Ion efflux and influenza infection trigger NLRP3 inflammasome signaling in human dendritic cells

Journal of Leukocyte Biology

Volumn 99, Issue 5, 2016, Pages 723-734

  Fernandez, Melissa Victoria ^a   Miller, Elizabeth ^b   Krammer, Florian ^b   Gopal, Ramya ^b   Greenbaum, Benjamin D ^b   Bhardwaj, Nina ^b  

^a NEW YORK UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b MOUNT SINAI SCHOOL OF MEDICINE   (United States)

Author keywords

Caspase 1; Cell death; IL 1 ; Interferon; Pyroptosis

Indexed keywords

CRYOPYRIN; INFLAMMASOME; INTERLEUKIN 18; INTERLEUKIN 1ALPHA; INTERLEUKIN 1BETA; NUCLEOTIDE BINDING OLIGOMERIZATION DOMAIN PROTEIN; AMANTADINE; IMIDAZOLE DERIVATIVE; INTERFERON; INTERLEUKIN 1; INTERLEUKIN 1BETA CONVERTING ENZYME; ION; NLRP3 PROTEIN, HUMAN; RESIQUIMOD; RIMANTADINE; TOLL LIKE RECEPTOR;

ARTICLE; CELL ACTIVATION; CELL DEATH; CELL ISOLATION; CONTROLLED STUDY; CYTOKINE PRODUCTION; DENDRITIC CELL; HUMAN; HUMAN CELL; INFLUENZA; INNATE IMMUNITY; ION TRANSPORT; POTASSIUM TRANSPORT; PRIORITY JOURNAL; PYROPTOSIS; SIGNAL TRANSDUCTION; APOPTOSIS; DRUG EFFECTS; IMMUNOLOGY; INFLUENZA A VIRUS; METABOLISM; MONOCYTE; PATHOLOGY; PHYSIOLOGY; UPREGULATION; VIROLOGY;

AMANTADINE; APOPTOSIS; CASPASE 1; DENDRITIC CELLS; HUMANS; IMIDAZOLES; INFLAMMASOMES; INFLUENZA A VIRUS; INTERFERONS; INTERLEUKIN-1; ION TRANSPORT; IONS; MONOCYTES; NLR FAMILY, PYRIN DOMAIN-CONTAINING 3 PROTEIN; RIMANTADINE; SIGNAL TRANSDUCTION; TOLL-LIKE RECEPTORS; UP-REGULATION;

EID: 84964734963     PISSN: 07415400     EISSN: 19383673     Source Type: Journal    
DOI: 10.1189/jlb.3A0614-313RRR     Document Type: Article

References (82)

