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Elucidating the Link Between Structure and Function of Ion Channels and Transporters with Voltage-Clamp and Patch-Clamp Fluorometry

  • Giovanni Zifarelli
  • Jana KuschEmail author
Protocol
Part of the Neuromethods book series (NM, volume 113)

Abstract

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. This implies that to gain a deep understanding of their dynamic behavior, structural and functional information need to be integrated. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but their correlation in time remains a challenging task. In this chapter, we illustrate how two related techniques, voltage-clamp fluorometry (VCF) and patch-clamp fluorometry (PCF), provide such a type of integrated information. They combine electrophysiological techniques, two-electrode voltage-clamp (VCF) and patch-clamp (PCF), with spectroscopic approaches to simultaneously detect conformational changes and ionic currents mediated by ion channels and transporters in a native membrane environment. The optical part is based on the environmental sensitivity of the fluorescence emission of probes attached at specific sites of ion channels and transporters. This allows the correlation between structural conformation and defined functional states. VCF and PCF have been applied to a variety of ion channel and transporter families to investigate several biophysical problems ranging from structural changes linked to activation by various stimuli to the analysis of the process of inactivation and deactivation. Additionally, these techniques allowed for reading out gating-dependent ligand binding and protein mobility. In this chapter, illustrating some typical examples of the application of VCF and PCF, we try to show their potential and flexibility and to highlight some of the technical caveats.

Key words

Ion channels Transporters Voltage-clamp fluorometry Patch-clamp fluorometry VCF PCF Gating Conformational changes Ligand binding Transport 

References

  1. 1.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer Academics/Plenum, New York, NYCrossRefGoogle Scholar
  2. 2.
    Mannuzzu LM, Moronne MM, Isacoff EY (1996) Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271:213–216PubMedCrossRefGoogle Scholar
  3. 3.
    Cha A, Bezanilla F (1997) Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. Neuron 19:1127–1140PubMedCrossRefGoogle Scholar
  4. 4.
    Kalstrup T, Blunck R (2013) Dynamics of internal pore opening in K(V) channels probed by a fluorescent unnatural amino acid. Proc Natl Acad Sci U S A 110:8272–8277PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Bhargava Y, Nicke A, Rettinger J (2013) Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization. Biochem Biophys Res Commun 438:295–300PubMedCrossRefGoogle Scholar
  6. 6.
    Claydon TW, Fedida D (2007) Voltage clamp fluorimetry studies of mammalian voltage-gated K(+) channel gating. Biochem Soc Trans 35:1080–1082PubMedCrossRefGoogle Scholar
  7. 7.
    Dempski RE, Friedrich T, Bamberg E (2009) Voltage clamp fluorometry: combining fluorescence and electrophysiological methods to examine the structure-function of the Na(+)/K(+)-ATPase. Biochim Biophys Acta 1787:714–720PubMedCrossRefGoogle Scholar
  8. 8.
    Pless SA, Lynch JW (2008) Illuminating the structure and function of Cys-loop receptors. Clin Exp Pharmacol Physiol 35:1137–1142PubMedCrossRefGoogle Scholar
  9. 9.
    Gandhi CS, Isacoff EY (2005) Shedding light on membrane proteins. Trends Neurosci 28:472–479PubMedCrossRefGoogle Scholar
  10. 10.
    Gandhi CS, Olcese R (2008) The voltage-clamp fluorometry technique. Methods Mol Biol 491:213–231PubMedCrossRefGoogle Scholar
  11. 11.
    Jiang Y, Lee A, Chen J et al (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423:33–41PubMedCrossRefGoogle Scholar
  12. 12.
    Doyle DA, Morais CJ, Pfuetzner RA et al (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77PubMedCrossRefGoogle Scholar
  13. 13.
    Noda M, Shimizu S, Tanabe T et al (1984) Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312:121–127PubMedCrossRefGoogle Scholar
  14. 14.
