US007857978B2

(12) United States Patent (10) Patent No.: US 7,857,978 B2 Jensen et a]. (45) Date of Patent: Dec. 28, 2010

(54) MEMBRANE FOR FILTERING OF WATER (58) Field of Classi?cation Search ...... 210/ 500.35, 210/500.36, 500.42, 641, 321.75, 257.2, (75) Inventors: Peter Holme Jensen, Copenhagen (DK); 210/500.2, 7, 490, 6504653, 655, 500.1, Danielle Keller, Odense (DK); Claus 210/500.29, 900; 435/17.1, 4; 436/524, 436/172, 82.05; 530/402; 425/450, 1.21, HéliX Nielsen, Taastrup (DK) 425/9, 32, 417, 489; 264/4.1, 4.3, 4.6, 41; 424/450; 977/7l3i7l4, 718; 422/101 (73) Assignee: A/S, Copehhagen N (DK) See application ?le for complete search history. ( * ) Notice: Subject to any disclaimer, the term of this (56) References Cited patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. U.S. PATENT DOCUMENTS 3,906,250 A 9/1975 Loeb (21) Appl. No.: 11/915,121 (Continued) (22) PCT Filed: May 19, 2006 FOREIGN PATENT DOCUMENTS EP 1885477 Bl 2/2010 (86) PCT No.: PCT/DK2006/000278 W0 WO 02/13955 2/2002 W0 WO 2004/099088 11/2004 § 371 (0X1)’ W0 WO 2007/033675 3/2007 (2), (4) Date: May 12, 2008 OTHER PUBLICATIONS Coury et al., Reconstintution of Water channel function of (87) PCT Pub. No.: WO2006/122566 l and 2 by expression in yeast secretory vesicles, 1998, The American Physiological Society, pp. F34 -F42 .* PCT Pub. Date: Nov. 23, 2006 (Continued) (65) Prior Publication Data Primary ExamineriTony G Soohoo Assistant ExamineriDavid C Mellon US 2009/0120874 A1 May 14, 2009 (74) Attorney, Agent, or FirmiKristina Bieker-Brady; Clark & Elbing LLP Related U.S. Application Data (57) ABSTRACT (60) Provisional application No. 60/ 683,466, ?led on May Disclosed are novel Water membranes comprising bilay 20, 2005, provisional application No. 60/718,890, ers incorporating functional aqua-porins. The lipid bilayers ?led on Sep. 20, 2005. are arranged in sandwich structures including hydrophilic or hydro-phobic support materials. Also disclosed are Water (30) Foreign Application Priority Data puri?cation devices/systems, including reverse osmosis ?l tering devices that include membranes having functional May 20, 2005 (DK) ...... PA 2005 00740 aquaporins. Methods of Water puri?cation and methods of Sep. 20, 2005 (DK) ...... PA 2005 01309 preparing the membranes are also disclosed. Further, the invention provides for a neW type of perforated, hydrophobic (51) Int. Cl. polymer ?lm and to membranes containing lipid bilayers B01D 69/02 (2006.01) having other transmembrane than aquaporins intro (52) U.S. Cl...... 210/652; 210/650; 210/651; duced. 210/653; 210/655; 210/500.1; 210/500.29; 210/641; 210/321.75; 422/101; 424/450 46 Claims, 14 Drawing Sheets

Supported lipid bilayer with incorporated Aquaporln molecules

Porous support of llpirl lrilayer, like mica, nurscovlte, mica time . polyeulfon, M0,, oellulose or other sunnort with IIWII'OIJIIIIIC surface.