1. Bhardwaj, N., Lau, L. L., Friedman, S. M., Crow, M. K., Steinman, R. M. (1989) Interleukin 1 production during accessory cell-dependent mitogenesis of T lymphocytes. J. Exp. Med. 169, 1121-1136.
2. Dinarello, C. A. (1996) Cytokines as mediators in the pathogenesis of septic shock. Curr. Top. Microbiol. Immunol. 216, 133-165.
3. Piper, S. C., Ferguson, J., Kay, L., Parker, L. C., Sabroe, I., Sleeman, M. A., Briend, E., Finch, D. K. (2013) The role of interleukin-1 and interleukin- 18 in pro-inflammatory and anti-viral responses to rhinovirus in primary bronchial epithelial cells. PLoS One 8, e63365.
4. Dinarello, C. A. (1999) IL-18: a TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J. Allergy Clin. Immunol. 103, 11-24.
5. Gross, O., Thomas, C. J., Guarda, G., Tschopp, J. (2011) The inflammasome: an integrated view. Immunol. Rev. 243, 136-151.
6. Martinon, F., Tschopp, J. (2004) Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell 117, 561-574.
7. Martinon, F., Tschopp, J. (2005) NLRs join TLRs as innate sensors of pathogens. Trends Immunol. 26, 447-454.
8. Fernandez, M. V., Miller, E. A., Bhardwaj, N. (2014) Activation and measurement of NLRP3 inflammasome activity using IL-1b in human monocyte-derived dendritic cells. J. Vis. Exp. 87, e51284.
9. Puren, A. J., Fantuzzi, G., Dinarello, C. A. (1999) Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc. Natl. Acad. Sci. USA 96, 2256-2261.
10. Pirhonen, J., Sareneva, T., Kurimoto, M., Julkunen, I., Matikainen, S. (1999) Virus infection activates IL-1 beta and IL-18 production in human macrophages by a caspase-1-dependent pathway. J. Immunol. 162, 7322-7329.
11. Martinon, F., Agostini, L., Meylan, E., Tschopp, J. (2004) Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr. Biol. 14, 1929-1934.
12. Kanneganti, T. D., Ozören, N., Body-Malapel, M., Amer, A., Park, J. H., Franchi, L., Whitfield, J., Barchet, W., Colonna, M., Vandenabeele, P., Bertin, J., Coyle, A., Grant, E. P., Akira, S., Núñez, G. (2006) Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature 440, 233-236.
13. Mariathasan, S., Weiss, D. S., Newton, K., McBride, J., O’Rourke, K., Roose-Girma, M., Lee, W. P., Weinrauch, Y., Monack, D. M., Dixit, V. M. (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228-232.
14. Eisenbarth, S. C., Colegio, O. R., O’Connor, W., Sutterwala, F. S., Flavell, R. A. (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453, 1122-1126.
15. Dostert, C., Pétrilli, V., Van Bruggen, R., Steele, C., Mossman, B. T., Tschopp, J. (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320, 674-677.
16. Pétrilli, V., Papin, S., Dostert, C., Mayor, A., Martinon, F., Tschopp, J. (2007) Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14, 1583-1589.
17. McWilliam, A. S., Nelson, D., Thomas, J. A., Holt, P. G. (1994) Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces. J. Exp. Med. 179, 1331-1336.
18. Xia, W., Pinto, C. E., Kradin, R. L. (1995) The antigen-presenting activities of Ia+ dendritic cells shift dynamically from lung to lymph node after an airway challenge with soluble antigen. J. Exp. Med. 181, 1275-1283.
19. Holt, P. G., Schon-Hegrad, M. A., Oliver, J., Holt, B. J., McMenamin, P. G. (1990) A contiguous network of dendritic antigen-presenting cells within the respiratory epithelium. Int. Arch. Allergy Appl. Immunol. 91, 155-159.
20. Holt, P. G., Haining, S., Nelson, D. J., Sedgwick, J. D. (1994) Origin and steady-state turnover of class II MHC-bearing dendritic cells in the epithelium of the conducting airways. J. Immunol. 153, 256-261.
21. Bender, A., Albert, M., Reddy, A., Feldman, M., Sauter, B., Kaplan, G., Hellman, W., Bhardwaj, N. (1998) The distinctive features of influenza virus infection of dendritic cells. Immunobiology 198, 552-567.
22. Lund, J. M., Alexopoulou, L., Sato, A., Karow, M., Adams, N. C., Gale, N. W., Iwasaki, A., Flavell, R. A. (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101, 5598-5603.
23. Ichinohe, T., Pang, I. K., Iwasaki, A. (2010) Influenza virus activates inflammasomes via its intracellular M2 ion channel. Nat. Immunol. 11, 404-410.
