Learning about Conformational changes in ion channels with Fluorescence

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1 Learning about Conformational changes in ion channels with Fluorescence Walter Sandtner,, Clark Hyde, Jerome Lacroix,, Carlos Villalba-Galea Galea,, Baron Chanda, Rikard Blunck Francisco Bezanilla Dept. Biochemistry and Molecular Biology University of Chicago

2 A voltage-gated Ion channel

3 Conformational changes detected at the macroscopic level Electrophysiological Methods OUT 1. Oocyte Cut-open Voltage clamp 2. Whole cell patch clamp 3. Bilayers voltage clamp 4. Liposomes Nernst clamp What do we record? IN Ionic Current Gating Current 40 ms 100 μa 1 μa The conduction of ions through the protein: -----the actual function---- The rearrangement of intrinsic charges in the protein: ----voltage dependent Conformational changes-----

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5 X-ray structure of Kv1.2 (pdb id 2A79) Long et al (Science, 2005) OPEN-INACTIVATED state

6 Tetramethylrhodamine maleimide

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9 The fluorescence change in M356C is due to quenching

10 Depolarization Induces a Conformational Change in the m2 Muscarinic Receptor Gating currents normalized gating charge V (mv) norm ΔF/F Fluorescence signal V (mv) Voltage dependent binding to Xenopus oocytes specific binding (fmol/oocyte) [3H] ACh (nm) resting potential depolarization

11 1.2 Na Channel normalized GNa V (mv)

12 Sodium Channel mv mv I g Q 2 Q 3 time Q 3

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15 Activation Deactivation OPEN II I III III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV I II III IV Inactivation Recovery from Inactivation

16 50 mv Comparison of fluorescence, gating and ionic traces of labeled mutants Gating charge Ionic current Fluorescence 10 mv -30 mv -70 mv 1 ms N DOMAIN I (S216C) S1 S2 S3 S4 S5 S6 P - DOMAIN II (S660C) DOMAIN III (L1115C) DOMAIN IV (S1436C) Domain I Domain II Domain III Domain IV S1 S2 S3 S4 S5 P S6 S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6 P P IFM C

17 Time course of F signal Of different domains -30 mv DII DIII DI DIV The S4s of the first three domains do not activate in a specific sequence. The activation of S4 in domain IV requires a prior activation of one of the three S4 segments.

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19 Electrochromic Effect Ground State Electrochromic Shift Δ1/λ = Δ1/λ solv (μ- e μ ) g E Cos α + Hyperpolarized Potential Depolarized Potential Photoexcitation + Fluorescence Excited State Wavelength

20 A C 40 mv Figure mv Norm F, Q Fluorescence Gating Charge Capacitance Charge Pulse Duration (ms) B Tau (ms) Time (ms) Voltage (mv) Slow Fluorescence Gating D Normalized Fluorescence and E Off /E On Fluorescence Signal Electrochromic Ratio Time (ms)

21 Mapping the electric Field with electrochromic probes Electric Field Strength(1x10 5 V/cm) N - - P278C - V345C K350C Q354C A359C + + R365C S1 S2 S3 S4 S5 S6 + + F425C P C bilayer C 345C 350C 354C 359C 365C 425C Di8 Residue No.

22 FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) Donor Acceptor hν d h d K et α R -6 hν a h a Donor Acceptor ex Wavelength e s

23 Forster theory: E=k ET /(k ET +k n ) k T (R) = 1 R 0 R τ D [ ] 6 Where R 0 =8.79 x 10-5 (J D q D n -4 κ 2 ) 1/6 Then, R=R 0 [(1-E)/E] 1/6 R=R o [τ DA /(τ D -τ DA )] 1/6

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25 Another method: Using hydrophobic ions as FRET acceptors (reference) in the bilayer Gating current trace of NO 2 Dipicrylamine (dpa) O 2N NH NO 2 Absorption of dpa 20 ms NO 2 O 2N Dipicrylamine NO 2 Emission of TMR mv -100 mv Charge voltage relationship of dpa in oocyte membranes Normalized Charge Wavelength (nm) Emission spectra of TMRM Absorbance spectra of DPA Overlap between DPA and TMRM Excitation spectra of TMRM Voltage (mv) Calculated R o between TMR and dpa is 20 A

26 Large movement Small movement depolarization repolarization

27 WT Individual experiments average WT 4% ΔF/F 20 ms 425C 425C 349C 349C 354C 363C - ABD 354C 363C 1% ΔF/F 20 ms 363C 367C 367C

28 Lanthanide-based Resonance Energy Transfer (LRET) LRET utilizes luminescent lanthanides (i.e. Tb, Eu ) with sensitizer and reaction group as fluorescent donors Lifetimes are easily measurable ( τ ~ ms) Lanthanides fluoresce isotropically, reducing errors from orientation factor κ 2 Sharply spiked emission spectrum and temporal elimination of direct acceptor excitation enables specific measurements of acceptor sensitized emission