Planar liniil bilayer membrane with Incorporated mumporlns. {if-l Auuaporinmulecule

n Plrospholipill molecule or other :unplu'philic lipid molecule US 7,857,978 B2 Page 2

US. PATENT DOCUMENTS Ilan et al., “The Mechanism of Proton Exclusion in 4,193,267 A 3/1980 Loeb Aquaporin Channels,” PROTEINS: Structure, Function, and 4,966,708 A 10/1990 Oklejas et a1. Bioinformatics 551223-228 (2004). 5,741,416 A 4/1998 Tempest, Jr. Jensen et al., “Electrostatic Tuning of Permeation and Selec 6,297,059 B1* 10/2001 Song et a1...... 436/501 tivity in Aquaporin Water Channels,” Biophys. .1. 8512884 7,563,370 B2 7/2009 Thorsen et a1. 2899 (2003). 7,566,402 B2 7/2009 Thorsen et a1. Leonenko et al., “Supported Planar Bilayer Formation by 7,713,544 B2* 5/2010 Chaikofet a1...... 424/450 Vesicle Fusion: The Interaction of PhospholipidVesicles With 2001/0034432 A1* 10/2001 Sodroskiet a1...... 530/350 Surfaces and the Effect of Gramicidin on Bilayer Properties 2002/0107215 A1* 8/2002 Brown et a1...... 514/44 Using Atomic Force Microscopy,” Biochim. Biophys. Acta 2003/0102263 A1 * 6/2003 Lopez et a1...... 210/639 2004/0049230 A1 3/2004 Montemagno et a1. 1509:131-147 (2000). 2007/0087328 A1* 4/2007 Sleytr et a1...... 435/4 Lin et al., “Amyloid [3 Forms Ion Channels: Implica 2007/0275480 A1 * 11/2007 Brander et a1...... 436/501 tions for AlZheimer’s Disease Pathophysiology,” FASEB .1. 2009/0007555 A1 1/2009 Jensen 15:2433-2444 (2001). 2009/0120874 A1 5/2009 Jensen et a1. Montal et al., “Formation of Biomolecular Membranes from 2010/0178592 A1 * 7/2010 Cinquin et a1...... 429/512 Lipid Monolayers and a Study of Their Electrical Properties,” Proc. Nat. Acad. Sci. USA. 69:3561-3566 (1972). OTHER PUBLICATIONS Murata et al., “Structural Determinants of Water Permeation Saparov et al., Water and Ion Permeation of Aquaporin-l in Through Aquaporin-l,” Nature 407:599-605 (2000). Planar Lipid Bilayers, Jun. 15, 2001, The Journal of Biologi Pohl et al., “The Effect of a Transmembrane Osmotic Flux on cal Chemistry, pp. 31515-31520.* the Ion Concentration Distribution in the Immediate Mem Becker et al., The World of the Cell, 2006, Pearson Benjamin brane Vicinity Measured by Microelectrodes,” Biophys. .1. Cummings, Sixth Edition, pp. 171-174,203.* 721171 1-1718 (1997). Tien et al., Planar Lipid Bilayers (BLMs) and their Applica Pohl et al., “Highly Selective Water Channel Activity Mea tions, 2003, Elsevior, Membrane Science and Technology sured by Voltage Clamp: Analysis of Planar Lipid Bilayers Series 7, pp. 381-382, 450-454, 807-819, 825-829.* Reconstituted With Puri?ed AqpZ,” Proc. Natl. Acad. Sci. Heyse et al.,“Emerging Techniques for Investigating Molecu USA. 98:9624-9629 (2001). lar Interactions at Lipid Membranes,” Biochimica et Preston et al., “Appearance of Water Channels in Xenopus Biophysica Acta. Mr. Reviews on Biomembranes, Oocytes Expressing Red Cell CHIP28 Protein,” Science 1376(3):319-338 (1998). 256:385-387 (1992). Mou et al., “Gramicidin A Aggregation in Supported Gel Reimhult et al., “Intact Vesicle Adsorption and Supported State Phosphatidylcholine Bilayers,” Biochemistry, 3513222 Biomembrane Formation from Vesicles in : In?u 3226 (1996). ence of Surface Chemistry, Vesicle SiZe, Temperature, and Reviakine I., Brisson A., “Formation of Supported Osmotic Pressure,” Langmuir 19:1681-1691 (2003). Phospholipid Bilayers from Unilamellar Vesicles Investi Ren et al., “Visualization of Water-Selective Pore by Electron gated by Atomic Force Microscopy,” Langmuir, 1611806 Crystallography in Vitreous Ice,” Proc. Natl. Acad. Sci. US. 1815 (2000). A. 98:1398-1403 (2001). Agre et al., “The Aquaporins, Blueprints for Cellular Plumb Rinia et al., “Visualization of Highly Ordered Striated ing Systems,” .1. Biol. Chem. 273:14659-14662 (1998). Domains Induced by Transmembrane Peptides in Supported Borgnia et al., “Cellular and Molecular Biology of the Phosphatidylcholine Bilayers,” Biochemistry 39:5852-5858 Aquaporin Water Channels,” Annu. Rev. Biochem. 68:425 (2000). 