    Armstrong CM, Bezanilla F (1974) Charge movement associated with the opening and closing of the activation gates of the Na channels. J Gen Physiol 63:533–552PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Bezanilla F (2008) How membrane proteins sense voltage. Nat Rev Mol Cell Biol 9:323–332PubMedCrossRefGoogle Scholar
  16. 16.
    Yang N, Horn R (1995) Evidence for voltage-dependent S4 movement in sodium channels. Neuron 15:213–218PubMedCrossRefGoogle Scholar
  17. 17.
    Perozo E, MacKinnon R, Bezanilla F et al (1993) Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. Neuron 11:353–358PubMedCrossRefGoogle Scholar
  18. 18.
    Stefani E, Bezanilla F (1998) Cut-open oocyte voltage-clamp technique. Methods Enzymol 293:300–318PubMedCrossRefGoogle Scholar
  19. 19.
    Bezanilla F (2000) The voltage sensor in voltage-dependent ion channels. Physiol Rev 80:555–592PubMedGoogle Scholar
  20. 20.
    Seoh SA, Sigg D, Papazian DM et al (1996) Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16:1159–1167PubMedCrossRefGoogle Scholar
  21. 21.
    Bezanilla F, Perozo E, Stefani E (1994) Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. Biophys J 66:1011–1021PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Cha A, Bezanilla F (1998) Structural implications of fluorescence quenching in the Shaker K+ channel. J Gen Physiol 112:391–408PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Posson DJ, Ge P, Miller C et al (2005) Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer. Nature 436:848–851PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Glauner KS, Mannuzzu LM, Gandhi CS et al (1999) Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813–817PubMedCrossRefGoogle Scholar
  25. 25.
    Cha A, Ruben PC, George AL Jr et al (1999) Voltage sensors in domains III and IV, but not I and II, are immobilized by Na + channel fast inactivation. Neuron 22:73–87PubMedCrossRefGoogle Scholar
  26. 26.
    Cha A, Snyder GE, Selvin PR et al (1999) Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402:809–813PubMedCrossRefGoogle Scholar
  27. 27.
    Tombola F, Pathak MM, Isacoff EY (2006) How does voltage open an ion channel? Annu Rev Cell Dev Biol 22:23–52PubMedCrossRefGoogle Scholar
  28. 28.
    Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309:897–903PubMedCrossRefGoogle Scholar
  29. 29.
    Long SB, Campbell EB, Mackinnon R (2005) Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309:903–908PubMedCrossRefGoogle Scholar
  30. 30.
    Ruta V, Chen J, MacKinnon R (2005) Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. Cell 123:463–475PubMedCrossRefGoogle Scholar
  31. 31.
    Borjesson SI, Elinder F (2008) Structure, function, and modification of the voltage sensor in voltage-gated ion channels. Cell Biochem Biophys 52:149–174PubMedCrossRefGoogle Scholar
  32. 32.
    Hoshi T, Zagotta WN, Aldrich RW (1991) Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 7:547–556PubMedCrossRefGoogle Scholar
  33. 33.
    Hoshi T, Armstrong CM (2013) C-type inactivation of voltage-gated K+ channels: pore constriction or dilation? J Gen Physiol 141:151–160PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Loots E, Isacoff EY (1998) Protein rearrangements underlying slow inactivation of the Shaker K+ channel. J Gen Physiol 112:377–389PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Loots E, Isacoff EY (2000) Molecular coupling of S4 to a K(+) channel’s slow inactivation gate. J Gen Physiol 116:623–636PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Olcese R, Latorre R, Toro L et al (1997) Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels. J Gen Physiol 110:579–589PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Claydon TW, Vaid M, Rezazadeh S et al (2007) A direct demonstration of closed-state inactivation of K+ channels at low pH. J Gen Physiol 129:437–455PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Smith PL, Yellen G (2002) Fast and slow voltage sensor movements in HERG potassium channels. J Gen Physiol 119:275–293PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Tan PS, Perry MD, Ng CA et al (2012) Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels. J Gen Physiol 140:293–306PubMedPubMedCentralCrossRefGoogle Scholar
  40. 40.