29 Tb Tb RSA Tb Donor only τ d = 1.6 ms RSC Fl 2 ms N N N CO 2 O C NH C O - Tb 3+ - CO 2 - CO2 NH NH O C CH 3 N O (CH 2 ) 2 N O O S - Protein Sensitized emission τ μ μ ad = 92 s, 566 s R SC=28 Å = 41 Å R SA RSA predicted from RSC using Pithagorean Theorem = 40 A

30 INVERTED CUT-OPEN FLUORESCENCE SETUP

31 A -90 mv 50 ms. 50 mv -120 mv 50 mv: τ ad= 173 μs Acquisition 10 ms 100 μs -120 mv: τ ad= 65 μs RSC A R SC Q Fit, R SC Fit, Q Q Pulse potential 0.0

32 The Reference results show in the that pore: upon depolarization distances LRET from channel toxin to: to toxin in the pore S4 decrease by 0.8 Å (361and 365 in S4) S3-S4 linker decrease by 2 Å (351,352,353) S3 Increase by 1 Å (333, 335) Conclusion: transmembrane displacement is about 2Å With S4 and S3 moving in opposite directions D. Posson, P. Selvin & C. Miller

33 LBT peptide YIDTNNDGWYEGDELLA LBT donor and 6-His acceptor groups in the Shaker K + Channel Genetically encoded LRET Normalized Intensity normalized intensity Tb 3+ + Cu 2+ time(ms) Tb 3+ only Time, ms Voltage-Sensing Domain Pore Domain Ni 2+ Cu 2+ S2 S3 S1 S4 S5 S6 K K K S6 6-His Position S5 S1 S1 S4 Transfer Efficiency 31% Distance Å 13.7 Transfer Efficiency -/- Distance Å - S2 14% % 15.8 Hexa-Histidine (6-His)Tag Lanthanide Binding Tag (LBT) N C S3 S5 11% 22% % -/

34 Lanthanide based Transfer-LRET using a genetically encoded tag Expression of modified channels in XL- LBT 1 Blue X + X HHHHHH HHHHHH Solubilization of membranes proteins in detergent HO Transfer O CO 2 H O Purification of His-tagged channels on cobalt resin O NH LBT LBT Lanthanide Binding Tag O N Fluorescein maleimide O Reconstitution of channels in proteoliposomes HHHHHH HHHHHH 10mM KCl Crown Ether Valinomycin Normalized fluorescence Intensity Liposomes 16 Oxonol VI F initia l

35 Example: Demonstration of rotational movement of the voltage sensor of KvAP Distance changes in KvAP LBT Open/Inactivated Closed LBT -3 LBT -2 S4 R Å LBT -1 ~4 Å LBT -2 S4 S4 R117 LBT -3 Distance (Å) Å 42 Å 35 Å Å 45 Å 33 Å LBT -3 LBT -2 LBT -1 acceptor acceptor These experiments are also done in oocytes under voltage clamp allowing better potential control and time resolution

36 Conformational changes detected at the single molecule level Optical Methods: fluorescence

37 SPR & SPCE Surface plasmon resonance (SPR) is used to excite fluorophores near a specially prepared thin metal surface on glass substrate Surface plasmon-coupled emission (SPCE) is used in the configuration below to capture the emission of the fluorophores excited by SPR as described by Lakowicz et al Reflecti vity Ө SPR (optimal) Ө critica Ө max (of objective) Excitation Angle (degrees) Figure 1. Schematic of the application of surface plasmon resonance to fluorescence in aqueous solution. The evanescent field strength decreases away from the metal surface. Molecules within the evanescent field are excited (stars). However, molecules within approximately 10 nm of the metal surface will be quenched and those beyond the evanescent field are not excited. Figure 2. Simulation of reflectance of 647 nm laser excitation off the surface of the plasmon chip. The dip is the angle at which photons are most efficiently absorbed and maximally excite surface plasmon. The critical angle for SF11glass at an H 2 O interface and the maximum acceptance angle for the Olympus APO 100x NA 1.65 objective are shown as dotted lines.

38 Surface-coupled plasmon emission Start of Acquisitio n Avg. Intensity Time (ms) End of Acquisitio n Avg. Intensity Time (ms)

39 I287 CONCLUSION The position of S4 at the level of R362 is constrained with respect to S1 and S2 in CLOSED state I241 Open R I287 R362 I241 Closed 0-13 MINIMAL MODEL Translation ~6.5 A Rotation ~180 o Tilt ~30 o

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41 Clark Hyde Walter Sandtner Jerome Lacroix Fabiana Campos Carlos Villalba-Galea Baron Chanda Dorine Starace Kwame Asamoah Rikard Blunck A. Cha D. Sigg S-A Seoh Collaborators Ana M. Correa P. Selvin Benoit Roux D. Posson C. Miller L. Loew G.Snyder R. Latorre, E. Stefani, D. Papazian