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

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Book cover Advanced Patch-Clamp Analysis for Neuroscientists

Part of the book series: Neuromethods ((NM,volume 113))

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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.

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References

  1. Lakowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer Academics/Plenum, New York, NY

    Book  Google Scholar 

  2. Mannuzzu LM, Moronne MM, Isacoff EY (1996) Direct physical measure of conformational rearrangement underlying potassium channel gating. Science 271:213–216

    Article  CAS  PubMed  Google Scholar 

  3. Cha A, Bezanilla F (1997) Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. Neuron 19:1127–1140

    Article  CAS  PubMed  Google Scholar 

  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 110:8272–8277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 438:295–300

    Article  CAS  PubMed  Google Scholar 

  6. Claydon TW, Fedida D (2007) Voltage clamp fluorimetry studies of mammalian voltage-gated K(+) channel gating. Biochem Soc Trans 35:1080–1082

    Article  CAS  PubMed  Google Scholar 

  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 1787:714–720

    Article  CAS  PubMed  Google Scholar 

  8. Pless SA, Lynch JW (2008) Illuminating the structure and function of Cys-loop receptors. Clin Exp Pharmacol Physiol 35:1137–1142

    Article  CAS  PubMed  Google Scholar 

  9. Gandhi CS, Isacoff EY (2005) Shedding light on membrane proteins. Trends Neurosci 28:472–479

    Article  CAS  PubMed  Google Scholar 

  10. Gandhi CS, Olcese R (2008) The voltage-clamp fluorometry technique. Methods Mol Biol 491:213–231

    Article  CAS  PubMed  Google Scholar 

  11. Jiang Y, Lee A, Chen J et al (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423:33–41

    Article  CAS  PubMed  Google Scholar 

  12. Doyle DA, Morais CJ, Pfuetzner RA et al (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77

    Article  CAS  PubMed  Google Scholar 

  13. Noda M, Shimizu S, Tanabe T et al (1984) Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312:121–127

    Article  CAS  PubMed  Google Scholar 

  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 63:533–552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bezanilla F (2008) How membrane proteins sense voltage. Nat Rev Mol Cell Biol 9:323–332

    Article  CAS  PubMed  Google Scholar 

  16. Yang N, Horn R (1995) Evidence for voltage-dependent S4 movement in sodium channels. Neuron 15:213–218

    Article  CAS  PubMed  Google Scholar 

  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 11:353–358

    Article  CAS  PubMed  Google Scholar 

  18. Stefani E, Bezanilla F (1998) Cut-open oocyte voltage-clamp technique. Methods Enzymol 293:300–318

    Article  CAS  PubMed  Google Scholar 

  19. Bezanilla F (2000) The voltage sensor in voltage-dependent ion channels. Physiol Rev 80:555–592

    CAS  PubMed  Google Scholar 

  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 16:1159–1167

    Article  CAS  PubMed  Google Scholar 

  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 66:1011–1021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cha A, Bezanilla F (1998) Structural implications of fluorescence quenching in the Shaker K+ channel. J Gen Physiol 112:391–408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 436:848–851

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Glauner KS, Mannuzzu LM, Gandhi CS et al (1999) Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402:813–817

    Article  CAS  PubMed  Google Scholar 

  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 22:73–87

    Article  CAS  PubMed  Google Scholar 

  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 402:809–813

    Article  CAS  PubMed  Google Scholar 

  27. Tombola F, Pathak MM, Isacoff EY (2006) How does voltage open an ion channel? Annu Rev Cell Dev Biol 22:23–52

    Article  CAS  PubMed  Google Scholar 

  28. Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309:897–903

    Article  CAS  PubMed  Google Scholar 

  29. Long SB, Campbell EB, Mackinnon R (2005) Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309:903–908

    Article  CAS  PubMed  Google Scholar 

  30. Ruta V, Chen J, MacKinnon R (2005) Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. Cell 123:463–475

    Article  CAS  PubMed  Google Scholar 

  31. Borjesson SI, Elinder F (2008) Structure, function, and modification of the voltage sensor in voltage-gated ion channels. Cell Biochem Biophys 52:149–174

    Article  PubMed  CAS  Google Scholar 

  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 7:547–556

    Article  CAS  PubMed  Google Scholar 

  33. Hoshi T, Armstrong CM (2013) C-type inactivation of voltage-gated K+ channels: pore constriction or dilation? J Gen Physiol 141:151–160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Loots E, Isacoff EY (1998) Protein rearrangements underlying slow inactivation of the Shaker K+ channel. J Gen Physiol 112:377–389

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Loots E, Isacoff EY (2000) Molecular coupling of S4 to a K(+) channel’s slow inactivation gate. J Gen Physiol 116:623–636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 110:579–589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 129:437–455

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Smith PL, Yellen G (2002) Fast and slow voltage sensor movements in HERG potassium channels. J Gen Physiol 119:275–293

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 140:293–306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 141:431–443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 107:22710–22715