    Wang Z, Dou Y, Goodchild SJ et al (2013) Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels. J Gen Physiol 141:431–443PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Osteen JD, Gonzalez C, Sampson KJ et al (2010) KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate. Proc Natl Acad Sci U S A 107:22710–22715PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Li Y, Gao J, Lu Z et al (2013) Intracellular ATP binding is required to activate the slowly activating K+ channel I(Ks). Proc Natl Acad Sci U S A 110:18922–18927PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Savalli N, Kondratiev A, Toro L et al (2006) Voltage-dependent conformational changes in human Ca(2+)- and voltage-activated K(+) channel, revealed by voltage-clamp fluorometry. Proc Natl Acad Sci U S A 103:12619–12624PubMedPubMedCentralCrossRefGoogle Scholar
  44. 44.
    Latorre R, Olcese R, Basso C et al (2003) Molecular coupling between voltage sensor and pore opening in the Arabidopsis inward rectifier K+ channel KAT1. J Gen Physiol 122:459–469PubMedPubMedCentralCrossRefGoogle Scholar
  45. 45.
    Bruening-Wright A, Larsson HP (2007) Slow conformational changes of the voltage sensor during the mode shift in hyperpolarization-activated cyclic-nucleotide-gated channels. J Neurosci 27:270–278PubMedCrossRefGoogle Scholar
  46. 46.
    Chanda B, Bezanilla F (2002) Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. J Gen Physiol 120:629–645PubMedPubMedCentralCrossRefGoogle Scholar
  47. 47.
    Qiu F, Rebolledo S, Gonzalez C et al (2013) Subunit interactions during cooperative opening of voltage-gated proton channels. Neuron 77:288–298PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Tombola F, Ulbrich MH, Kohout SC et al (2010) The opening of the two pores of the Hv1 voltage-gated proton channel is tuned by cooperativity. Nat Struct Mol Biol 17:44–50PubMedCrossRefGoogle Scholar
  49. 49.
    Passero CJ, Okumura S, Carattino MD (2009) Conformational changes associated with proton-dependent gating of ASIC1a. J Biol Chem 284:36473–36481PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Bonifacio G, Lelli CI, Kellenberger S (2014) Protonation controls ASIC1a activity via coordinated movements in multiple domains. J Gen Physiol 143:105–118PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Dahan DS, Dibas MI, Petersson EJ et al (2004) A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site. Proc Natl Acad Sci U S A 101:10195–10200PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Lester HA, Dibas MI, Dahan DS et al (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27:329–336PubMedCrossRefGoogle Scholar
  53. 53.
    Li P, Khatri A, Bracamontes J et al (2010) Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids. Mol Pharmacol 77:539–546PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Khatri A, Sedelnikova A, Weiss DS (2009) Structural rearrangements in loop F of the GABA receptor signal ligand binding, not channel activation. Biophys J 96:45–55PubMedCrossRefGoogle Scholar
  55. 55.
    Muroi Y, Theusch CM, Czajkowski C et al (2009) Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA. Biophys J 96:499–509PubMedPubMedCentralCrossRefGoogle Scholar
  56. 56.
    Muroi Y, Czajkowski C, Jackson MB (2006) Local and global ligand-induced changes in the structure of the GABA(A) receptor. Biochemistry 45:7013–7022PubMedCrossRefGoogle Scholar
  57. 57.
    Pless SA, Dibas MI, Lester HA et al (2007) Conformational variability of the glycine receptor M2 domain in response to activation by different agonists. J Biol Chem 282:36057–36067PubMedCrossRefGoogle Scholar
  58. 58.
    Han L, Talwar S, Lynch JW (2013) The relative orientation of the TM3 and TM4 domains varies between alpha1 and alpha3 glycine receptors. ACS Chem Neurosci 4:248–254PubMedCrossRefGoogle Scholar
  59. 59.