458 (1999). Saparov et al., “Water and Ion Permeation of Aquaporin-l in Brian et al., “Allogeneic Stimulation of Cytotoxic T Cells by Planar Lipid Bilayers,” .1. Biol. Chem. 276:31515-31520 Supported Planar Membranes,” Proc. Natl. Acad. Sci. USA. (2001). 81:6159-6163 (1984). Simonsen et al., “Structure of Spin-Coated Lipid Films and Burykin et al., “What Really Prevents Proton Transport Domain Formation in Supported Membranes Formed by through Aquaporin? Charge Self-Energy versus Proton Wire Hydration,” Langmuir 20:9720-9728 (2004). Proposals,” Biophys. .1. 85:3696-3706 (2003). Sui et al., “Structural Basis of Water-Speci?c Transport Chakrabarti et al., “Molecular Basis of Proton Blockage in Through the AQPl Water Channel,” Nature 414:872-878 Aquaporins,” Structure 12:65-74 (2004). (2001). Dainty et al., “Unstirred Layers’ in Frog Skin,” .1. Physiol. Tajkhorshid et al., “Control of the Selectivity of the 182166-78 (1966). Aquaporin Water Channel Family by Global Orientation Tun de Groot et al., “Water Permeation Across Biological Mem ing,” Science 296:525-530 (2002). branes: Mechanism and Dynamics of Aquaporin-l and Tokumasu et al., “Nanoscopic Lipid Domain Dynamics GlpF,” Science 294:2353-2357 (2001). Revealed by Atomic Force Microscopy,” Biophys. .1. de Groot et al., “The Mechanism of Proton Exclusion in the 84:2609-2618 (2003). Aquaporin-l Water Channel,” .1. Mol. Biol. 333:279-293 van Kan et al., “The Peptide Antibiotic Clavanin A Interacts (2003). Strongly and Speci?cally With Lipid Bilayers,” Biochemistry Fettiplace et al., “Water Permeability of Lipid Membranes,” 42:11366-11372 (2003). Physiological Reviews 601510-550 (1980). Webber et al., “Hydrodynamic Studies of Adsorbed Diblock Fu et al., “Structure of a Glycerol-Conducting Channel and Copolymers in Porous Membranes,” Macromolecules the Basis for Its Selectivity,” Science 290:481-486 (2000). 23:1026-1034 (1990). Heymann et al., “Aquaporins: Phylogeny, Structure, and Zeidel et al ., “Reconstitution of Functional Water Channels in Physiology of Water Channels,” News Physiol. Sci. 14: 187 Liposomes Containing Puri?ed Red Cell CHIP28 Protein,” 1 93 (1 999). Biochemistry 31 17436-7440 (1992). US 7,857,978 B2 Page 3

Zhu et al., “Theory and Simulation of Water Permeation in Written Opinion of the International Searching Authority Aquaporin-1,”Bi0phys_ J, 8650-57 (2004), from PCT/DK2006/000278, completed Jan. 8, 2007, com International Search Report from PCT/DK2006/000278, Pleted 12111293001 Completed Jan 8 2007 mailed Jan 29 2007_ Thesis of Danielle Keller, “Chapter 4: Reconstitution of International Preliminary Report on Patentability from PCT/ Cytochrome C oxidase’” pp’ 41-45’ 2005' DK2006/000278, completed Aug. 23, 2007. * Cited by examiner

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3.mm US 7,857,978 B2 1 2 MEMBRANE FOR FILTERING OF WATER Desalination devices that use membrane elements (for example: R0 or NF) alWays create tWo streams of Water as the CROSS-REFERENCE TO RELATED Water exits the element: desalinated product Water (Which has APPLICATIONS passed through the membrane), and a Waste brine (that has ?oWed across the membrane surface). This Waste brine This application is the National Stage of International stream is necessary to ?ush salts and minerals aWay from the Application No. PCT/DK2006/000278, ?led May 19, 2006, membrane to prevent them from accumulating and fouling Which claims bene?t of Denmark Application Nos. PA 2005 the membrane surface. If a buildup of salts and minerals in the 00740, ?led May 20, 2005, and PA 2005 01309, ?led Sep. 20, feed-Water to a membrane occurs continuously, dissolved 2005, and US. ProvisionalApplication Ser. Nos. 60/683,466, substances can precipitate and form a solid ?lm, fouling the ?led May 20, 2005, and 60/718,890, ?led Sep. 20, 2005. surface of the membrane. In addition, colloidal and particu late contaminants can also adhere to the membrane surface FIELD OF THE INVENTION and cause fouling. With many Water-borne contaminants, if a membrane becomes irreversibly scaled, or fouled, it