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 110:18922–18927

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 103:12619–12624

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 122:459–469

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 27:270–278

    Article  CAS  PubMed  Google Scholar 

  46. Chanda B, Bezanilla F (2002) Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation. J Gen Physiol 120:629–645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Qiu F, Rebolledo S, Gonzalez C et al (2013) Subunit interactions during cooperative opening of voltage-gated proton channels. Neuron 77:288–298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 17:44–50

    Article  CAS  PubMed  Google Scholar 

  49. Passero CJ, Okumura S, Carattino MD (2009) Conformational changes associated with proton-dependent gating of ASIC1a. J Biol Chem 284:36473–36481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bonifacio G, Lelli CI, Kellenberger S (2014) Protonation controls ASIC1a activity via coordinated movements in multiple domains. J Gen Physiol 143:105–118

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 101:10195–10200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lester HA, Dibas MI, Dahan DS et al (2004) Cys-loop receptors: new twists and turns. Trends Neurosci 27:329–336

    Article  CAS  PubMed  Google Scholar 

  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 77:539–546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 96:45–55

    Article  CAS  PubMed  Google Scholar 

  55. Muroi Y, Theusch CM, Czajkowski C et al (2009) Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA. Biophys J 96:499–509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Muroi Y, Czajkowski C, Jackson MB (2006) Local and global ligand-induced changes in the structure of the GABA(A) receptor. Biochemistry 45:7013–7022

    Article  CAS  PubMed  Google Scholar 

  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 282:36057–36067

    Article  CAS  PubMed  Google Scholar 

  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 4:248–254

    Article  CAS  PubMed  Google Scholar 

  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 109:11396–11401

    Article  PubMed  PubMed Central  Google Scholar 

  60. Shin JM, Munson K, Sachs G (2011) Gastric H+, K + -ATPase. Compr Physiol 1:2141–2153

    PubMed  Google Scholar 

  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 20:431–439

    Article  CAS  PubMed  Google Scholar 

  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 114:245–256

    Article  PubMed  Google Scholar 

  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 100:964–969

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 986:31–38

    Article  CAS  PubMed  Google Scholar 

  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:e33645

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  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 288:36492–36501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 101:3951–3956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 287:31561–31573

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Patti M, Forster IC (2014) Correlating charge movements with local conformational changes of a Na(+)-coupled cotransporter. Biophys J 106:1618–1629

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 127:539–555

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 92:2403–2411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Meinild AK, Hirayama BA, Wright EM et al (2002) Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter. Biochemistry 41:1250–1258

    Article  CAS  PubMed  Google Scholar 

  73. Meinild AK, Loo DD, Skovstrup S et al (2009) Elucidating conformational changes in the gamma-aminobutyric acid transporter-1. J Biol Chem 284:16226–16235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 592:899–914

    Article  CAS  PubMed  Google Scholar 

  75. Kusch J, Zifarelli G (2014) Patch-clamp fluorometry: electrophysiology meets fluorescence. Biophys J 106:1250–1257

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Zheng J, Zagotta WN (2000) Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron 28:369–374

    Article  CAS  PubMed  Google Scholar 

  77. Kenworthy AK (2001) Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24:289–296

    Article  CAS  PubMed  Google Scholar 

  78. Giraldez T, Hughes TE, Sigworth FJ (2005) Generation of functional fluorescent BK channels by random insertion of GFP variants. J Gen Physiol 126:429–438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 278:5021–5028

    Article  CAS  PubMed  Google Scholar 

  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 281:19233–19240

    Article  CAS  PubMed  Google Scholar 

  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 87:844–857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 280:12474–12485

    Article  CAS  PubMed  Google Scholar 

  83. Fisher JA, Girdler G, Khakh BS (2004) Time-resolved measurement of state-specific P2X2 ion channel cytosolic gating motions. J Neurosci 24:10475–10487

    Article  CAS  PubMed  Google Scholar 

  84. Ambrose EJ (1956) A surface contact microscope for the study of cell movements. Nature 178:1194

    Article  CAS  PubMed  Google Scholar 

  85. Axelrod D (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89:141–145

    Article  CAS  PubMed  Google Scholar 

  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 276:15337–15344

    Article  PubMed  Google Scholar 

  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 107:7083–7088

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Triller A, Choquet D (2008) New concepts in synaptic biology derived from single-molecule imaging. Neuron 59:359–374

    Article  CAS  PubMed  Google Scholar 

  89. Walling MA, Novak JA, Shepard JR (2009) Quantum dots for live cell and in vivo imaging. Int J Mol Sci 10:441–491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Michalet X, Pinaud FF, Bentolila LA et al (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Richler E, Shigetomi E, Khakh BS (2011) Neuronal P2X2 receptors are mobile ATP sensors that explore the plasma membrane when activated. J Neurosci 31:16716–16730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Zheng J, Zagotta WN (2003) Patch-clamp fluorometry recording of conformational rearrangements of ion channels. Sci STKE 2003:PL7

