Glycerol facilitator GlpF and the associated aquaporin family of channelsIntroductionOne of the earliest recognized transmembrane channelsGlycerol conductance and the three-dimensional structure of GlpFChannel selectivityStereoselectivity among linear alditolsSimulated rates through the channelSimulation and rates of water passageInsulation from proton conductance?Against passing ionsSelectivity: glycerol versus waterConclusionsReferences and recommended readingGlycerol facilitator GlpF and the associated aquaporinfamily of channelsRobert M Stroud, Larry JW Miercke, Joseph O’Connell,Shahram Khademi, John K Lee, Jonathan Remis, William Harries,Yaneth Robles and David AkhavanThe aqua (glycero) porins conduct water (and glycerol) across cellmembranes. The structure of these channels reveals a tripathicchannel that supports a hydrophobicsurface and, opposite to this,a line of eight hydrogen-bond acceptors and four hydrogen-bonddonors. The eight carbonyls act as acceptors for water (or glycerolOH) molecules. The central water molecule in the channel isoriented to polarize hydrogen atoms outward from the center. Thisarrangement suggests how the structure prevents the potentiallylethal conduction of protons across the membrane. The structurealso suggests the mechanism behind the selectivity ofaquaglyceroporins for glycerol, the basis for enantioselectivityamong alditols, and the basis for the prevention of any leakage ofthe electrochemical gradient.AddressesDepartment of Biochemistry & Biophysics, School of Medicine, 600 16thStreet, University of California San Francisco, CA 94143-2240, USAe-mail: [email protected] Opinion in Structural Biology 2003, 13:424–431This review comes from a themed issue onMembranesEdited by Eric Gouaux and Stephen H White0959-440X/$ – see front matterß 2003 Elsevier Ltd. All rights reserved.DOI 10.1016/S0959-440X(03)00114-3AbbreviationAQP aquaporinIntroductionEvery cell is surrounded by an insulating membrane. It isessential that cells insulate themselves from the high elec-trochemical gradient of the 100,000 volt/cm electric fieldacross the membrane. Every cell also has to capture meta-bolic nutrients. How cells bring in the essential nutrientsand how the cell membrane absolutely excludes leakage ofeven protons out of the cell is a fundamental question inmembrane biology. The structures of channels in theaquaporin (AQP) family open the way to address the largerissue of how strict membrane selectivity is determined.One of the earliest recognizedtransmembrane channelsOne hundred years ago, Alfred Fischer deduced thatglycerol channels existed. When certain species of patho-genic bacteria were placed in hyperosmotic solutions,about half of them failed to undergo lysis in glycerolsolutions [1]. Fischer correctly concluded that this sub-group of pathogens had to have membranes that werehighly permeable to glycerol to relieve the hyperosmoticstress.Beginning in the mid 1960s, a brilliant series of geneticanalyses pioneered by ECC Lin defined the nature of andthe conducting specificity of GlpF [2]. GlpF is a highlyselective transmembrane channel that conducts glycerol,water and small uncharged organic molecules. Once insidethe bacterial cell, glycerol is rapidly phosphorylated byglycerol kinase to produce glycerol 3-phosphate. GlpFconducts urea, glycine andDL-glyceraldehyde in additionto glycerol. It is also stereo and enantio selective in theconductance of linear carbohydrates (called alditols) [2].Aldoses — cyclized alditols — are not conducted throughthe GlpF channel. The structure of the GlpF channelshows that it is indeed too small to conduct cyclic mole-cules, beautifully demonstrating its stereo and enantioselectivity [3,4].GlpF is now known to be a member of the AQP channelfamily. The first member of this family to be identifiedwas the human water channel AQP1 from red blood cellsand renal tubules [5]. Peter Agre and colleagues firstidentified the cDNA of this water-conducting channel[6], and named the family ‘aquaporins’ or AQPs [7]. AQPscomprise functionally distinct subgroups that includetransmembrane water-conducting channels, which con-duct water but do not conduct linear alcohols or glycerol(aquaporins), and channels that conduct water and alsoconduct glycerol (aquaglyceroporins). These latter chan-nels also conduct urea,DL-glyceraldehyde, alditols andother small organic molecules [8–10].The AQP family arose by tandem intragenic duplication[11] to give proteins that have an internal repeat; the N-terminal segment displays 20% conservation with theC-terminal segment [12] (Figures 1 and 2). This duplica-tion event appeared early in evolution, as bacteria suchas Escherichia coli contain both a water- and glycerol-conducting channel (GlpF), and a separate water channel(AQPZ). Each segment contains a conserved -Asn-Pro-Ala- (NPA) signature sequence near its center and severalother conserved residues, including Glu14 and Glu152near the beginning of each segment.424Current Opinion in Structural Biology 2003, 13:424–431 www.current-opinion.comEleven human AQPs, numbered AQP0 to AQP10, playkey roles in human physiology. In humans, AQP3, AQP7,AQP9 and AQP10 are aquaglyceroporins [13,14]. Theatomic structures of GlpF and AQP1 offer clues to themechanisms of AQP selectivity [3,15,16].Both intuition and molecular mechanics can clearly beflawed, making it crucial to understand the limitations ofeach. But, in principle, molecular mechanics can be usedto identify and subsequently evaluate the contributions ofeach factor to selectivity by ‘turning them off’ in asimulation [17]. The factors refer to such aspects aschannel amino acid identity and charge nature, aminoacid proximity to the channel, the hydrophilic/hydro-phobic nature of the channel residues, and the spatialorientation of specific residues that provide hydrogenacceptors/donors for the water and glycerol moleculesthat traverse the channel. For these membrane channels,such simulations have been particularly instructive andserve to direct the next round of experiment.AQPs are all based on arrangements of four channels astetramers that seem to stick together well, even indetergent-based purifications, as they do in their hostmembrane [18] (Figure 3). Within each monomer, aright-hand twisted arrangement of six a helices andtwo half-spanning a helices (M1–M8) surrounds thechannel [14].Thiswasfirst visualized by electroncrystallography