cannot be cleaned and must be replaced. This characteristic of mem The present invention relates to a novel membrane com brane processes poses a signi?cant problem in reducing Waste prising functional aquaporin channels or tetramers suitable e?luent especially in point of use (POU) Water treatment for ?ltering pure Water and/or glycerol, a ?ltering device/ puri?cation system comprising such membrane, and methods systems that are typically compact and built as economically as possible. of using the same for producing ultra pure Water and for 20 Ion exchange devices are also used to soften so called “hard extracting excess Water from aqueous compositions. The Water”. The problem With ion exchange Water softening sys invention also relates to novel hydrophobic polymer ?lms. tems is that they remove the hardness components of Water (calcium and magnesium ions) by exchanging them for BACKGROUND OF THE INVENTION sodium ions in order to create What is called “soft Water”. 25 When regeneration of the ion exchange media takes place, a Various Water treatment systems and methods have tradi concentrated Water stream of sodium, chloride, calcium and tionally been developed for purifying natural and polluted magnesium ions goes into the seWer system creating an envi Water sources to obtain puri?ed Water, Which is suitable for ronmental Waste disposal problem. An example of a Water human and/or animal consumption. In addition, ultra pure puri?cation system of such type is described in US. Pat. No. Water is in high demand from the semiconductor and phar 30 5,741,416 for “Water puri?cation system having plural pairs maceutical industry. The production of ultra pure Water of ?lters and an ozone contact chamber”, disclosing a Water demands more specialized ?lters and chemical treatment of puri?cation system that is effective to oxidize organic con the Water source. A number of techniques are used, such as taminants and to destroy most of the bacteria, viruses, and membrane ?ltration, ion exchangers, sub micron particle ?l other microbes in such Water stream. Systems involving ters or nano -?lters, ultraviolet light and ozone treatment. The 35 dialysis membranes that are selective for monovalent cations produced Water is extremely pure and contains no to very loW have also been disclosed in WO 2004/099088. concentrations of salts, organic components, dissolved gases There is thus a continuing need for Water puri?cation sys such as oxygen, suspended solids, and microorganisms such tems for treatment of Water that is or may be contaminated as viruses and bacteria. HoWever, because of factors such as With chemical, biological and/or radiological contaminants the continuing miniaturization in the semiconductor industry, 40 both for normal household purposes as Well as for advanced the speci?cations for ultra pure Water become increasingly research, industrial and pharmaceutical purposes. stricter. Since contamination or threats of contamination of Water Traditionally, Water is puri?ed or treated through a variety are frequently of a highly local character, eg on a ship or a in of available Water treatment devices designed both for com remote village or a camp, there is a need for a ?xed or portable munal and for point-of-use applications, e. g. based on the 45 Water puri?cation system that can be rapidly and easily folloWing technologies: activated carbon for organic deployed at a location of actual or potential contamination. Of removal: ultraviolet light disinfection: ion exchange for hard particular relevance is a system that can effectively remove ness removal (Water softening), and membrane desalination contaminants from an actually or potentially contaminated such as reverse osmosis (R0) or nano?ltration (NF). HoW Water supply, such as sea Water, to produce treated Water that ever, nano?ltration is relatively neW in the ?eld of Water 50 is suitable for human consumption. treatment technology. An NF membrane produces soft Water Since the discovery of the aquaporin Water transport pro by