), monocarboxylates (lactate, B-hydroxybutyrate) (10-12) and ammonia (13) in a mercury-and phloretin sensitive manner (12). The AQP9-mediated transportation of monocarboxylates increases significantly in acidic conditions with pH <5.5, suggesting that these agents are primarily permeating in their protonated form (11,268). Other solutes have been demonstrated not to be transported through AQP9, including ions, cyclic sugars (Dglucose, D-mannose and myoinositol), the nucleosine uridine, glutamine and glycine (11).…”

“…The AQP9-mediated transportation of monocarboxylates increases significantly in acidic conditions with pH <5.5, suggesting that these agents are primarily permeating in their protonated form (11,268). Other solutes have been demonstrated not to be transported through AQP9, including ions, cyclic sugars (Dglucose, D-mannose and myoinositol), the nucleosine uridine, glutamine and glycine (11). The particularly high permeability through AQP9 can be attributed to a an oval shaped pore with dimensions 7x12 Å compared to a round pore in GlpF with a diameter of 7 Å.…”

“…Although not previously related to PD, several lines of evidence argue for a link between these. AQP9 is an aquaglyceroporin with particularly broad substance permeability, transporting water, glycerol, monocarboxylates and arsenite, among others (10)(11)(12)(13)(14). This water channel has recently been found to be selectively expressed in dopaminergic plasma membranes as well as mitochondrial inner membranes in the SN (15)(16)(17), indicating that it may serve as a toxin influx route into these cells, thereby accounting for an increased susceptibility to cell death.…”