24. Diebold, S. S., Kaisho, T., Hemmi, H., Akira, S., Reis e Sousa, C. (2004) Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529-1531.
25. Schmitz, N., Kurrer, M., Bachmann, M. F., Kopf, M. (2005) Interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection. J. Virol. 79, 6441-6448.
26. Ichinohe, T., Lee, H. K., Ogura, Y., Flavell, R., Iwasaki, A. (2009) Inflammasome recognition of influenza virus is essential for adaptive immune responses. J. Exp. Med. 206, 79-87.
27. Cill óniz, C., Shinya, K., Peng, X., Korth, M. J., Proll, S. C., Aicher, L. D., Carter, V. S., Chang, J. H., Kobasa, D., Feldmann, F., Strong, J. E., Feldmann, H., Kawaoka, Y., Katze, M. G. (2009) Lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes. PLoS Pathog. 5, e1000604.
28. La Gruta, N. L., Kedzierska, K., Stambas, J., Doherty, P. C. (2007) A question of self-preservation: immunopathology in influenza virus infection. Immunol. Cell Biol. 85, 85-92.
29. O’Neill, D. W., Bhardwaj, N. (2005) Differentiation of peripheral blood monocytes into dendritic cells. Curr. Protoc. Immunol. Chapter 22, Unit 22F 4.
30. Sabado, R. L., Miller, E., Spadaccia, M., Vengco, I., Hasan, F., Bhardwaj, N. (2013) Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy. J. Vis. Exp. 78, e50085.
31. Bhardwaj, N., Bender, A., Gonzalez, N., Bui, L. K., Garrett, M. C., Steinman, R. M. (1994) Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells. J. Clin. Invest. 94, 797-807.
32. Bender, A., Bui, L. K., Feldman, M. A., Larsson, M., Bhardwaj, N. (1995) Inactivated influenza virus, when presented on dendritic cells, elicits human CD8+ cytolytic T cell responses. J. Exp. Med. 182, 1663-1671.
33. Duramad, O., Fearon, K. L., Chan, J. H., Kanzler, H., Marshall, J. D., Coffman, R. L., Barrat, F. J. (2003) IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences. Blood 102, 4487-4492.
34. Barrat, F. J., Meeker, T., Gregorio, J., Chan, J. H., Uematsu, S., Akira, S., Chang, B., Duramad, O., Coffman, R. L. (2005) Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J. Exp. Med. 202, 1131-1139.
35. Ade, N., Antonios, D., Kerdine-Romer, S., Boisleve, F., Rousset, F., Pallardy, M. (2007) NF-kappaB plays a major role in the maturation of human dendritic cells induced by NiSO(4) but not by DNCB. Toxicol. Sci. 99, 488-501.
36. Ardeshna, K. M., Pizzey, A. R., Devereux, S., Khwaja, A. (2000) The PI3 kinase, p38 SAP kinase, and NF-kappaB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells. Blood 96, 1039-1046.
37. Renn, C. N., Sanchez, D. J., Ochoa, M. T., Legaspi, A. J., Oh, C. K., Liu, P. T., Krutzik, S. R., Sieling, P. A., Cheng, G., Modlin, R. L. (2006) TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response. J. Immunol. 177, 298-305.
38. Gorden, K. B., Gorski, K. S., Gibson, S. J., Kedl, R. M., Kieper, W. C., Qiu, X., Tomai, M. A., Alkan, S. S., Vasilakos, J. P. (2005) Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J. Immunol. 174, 1259-1268.
39. Muñoz-Planillo, R., Kuffa, P., Martínez-Colón, G., Smith, B. L., Rajendiran, T. M., Núñez, G. (2013) K⁺ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38, 1142-1153.
40. Netea, M. G., Nold-Petry, C. A., Nold, M. F., Joosten, L. A., Opitz, B., van der Meer, J. H., van de Veerdonk, F. L., Ferwerda, G., Heinhuis, B., Devesa, I., Funk, C. J., Mason, R. J., Kullberg, B. J., Rubartelli, A., van der Meer, J. W., Dinarello, C. A. (2009) Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages. Blood 113, 2324-2335.
41. Vergani, A., Fotino, C., D’Addio, F., Tezza, S., Podetta, M., Gatti, F., Chin, M., Bassi, R., Molano, R. D., Corradi, D., Gatti, R., Ferrero, M. E., Secchi, A., Grassi, F., Ricordi, C., Sayegh, M. H., Maffi, P., Pileggi, A., Fiorina, P. (2013) Effect of the purinergic inhibitor oxidized ATP in a model of islet allograft rejection. Diabetes 62, 1665-1675.
42. Murgia, M., Hanau, S., Pizzo, P., Rippa, M., Di Virgilio, F. (1993) Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor. J. Biol. Chem. 268, 8199-8203.
43. Di Virgilio, F. (2005) Purinergic mechanism in the immune system: a signal of danger for dendritic cells. Purinergic Signal. 1, 205-209.