    Lorinczi E, Bhargava Y, Marino SF et al (2012) Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor. Proc Natl Acad Sci U S A 109:11396–11401PubMedPubMedCentralCrossRefGoogle Scholar
  60. 60.
    Shin JM, Munson K, Sachs G (2011) Gastric H+, K + -ATPase. Compr Physiol 1:2141–2153PubMedGoogle Scholar
  61. 61.
    Bublitz M, Poulsen H, Morth JP et al (2010) In and out of the cation pumps: p-type ATPase structure revisited. Curr Opin Struct Biol 20:431–439PubMedCrossRefGoogle Scholar
  62. 62.
    van der Hijden HT, Grell E, de Pont JJ et al (1990) Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H + K(+)-ATPase. J Membr Biol 114:245–256PubMedCrossRefGoogle Scholar
  63. 63.
    Geibel S, Kaplan JH, Bamberg E et al (2003) Conformational dynamics of the Na+/K + -ATPase probed by voltage clamp fluorometry. Proc Natl Acad Sci U S A 100:964–969PubMedPubMedCentralCrossRefGoogle Scholar
  64. 64.
    Geibel S, Zimmermann D, Zifarelli G et al (2003) Conformational dynamics of Na+/K + - and H+/K + -ATPase probed by voltage clamp fluorometry. Ann N Y Acad Sci 986:31–38PubMedCrossRefGoogle Scholar
  65. 65.
    Durr KL, Tavraz NN, Friedrich T (2012) Control of gastric H, K-ATPase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in Xenopus oocytes. PLoS One 7:e33645PubMedPubMedCentralCrossRefGoogle Scholar
  66. 66.
    Hotzy J, Schneider N, Kovermann P et al (2013) Mutating a conserved proline residue within the trimerization domain modifies Na + binding to excitatory amino acid transporters and associated conformational changes. J Biol Chem 288:36492–36501PubMedPubMedCentralCrossRefGoogle Scholar
  67. 67.
    Larsson HP, Tzingounis AV, Koch HP et al (2004) Fluorometric measurements of conformational changes in glutamate transporters. Proc Natl Acad Sci U S A 101:3951–3956PubMedPubMedCentralCrossRefGoogle Scholar
  68. 68.
    Egenberger B, Gorboulev V, Keller T et al (2012) A substrate binding hinge domain is critical for transport-related structural changes of organic cation transporter 1. J Biol Chem 287:31561–31573PubMedPubMedCentralCrossRefGoogle Scholar
  69. 69.
    Patti M, Forster IC (2014) Correlating charge movements with local conformational changes of a Na(+)-coupled cotransporter. Biophys J 106:1618–1629PubMedPubMedCentralCrossRefGoogle Scholar
  70. 70.
    Virkki LV, Murer H, Forster IC (2006) Voltage clamp fluorometric measurements on a type II Na + -coupled Pi cotransporter: shedding light on substrate binding order. J Gen Physiol 127:539–555PubMedPubMedCentralCrossRefGoogle Scholar
  71. 71.
    Gagnon DG, Frindel C, Lapointe JY (2007) Voltage-clamp fluorometry in the local environment of the C255-C511 disulfide bridge of the Na+/glucose cotransporter. Biophys J 92:2403–2411PubMedPubMedCentralCrossRefGoogle Scholar
  72. 72.
    Meinild AK, Hirayama BA, Wright EM et al (2002) Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter. Biochemistry 41:1250–1258PubMedCrossRefGoogle Scholar
  73. 73.
    Meinild AK, Loo DD, Skovstrup S et al (2009) Elucidating conformational changes in the gamma-aminobutyric acid transporter-1. J Biol Chem 284:16226–16235PubMedPubMedCentralCrossRefGoogle Scholar
  74. 74.
    Sakata S, Okamura Y (2014) Phosphatase activity of the voltage-sensing phosphatase, VSP, shows graded dependence on the extent of activation of the voltage sensor. J Physiol 592:899–914PubMedCrossRefGoogle Scholar
  75. 75.