retaining the hardness creating divalent ions present in teins, Which are distinguished by their ability to selectively Water. An NF membrane alloWs a high percentage of monova transport H2O molecules across biological membranes, there lent ions such as sodium and chloride to pass through, While has been a certain interest in devising an arti?cial Water it retains a high percentage of the divalent ions. It is the 55 membrane incorporating these proteins, cf. US Patent Appli monovalent ions that create osmotic pres sure that requires the cation No. 20040049230 “Biomimetic membranes” Which moderate to high pressures necessary to pump Water through aims to describe hoW Water transport proteins are embedded an RO membrane. Therefore, nano?lter membranes require in a membrane to enable Water puri?cation. The preferred much less pressure to pump Water across the membrane form described has the form of a conventional ?lter disk. To because hydraulic driving force does not have to overcome 60 fabricate such a disk, a 5 nm thick monolayer of synthetic the effect of osmotic pres sure derived from monovalent ions. triblock copolymer and protein is deposited on the surface of Generally speaking, RO membranes used for residential and a 25 mm commercial ultra?ltration disk using a Langmuir commercial Water treatment applications remove all dis Blodgett trough. The monolayer on the disk is then cross solved solids by approximately 98%. While nano?lter mem linked using UV light to the polymer to increase its durability. branes remove divalent ions (hardness components: calcium 65 The device may be assayed by ?tting it in a chamber that and magnesium) by approximately 90% and monovalent ions forces pressurized source Water across the membrane. HoW (sodium chloride) by approximately 50%. ever, there is no guidance as to hoW one should select a US 7,857,978 B2 3 4 synthetic triblock copolymer nor is there any data in support The invention further relates to a method of preparing a of the actual function of the embedded aquaporin. Water membrane comprising the steps of It has been suggested that a Water puri?cation technology a) obtaining lipid micro-vesicles containing aquaporin Water could be created by expressing the aquaporin protein into channels comprising at least 0.1% mol/mol of said micro lipid bilayer vesicles and cast these membranes on porous vesicles, supports, cf. James R. SWaITZ, home page. b) fusing said vesicles into a planar lipid bilayer on an essen The invention primarily aims at developing an industrial tially planar, permeable support having a hydrophilic sur Water ?ltration membrane and device comprising aquaporins face, Wherein the aquaporin protein covers at least 1% of incorporated into a membrane capable of purifying Water the bilayer area, With the highest purity, eg 100%. No techniques or ?lters c) optionally repeating step b) to obtain multiple fused bilay knoWn today can perform this task. ers, d) depositing a second essentially planar, permeable support SUMMARY OF THE INVENTION having a hydrophilic surface on the lipid bilayer obtained in step b) or step c) to obtain a sandWich structure, and The present invention relates in one aspect to a membrane e) optionally enclosing the obtained sandWich structure in a for ?ltering of Water, Which membrane utiliZes aquaporin permeable stabiliZing membrane. Water transport proteins that have been reconstituted in lipid The invention also relates to a method of preparing a Water vesicles, and transformed into a supported layer to form a membrane, comprising the steps of Water ?ltering membrane using a method such as the Lang a) obtaining lipid micro-vesicles containing aquaporin Water muir-Blodgett method. 