44. Baroni, M., Pizzirani, C., Pinotti, M., Ferrari, D., Adinolfi, E., Calzavarini, S., Caruso, P., Bernardi, F., Di Virgilio, F. (2007) Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles. FASEB J. 21, 1926-1933.
45. Hari, A., Zhang, Y., Tu, Z., Detampel, P., Stenner, M., Ganguly, A., Shi, Y. (2014) Activation of NLRP3 inflammasome by crystalline structures via cell surface contact. Sci. Rep. 4, 7281.
46. Riteau, N., Baron, L., Villeret, B., Guillou, N., Savigny, F., Ryffel, B., Rassendren, F., Le Bert, M., Gombault, A., Couillin, I. (2012) ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation. Cell Death Dis. 3, e403.
47. Schorn, C., Frey, B., Lauber, K., Janko, C., Strysio, M., Keppeler, H., Gaipl, U. S., Voll, R. E., Springer, E., Munoz, L. E., Schett, G., Herrmann, M. (2011) Sodium overload and water influx activate the NALP3 inflammasome. J. Biol. Chem. 286, 35-41.
48. Fink, S. L., Cookson, B. T. (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun. 73, 1907-1916.
49. Rayamajhi, M., Zhang, Y., Miao, E. A. (2013) Detection of pyroptosis by measuring released lactate dehydrogenase activity. Methods Mol. Biol. 1040, 85-90.
50. Lamkanfi, M. (2011) Emerging inflammasome effector mechanisms. Nat. Rev. Immunol. 11, 213-220.
51. Labbé, K., Saleh, M. (2011) Pyroptosis: a caspase-1-dependent programmed cell death and a barrier to infection. In The Inflammasomes (I. Couillin, V. Pétrilli, F. Martinon, eds.), Springer Basel AG, Basel, Switzerland, 17-36.
52. Compan, V., Baroja-Mazo, A., López-Castejón, G., Gomez, A. I., Martínez, C. M., Angosto, D., Montero, M. T., Herranz, A. S., Bazán, E., Reimers, D., Mulero, V., Pelegrín, P. (2012) Cell volume regulation modulates NLRP3 inflammasome activation. Immunity 37, 487-500.
53. Williams, O. (2004) Flow cytometry-based methods for apoptosis detection in lymphoid cells. Methods Mol. Biol. 282, 31-42.
54. Lamkanfi, M., Mueller, J. L., Vitari, A. C., Misaghi, S., Fedorova, A., Deshayes, K., Lee, W. P., Hoffman, H. M., Dixit, V. M. (2009) Glyburide inhibits the cryopyrin/Nalp3 inflammasome. J. Cell Biol. 187, 61-70.
55. Feldmeyer, L., Keller, M., Niklaus, G., Hohl, D., Werner, S., Beer, H. D. (2007) The inflammasome mediates UVB-induced activation and secretion of interleukin-1beta by keratinocytes. Curr. Biol. 17, 1140-1145.
56. Nasti, T. H., Timares, L. (2012) Inflammasome activation of IL-1 family mediators in response to cutaneous photodamage. Photochem. Photobiol. 88, 1111-1125.
57. Fernandez-Sesma, A., Marukian, S., Ebersole, B. J., Kaminski, D., Park, M. S., Yuen, T., Sealfon, S. C., García-Sastre, A., Moran, T. M. (2006) Influenza virus evades innate and adaptive immunity via the NS1 protein. J. Virol. 80, 6295-6304.
58. Hofmann, P., Sprenger, H., Kaufmann, A., Bender, A., Hasse, C., Nain, M., Gemsa, D. (1997) Susceptibility of mononuclear phagocytes to influenza A virus infection and possible role in the antiviral response. J. Leukoc. Biol. 61, 408-414.
59. Fesq, H., Bacher, M., Nain, M., Gemsa, D. (1994) Programmed cell death (apoptosis) in human monocytes infected by influenza A virus. Immunobiology 190, 175-182.
60. Epand, R. M., Epand, R. F. (2002) Thermal denaturation of influenza virus and its relationship to membrane fusion. Biochem. J. 365, 841-848.
61. Ruigrok, R. W., Martin, S. R., Wharton, S. A., Skehel, J. J., Bayley, P. M., Wiley, D. C. (1986) Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes. Virology 155, 484-497.
62. Guarda, G., Braun, M., Staehli, F., Tardivel, A., Mattmann, C., Förster, I., Farlik, M., Decker, T., Du Pasquier, R. A., Romero, P., Tschopp, J. (2011) Type I interferon inhibits interleukin-1 production and inflammasome activation. Immunity 34, 213-223.
63. Ichinohe, T., Pang, I. K., Kumamoto, Y., Peaper, D. R., Ho, J. H., Murray, T. S., Iwasaki, A. (2011) Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc. Natl. Acad. Sci. USA 108, 5354-5359.
64. Allen, I. C., Scull, M. A., Moore, C. B., Holl, E. K., McElvania-TeKippe, E., Taxman, D. J., Guthrie, E. H., Pickles, R. J., Ting, J. P. (2009) The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity 30, 556-565.