    Kusch J, Zifarelli G (2014) Patch-clamp fluorometry: electrophysiology meets fluorescence. Biophys J 106:1250–1257PubMedPubMedCentralCrossRefGoogle Scholar
  76. 76.
    Zheng J, Zagotta WN (2000) Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron 28:369–374PubMedCrossRefGoogle Scholar
  77. 77.
    Kenworthy AK (2001) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289–296PubMedCrossRefGoogle Scholar
  78. 78.
    Giraldez T, Hughes TE, Sigworth FJ (2005) Generation of functional fluorescent BK channels by random insertion of GFP variants. J Gen Physiol 126:429–438PubMedPubMedCentralCrossRefGoogle Scholar
  79. 79.
    Kobrinsky E, Schwartz E, Abernethy DR et al (2003) Voltage-gated mobility of the Ca2+ channel cytoplasmic tails and its regulatory role. J Biol Chem 278:5021–5028PubMedCrossRefGoogle Scholar
  80. 80.
    Kobrinsky E, Stevens L, Kazmi Y et al (2006) Molecular rearrangements of the Kv2.1 potassium channel termini associated with voltage gating. J Biol Chem 281:19233–19240PubMedCrossRefGoogle Scholar
  81. 81.
    Kobrinsky E, Kepplinger KJF, Yu A et al (2004) Voltage-gated rearrangements associated with differential beta-subunit modulation of the L-type Ca(2+) channel inactivation. Biophys J 87:844–857PubMedPubMedCentralCrossRefGoogle Scholar
  82. 82.
    Kobrinsky E, Tiwari S, Maltsev VA et al (2005) Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions. J Biol Chem 280:12474–12485PubMedCrossRefGoogle Scholar
  83. 83.
    Fisher JA, Girdler G, Khakh BS (2004) Time-resolved measurement of state-specific P2X2 ion channel cytosolic gating motions. J Neurosci 24:10475–10487PubMedCrossRefGoogle Scholar
  84. 84.
    Ambrose EJ (1956) A surface contact microscope for the study of cell movements. Nature 178:1194PubMedCrossRefGoogle Scholar
  85. 85.
    Axelrod D (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89:141–145PubMedCrossRefGoogle Scholar
  86. 86.
    van der Wal J, Habets R, Várnai P et al (2001) Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J Biol Chem 276:15337–15344PubMedCrossRefGoogle Scholar
  87. 87.
    Yang F, Cui Y, Wang K et al (2010) Thermosensitive TRP channel pore turret is part of the temperature activation pathway. Proc Natl Acad Sci U S A 107:7083–7088PubMedPubMedCentralCrossRefGoogle Scholar
  88. 88.
    Triller A, Choquet D (2008) New concepts in synaptic biology derived from single-molecule imaging. Neuron 59:359–374PubMedCrossRefGoogle Scholar
  89. 89.
    Walling MA, Novak JA, Shepard JR (2009) Quantum dots for live cell and in vivo imaging. Int J Mol Sci 10:441–491PubMedPubMedCentralCrossRefGoogle Scholar
  90. 90.
    Michalet X, Pinaud FF, Bentolila LA et al (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544PubMedPubMedCentralCrossRefGoogle Scholar
  91. 91.
    Richler E, Shigetomi E, Khakh BS (2011) Neuronal P2X2 receptors are mobile ATP sensors that explore the plasma membrane when activated. J Neurosci 31:16716–16730PubMedPubMedCentralCrossRefGoogle Scholar
  92. 92.
    Zheng J, Zagotta WN (2003) Patch-clamp fluorometry recording of conformational rearrangements of ion channels. Sci STKE 2003:PL7PubMedGoogle Scholar
  93. 93.
    Zheng J (2006) Patch fluorometry: shedding new light on ion channels. Physiology (Bethesda) 21:6–12CrossRefGoogle Scholar
  94. 94.