20 channels comprising at least 0.1% mol/mol of said micro Advantages of the Water membranes of the invention vesicles, include e?icient desalination of sea-Water (97-98% of the b) fusing said vesicles into planar lipid bilayers assembled earth Water is seaWater) Without the need for desalination around an essentially planar, permeable support having a chemicals and the provision of transportable desalination hydrophobic surface, Wherein the aquaporin protein covers ?lters (a “coffee ?lter”-like device capable of separating 25 at least 1% of the bilayer area, and Water and salt), ef?cient Water puri?cation for the semi con c) optionally enclosing the obtained sandWich structure in a ductor industry, robust household Water/ drinking Water puri permeable stabilising membrane. ?cation, and Water puri?cation Without use of electricity, for The invention further relates to a reverse osmosis Water instance in third World countries. ?ltering device comprising, as a reverse osmosis ?ltering Thus, the invention relates in one aspect to a Water mem 30 membrane, a Water membrane (e. g. a Water membrane of the brane comprising a sandWich construction having at least tWo invention) comprising functional aquaporin Water channels. permeable support layers separated by at least one lipid The invention also relates to a Water ?ltering device for bilayer comprising functional aquaporin Water channels. In extracting and recovering Water from body ?uids, such as this Way the permeable or porous support Will alloW Water urine, milk and sWeat/perspiration, comprising a Water mem molecules to penetrate through the support to reach the at 35 brane comprising functional aquaporin Water channels. least one lipid bilayer deposited betWeen the support layers. In addition, the present invention relates to a method of The lipid bilayer(s) comprising dispersed functional aqua preparing pure Water resulting from ?ltering a natural or porin channels Will then ?ltrate only Water, or, in case the polluted Water source through the Water membrane of the aquaporin is a GLpF channel, also glycerol, to the opposite invention. Said pure Water is characterized by the absence of porous support layer resulting in a ?ltrate consisting of pure 40 pollutants, such as dissolved substances or particles. The Water. Preferably this ?ltered Water is ultra pure Water invention furthermore relates to a method of obtaining puri (UPW), Which is highly puri?ed Water, loW in ions, particles, ?ed Water by ?ltering a Water source using a reverse osmosis organic matter and colloids. The Water membrane of the membrane comprising functional aquaporin channels. invention represents a neW generation of reverse osmosis Further, a different aspect of the invention relates to a membranes utiliZing the most selective Water transport chan 45 hydrophobic polymer ?lm, Which is described in detail beloW. nels knoWn. Finally, the general design of the Water membranes of the In the present context, a “Water membrane” denotes a present invention is also believed to be applicable to mem structure Which alloWs the passage of Water, Whereas most branes for other purposes, Where other transmembrane pro other materials or substances are not alloWed passage at the teins than aquaporins have been incorporated in membranes same time. Preferred Water membranes of the invention a 50 otherWise designed as the Water membranes of the present essentially only permeable for Water (and in some cases glyc invention. Such membranes are also part of the present inven erol), Whereas solutes and other solvents are not alloWed tion, and such membranes are in all aspects except from the passage. choice of identical to the membranes In a second aspect, the present invention relates to a Water disclosed herein, and all disclosures concerning such mem membrane comprising a sandWich construction having at 55 branes apply mutatis mutatndis to membranes containing least tWo lipid monolayers, Which, When assembled into one other transmembrane proteins than aquaporins. bilayer, comprises functional aquaporin Water channels, said Transmembrane proteins different from aquaporins suit at least tWo lipid monolayers being separated by at least one able for inclusion in the membranes of the present invention permeable support layer. In this embodiment, the permeable are for instance selected