65. Thomas, P. G., Dash, P., Aldridge, Jr., J. R., Ellebedy, A. H., Reynolds, C., Funk, A. J., Martin, W. J., Lamkanfi, M., Webby, R. J., Boyd, K. L., Doherty, P. C., Kanneganti, T. D. (2009) The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of caspase-1. Immunity 30, 566-575.
66. Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. (2007) Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 179, 1913-1925.
67. Zhou, R., Tardivel, A., Thorens, B., Choi, I., Tschopp, J. (2010) Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat. Immunol. 11, 136-140.
68. McDermott, M. F., Tschopp, J. (2007) From inflammasomes to fevers, crystals and hypertension: how basic research explains inflammatory diseases. Trends Mol. Med. 13, 381-388.
69. Morales-García, G., Falfán-Valencia, R., García-Ramírez, R. A., Camarena, Á., Ramirez-Venegas, A., Castillejos-López, M., Pérez-Rodríguez, M., González-Bonilla, C., Grajales-Muñíz, C., Borja-Aburto, V., Mejía-Aranguré, J. M. (2012) Pandemic influenza A/H1N1 virus infection and TNF, LTA, IL1B, IL6, IL8, and CCL polymorphisms in Mexican population: a case-control study. BMC Infect. Dis. 12, 299.
70. Liu, Y., Li, S., Zhang, G., Nie, G., Meng, Z., Mao, D., Chen, C., Chen, X., Zhou, B., Zeng, G. (2013) Genetic variants in IL1A and IL1B contribute to the susceptibility to 2009 pandemic H1N1 influenza A virus. BMC Immunol. 14, 37.
71. Hernandez, J. C., Latz, E., Urcuqui-Inchima, S. (2014) HIV-1 induces the first signal to activate the NLRP3 inflammasome in monocyte-derived macrophages. Intervirology 57, 36-42.
72. Pontillo, A., Silva, L. T., Oshiro, T. M., Finazzo, C., Crovella, S., Duarte, A. J. (2012) HIV-1 induces NALP3-inflammasome expression and interleukin-1b secretion in dendritic cells from healthy individuals but not from HIV-positive patients. AIDS 26, 11-18.
73. Hale, B. G., Randall, R. E., Ortín, J., Jackson, D. (2008) The multifunctional NS1 protein of influenza A viruses. J. Gen. Virol. 89, 2359-2376.
74. Pontillo, A., Santillo, B. T., Duarte, A. J., Oshiro, T. M. (2013) Differential inflammasome expression and IL-1b secretion in monocyte-derived dendritic cells differentiated with IL-4 or IFN-α. AIDS Res. Ther. 10, 35.
75. Sabado, R. L., O’Brien, M., Subedi, A., Qin, L., Hu, N., Taylor, E., Dibben, O., Stacey, A., Fellay, J., Shianna, K. V., Siegal, F., Shodell, M., Shah, K., Larsson, M., Lifson, J., Nadas, A., Marmor, M., Hutt, R., Margolis, D., Garmon, D., Markowitz, M., Valentine, F., Borrow, P., Bhardwaj, N. (2010) Evidence of dysregulation of dendritic cells in primary HIV infection. Blood 116, 3839-3852.
76. Pothlichet, J., Meunier, I., Davis, B. K., Ting, J. P., Skamene, E., von Messling, V., Vidal, S. M. (2013) Type I IFN triggers RIG-I/TLR3/NLRP3- dependent inflammasome activation in influenza A virus infected cells. PLoS Pathog. 9, e1003256.
77. Wang, C., Takeuchi, K., Pinto, L. H., Lamb, R. A. (1993) Ion channel activity of influenza A virus M2 protein: characterization of the amantadine block. J. Virol. 67, 5585-5594.
78. Hu, F., Luo, W., Cady, S. D., Hong, M. (2011) Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness. Biochim. Biophys. Acta 1808, 415-423.
79. Suzuki, H., Saito, R., Masuda, H., Oshitani, H., Sato, M., Sato, I. (2003) Emergence of amantadine-resistant influenza A viruses: epidemiological study. J. Infect. Chemother. 9, 195-200.
80. Shiraishi, K., Mitamura, K., Sakai-Tagawa, Y., Goto, H., Sugaya, N., Kawaoka, Y. (2003) High frequency of resistant viruses harboring different mutations in amantadine-treated children with influenza. J. Infect. Dis. 188, 57-61.
81. Pinto, L. H., Dieckmann, G. R., Gandhi, C. S., Papworth, C. G., Braman, J., Shaughnessy, M. A., Lear, J. D., Lamb, R. A., DeGrado, W. F. (1997) A functionally defined model for the M2 proton channel of influenza A virus suggests a mechanism for its ion selectivity. Proc. Natl. Acad. Sci. USA 94, 11301-11306.
82. Zhou, H. X. (2011) A theory for the proton transport of the influenza virus M2 protein: extensive test against conductance data. Biophys. J. 100, 912-921.