    Biskup C, Kusch J, Schulz E et al (2007) Relating ligand binding to activation gating in CNGA2 channels. Nature 446:440–443PubMedCrossRefGoogle Scholar
  95. 95.
    Craven KB, Zagotta WN (2006) CNG and HCN channels: two peas, one pod. Annu Rev Physiol 68:375–401PubMedCrossRefGoogle Scholar
  96. 96.
    Lehrer SS (1971) Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10:3254–3263PubMedCrossRefGoogle Scholar
  97. 97.
    Chanda B, Asamoah OK, Blunck R et al (2005) Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement. Nature 436:852–856PubMedCrossRefGoogle Scholar
  98. 98.
    Taraska JW, Zagotta WN (2007) Structural dynamics in the gating ring of cyclic nucleotide-gated ion channels. Nat Struct Mol Biol 14:854–860PubMedCrossRefGoogle Scholar
  99. 99.
    Miranda P, Contreras JE, Plested AJ et al (2013) State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels. Proc Natl Acad Sci U S A 110:5217–5222PubMedPubMedCentralCrossRefGoogle Scholar
  100. 100.
    Zheng J, Varnum MD, Zagotta WN (2003) Disruption of an intersubunit interaction underlies Ca2 + -calmodulin modulation of cyclic nucleotide-gated channels. J Neurosci 23:8167–8175PubMedGoogle Scholar
  101. 101.
    Trudeau MC, Zagotta WN (2004) Dynamics of Ca2 + -calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry. J Gen Physiol 124:211–223PubMedPubMedCentralCrossRefGoogle Scholar
  102. 102.
    Nache V, Eick T, Schulz E et al (2013) Hysteresis of ligand binding in CNGA2 ion channels. Nat Commun 4:2864PubMedCentralCrossRefGoogle Scholar
  103. 103.
    Nache V, Zimmer T, Wongsamitkul N et al (2012) Differential regulation by cyclic nucleotides of the CNGA4 and CNGB1b subunits in olfactory cyclic nucleotide-gated channels. Sci Signal 5:ra48PubMedCrossRefGoogle Scholar
  104. 104.
    Kusch J, Biskup C, Thon S et al (2010) Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels. Neuron 67:75–85PubMedCrossRefGoogle Scholar
  105. 105.
    Kusch J, Thon S, Schulz E et al (2012) How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels. Nat Chem Biol 8:162–169CrossRefGoogle Scholar
  106. 106.
    Colquhoun D (1998) Binding, gating, affinity and efficacy: the interpretation of structure-activity relationships for agonists and of the effects of mutating receptors. Br J Pharmacol 125:924–947PubMedCrossRefGoogle Scholar
  107. 107.
    Marni F, Wu S, Shah GM et al (2012) Normal-mode-analysis-guided investigation of crucial intersubunit contacts in the cAMP-dependent gating in HCN channels. Biophys J 103:19–28PubMedPubMedCentralCrossRefGoogle Scholar
  108. 108.
    Wu S, Gao W, Xie C et al (2012) Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel. J Gen Physiol 140:29–39PubMedPubMedCentralCrossRefGoogle Scholar
  109. 109.
    Wu S, Vysotskaya ZV, Xu X et al (2011) State-dependent cAMP binding to functioning HCN channels studied by patch-clamp fluorometry. Biophys J 100:1226–1232PubMedPubMedCentralCrossRefGoogle Scholar
  110. 110.
    Xu X, Marni F, Wu S et al (2012) Local and global interpretations of a disease-causing mutation near the ligand entry path in hyperpolarization-activated cAMP-gated channel. Structure 20:2116–2123PubMedCrossRefGoogle Scholar
  111. 111.
    Grandl J, Sakr E, Kotzyba-Hibert F et al (2007) Fluorescent epibatidine agonists for neuronal and muscle-type nicotinic acetylcholine receptors. Angew Chem Int Ed Engl 46:3505–3508PubMedCrossRefGoogle Scholar
  112. 112.