from, but not limited to, any trans support layer thus separates tWo lipid monolayers Which are 60 membrane protein found in the Transporter Classi?cation capable of forming lipid bilayers When the support layer Database (TCDB). TCDB is accessible at the TCDB Website. includes perforations/punctures. Examples of transmembrane proteins included in the A further aspect of the invention relates to a Water ?ltering present invention from TCDB are: device comprising the Water membrane of the invention, Aerolysin channel-forming toxin optionally enclosed in the stabiliZing membrane, Which has 65 Agrobacterial target-host cell-membrane anion channel been mounted in a housing having an inlet for aqueous liquid a-Hemolysin channel-forming toxin to be puri?ed and an outlet for puri?ed Water. Alamethicin channel US 7,857,978 B2 6 Alginate export porin H"- or Na+-translocating bacterial MotAB ?agellar motor/ Amoebapore ExbBD outer-membrane transport Amphipathic peptide mastoparan Helicobacler outer membrane porin Amyloid b-protein peptide HP1 holin Animal inward-recti?er K+ channel In?uenza Virus matrix-2 channel Annexin Insect defensin Apoptosis regulator Intracellular ArpQ holin 111 holin AS-48 jAdh holin ATP-gated cation channel jU53 holin Autotransporter Lactacin X Bacillus sublilisj29 holin Lacticin 481 Bacterial type III-target cell pore Lactocin S Bactericidal permeability-increasing protein Lactococcin 972 Bacteriocin AS-48 cyclic polypeptide Lactococcin A Bacteriorhodopsin Lactococcin G Beticolin channel Large-conductance mechanosensitive BlyA holin lholin S Botulinum and tetanus toxin Ligand-gated ion channel of neurotransmitter receptors Brucella-Rhizobium porin 20 LrgA holin Campylobaclerjejuni major outer membrane porin LydA holin Cathilicidin Magainin cation channel Major intrinsic protein Cation-channel-forming heat-shock protein 70 Melittin Cecropin 25 Metal-ion transporter (channel) Channel-forming Bacillus anthrax protective antigen Microcin E492 Channel-forming ceramide Mitochondrial and plastid porin Channel-forming colicin Channel-forming colicin V Nisin Channel-forming d-endotoxin insecticidal crystal protein 30 Nonselective cation channel-1 Channel-forming e-toxin Nonselective cation channel-2 Channel-forming leukocidin cytotoxin Nucleoside-speci?c channel-forming outer-membrane porin Chlamydial porin OmpA-OmpF porin Chloride channel OmpG porin membrane anion-channel-former 35 Organellar chloride channel Chloroplast outer-membrane solute channel Outer-bacterial-membrane secretin Cholesterol-binding, thiol-activated Outer-membrane auxiliary protein Clostridial cytotoxin Outer-membrane factor Complement protein C9 Outer-membrane ?mbrial usher porin Complexed polyhydroxybutyrate-Ca2+ channel 40 Outer-membrane porin Corynebacterial porin Outer-membrane receptor Cph1 holin P2 holin TM C-type natriuretic peptide P21 holin S Cyanobacterial porin Pediocin Cyclodextrin porin 45 Pho spholemman Cytohemolysin Pilosulin Cytotoxic amylin Plant defensin Defensin Plant plasmodesmata Dermaseptin Plant thionine Diphtheria toxin 50 Plantaricin EF Divergicin A Plantaricin J K Earthworm lysenin toxin Plastid outer-envelope porin of 16 kDa Envelope Virus E1 channel Plastid outer-envelope porin of 21 kDa Epithelial chloride channel Plastid outer-envelope porin of 24 kDa 55 Polycystin cation channel Epithelial Na+ channel Polyglutamine ion channel FadL outer-membrane protein Pore-forming equinatoxin Fusobacterial outer-membrane porin Pore-forming hemolysin E Gap-junction-forming Pore-forming RIX toxin Gap-junction-forming 60 PRDl holin M General bacterial porin Prion peptide fragment Glucose-selective OprB porin Pseudomanas syringae HrpZ target-host cell-membrane Glutamate-gated ion channel of neurotransmitter receptors Pseudomonas OprP porin gp91Ph°x phagocyte NADPH-oxidase-associated cyt b558 Raf?nose porin H+-channel 65 Rhodobacler PorCa porin Gramicidin A channel Ryanodine-inositol-l,4,5-trisphosphate receptor Ca2+ chan H"- or Na+-translocating bacterial ?agellar motor nel