    PubMed  Google Scholar 

  93. Zheng J (2006) Patch fluorometry: shedding new light on ion channels. Physiology (Bethesda) 21:6–12

    Article  CAS  Google Scholar 

  94. Biskup C, Kusch J, Schulz E et al (2007) Relating ligand binding to activation gating in CNGA2 channels. Nature 446:440–443

    Article  CAS  PubMed  Google Scholar 

  95. Craven KB, Zagotta WN (2006) CNG and HCN channels: two peas, one pod. Annu Rev Physiol 68:375–401

    Article  CAS  PubMed  Google Scholar 

  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 10:3254–3263

    Article  CAS  PubMed  Google Scholar 

  97. Chanda B, Asamoah OK, Blunck R et al (2005) Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement. Nature 436:852–856

    Article  CAS  PubMed  Google Scholar 

  98. Taraska JW, Zagotta WN (2007) Structural dynamics in the gating ring of cyclic nucleotide-gated ion channels. Nat Struct Mol Biol 14:854–860

    Article  CAS  PubMed  Google Scholar 

  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 110:5217–5222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Zheng J, Varnum MD, Zagotta WN (2003) Disruption of an intersubunit interaction underlies Ca2 + -calmodulin modulation of cyclic nucleotide-gated channels. J Neurosci 23:8167–8175

    CAS  PubMed  Google Scholar 

  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 124:211–223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Nache V, Eick T, Schulz E et al (2013) Hysteresis of ligand binding in CNGA2 ion channels. Nat Commun 4:2864

    Article  PubMed Central  CAS  Google Scholar 

  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:ra48

    Article  PubMed  CAS  Google Scholar 

  104. Kusch J, Biskup C, Thon S et al (2010) Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels. Neuron 67:75–85

    Article  CAS  PubMed  Google Scholar 

  105. Kusch J, Thon S, Schulz E et al (2012) How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels. Nat Chem Biol 8:162–169

    Article  CAS  Google Scholar 

  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 125:924–947

    Article  CAS  PubMed  Google Scholar 

  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 103:19–28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 140:29–39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 100:1226–1232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 20:2116–2123

    Article  CAS  PubMed  Google Scholar 

  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 46:3505–3508

    Article  CAS  PubMed  Google Scholar 

  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 12:2431–2434

    Article  CAS  PubMed  Google Scholar 

  113. Miller C (1986) Ion channel reconstitution. Plenum Press, New York, NY

    Book  Google Scholar 

  114. Ide T, Takeuchi Y, Aoki T et al (2002) Simultaneous optical and electrical recording of a single ion-channel. Jpn J Physiol 52:429–434

    Article  CAS  PubMed  Google Scholar 

  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 265:595–599

    Article  CAS  PubMed  Google Scholar 

  116. Borisenko V, Lougheed T, Hesse J et al (2003) Simultaneous optical and electrical recording of single gramicidin channels. Biophys J 84:612–622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 69:3561–3566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 85:1826–1838

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Valeur B (2001) Molecular fluorescence: principles and applications. Wiley-VCH Verlag GmbH, Berlin

    Book  Google Scholar 

  120. Ormo M, Cubitt AB, Kallio K et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395

    Article  CAS  PubMed  Google Scholar 

  121. Islas LD, Zagotta WN (2006) Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence. J Gen Physiol 128:337–346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. 41:2475–2484

    Google Scholar 

  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 6:532–537

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006:re2

    PubMed  Google Scholar 

  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 105:2724–2732

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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 7:309–311

    Article  CAS  PubMed  Google Scholar 

  127. Jensen MO, Jogini V, Borhani DW et al (2012) Mechanism of voltage gating in potassium channels. Science 336:229–233

    Article  CAS  PubMed  Google Scholar 

  128. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909

    Article  CAS  PubMed  Google Scholar 

  129. Taraska JW, Zagotta WN (2010) Fluorescence applications in molecular neurobiology. Neuron 66:170–189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Magidson V, Khodjakov A (2013) Circumventing photodamage in live-cell microscopy. Methods Cell Biol 114:545–560

    Article  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Physiology, Anatomy and Genetics, University of Oxford, Sherrington Rd, OX1 3PT, Oxford, UK

    Giovanni Zifarelli

  2. Institut für Physiologie II, Universitätsklinikum Jena, Kollegiengasse 9, 07743, Jena, Germany

    Jana Kusch

Authors
  1. Giovanni Zifarelli
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  2. Jana Kusch
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Correspondence to Jana Kusch .

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Editors and Affiliations

  1. The Leslie and Susan Gonda Multidisciplinary Brain Research Center, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

    Alon Korngreen

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Zifarelli, G., Kusch, J. (2016). Elucidating the Link Between Structure and Function of Ion Channels and Transporters with Voltage-Clamp and Patch-Clamp Fluorometry. In: Korngreen, A. (eds) Advanced Patch-Clamp Analysis for Neuroscientists. Neuromethods, vol 113. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3411-9_4

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  • DOI: https://doi.org/10.1007/978-1-4939-3411-9_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3409-6

  • Online ISBN: 978-1-4939-3411-9

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