    Schmauder R, Kosanic D, Hovius R et al (2011) Correlated optical and electrical single-molecule measurements reveal conformational diffusion from ligand binding to channel gating in the nicotinic acetylcholine receptor. Chembiochem 12:2431–2434PubMedCrossRefGoogle Scholar
  113. 113.
    Miller C (1986) Ion channel reconstitution. Plenum Press, New York, NYCrossRefGoogle Scholar
  114. 114.
    Ide T, Takeuchi Y, Aoki T et al (2002) Simultaneous optical and electrical recording of a single ion-channel. Jpn J Physiol 52:429–434PubMedCrossRefGoogle Scholar
  115. 115.
    Ide T, Yanagida T (1999) An artificial lipid bilayer formed on an agarose-coated glass for simultaneous electrical and optical measurement of single ion channels. Biochem Biophys Res Commun 265:595–599PubMedCrossRefGoogle Scholar
  116. 116.
    Borisenko V, Lougheed T, Hesse J et al (2003) Simultaneous optical and electrical recording of single gramicidin channels. Biophys J 84:612–622PubMedPubMedCentralCrossRefGoogle Scholar
  117. 117.
    Montal M, Mueller P (1972) Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A 69:3561–3566PubMedPubMedCentralCrossRefGoogle Scholar
  118. 118.
    Harms GS, Orr G, Montal M et al (2003) Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy. Biophys J 85:1826–1838PubMedPubMedCentralCrossRefGoogle Scholar
  119. 119.
    Valeur B (2001) Molecular fluorescence: principles and applications. Wiley-VCH Verlag GmbH, BerlinCrossRefGoogle Scholar
  120. 120.
    Ormo M, Cubitt AB, Kallio K et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395PubMedCrossRefGoogle Scholar
  121. 121.
    Islas LD, Zagotta WN (2006) Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence. J Gen Physiol 128:337–346PubMedPubMedCentralCrossRefGoogle Scholar
  122. 122.
    Mansoor SE, McHaourab HS, Farrens DL (2002) Mapping proximity within proteins using fluorescence spectroscopy. A study of T4 lysozyme showing that tryptophan residues quench bimane fluorescence. Biochemistry. 41:2475–2484Google Scholar
  123. 123.
    Taraska JW, Puljung MC, Olivier NB et al (2009) Mapping the structure and conformational movements of proteins with transition metal ion FRET. Nat Methods 6:532–537PubMedPubMedCentralCrossRefGoogle Scholar
  124. 124.
    Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006:re2PubMedGoogle Scholar
  125. 125.
    Jarecki BW, Zheng S, Zhang L et al (2013) Tethered spectroscopic probes estimate dynamic distances with subnanometer resolution in voltage-dependent potassium channels. Biophys J 105:2724–2732PubMedPubMedCentralCrossRefGoogle Scholar
  126. 126.
    Blaustein RO, Cole PA, Williams C et al (2000) Tethered blockers as molecular ‘tape measures’ for a voltage-gated K+ channel. Nat Struct Biol 7:309–311PubMedCrossRefGoogle Scholar
  127. 127.
    Jensen MO, Jogini V, Borhani DW et al (2012) Mechanism of voltage gating in potassium channels. Science 336:229–233PubMedCrossRefGoogle Scholar
  128. 128.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909PubMedCrossRefGoogle Scholar
  129. 129.
    Taraska JW, Zagotta WN (2010) Fluorescence applications in molecular neurobiology. Neuron 66:170–189PubMedPubMedCentralCrossRefGoogle Scholar
  130. 130.
    Magidson V, Khodjakov A (2013) Circumventing photodamage in live-cell microscopy. Methods Cell Biol 114:545–560PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Physiology, Anatomy and GeneticsUniversity of OxfordOxfordUK
  2. 2.Institut für Physiologie IIUniversitätsklinikum JenaJenaGermany

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