1 / 52

Erice School of Biophysics 3-10 April 2002

Erice School of Biophysics 3-10 April 2002 NE, 5-HT, and DA Transporters: electrophysiology and micro-fluorometry.

keisha
Download Presentation

Erice School of Biophysics 3-10 April 2002

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Erice School of Biophysics 3-10 April 2002 NE, 5-HT, and DA Transporters: electrophysiology and micro-fluorometry Louis J. DeFelice Department of Pharmacology Center for Molecular Neuroscience Vanderbilt University Medical Center Nashville TN 37232-6600

  2. Aurelio Galli, Ph.D.NET Assistant Professor of Pharmacology at UT San Antonio Christine Petersen, Ph.D. SERT Instructor at Vanderbilt University under Jeff Balser Lucia Carvelli, Ph.D. DAT Postdoctoral Fellow at Vanderbilt University Scott Ramsey, Ph.D. SERT Postdoctoral Fellow at Harvard University under David Clapham Joel Schwartz, NET Neuroscience graduate student at Vanderbilt University Scott Adams, SERT Physics graduate student at Vanderbilt University Randy Blakely, Ph.D. tutte le cose Allan D. Bass Professor of Pharmacology Director,Center for Molecular Neuroscience Vanderbilt University Medical Center

  3. Synaptic Transmission neuron synapse (signals must turn off as well as on)

  4. alternating access of co-transport Na [NE]i / [NE]o = [Na]o / [Na]i

  5. cell-attached patch with many transporters 4 mM NE -80 mV hNET-transfected HEK cell

  6. 4 mM NE -80 mV cell-detached patch with few transporters

  7. alternating access model modified to include a channel mode of conduction Na [NE]i / [NE]o = [Na]o / [Na]i

  8. By the AA model, one million transporters working at once would generate only 0.16 pA. But which ions carry the measured current?

  9. To answer that question, we turn to amperometry. inside-out patch 4 mM NE 66,000 NE

  10. NE-induced current and NE uptake are correlated 6000 NE

  11. channel mode of conduction modified to include NE NE Na NE NE NE ? NE [NE]i / [NE]o = [Na]o / [Na]i

  12. Single-File Channel and Alternating Access are Difficult to Distinguish Experimentally Single-File Channels can couple ionic gradients to substrate gradients exhibit saturating substrate fluxes Especially if experimental condition [I]out>>[S]out gate complicates kinetic measurements Experiments must • measure equilibrium gradients, over wide So • eliminate gating

  13. Experimental Protocols

  14. Unidirectional 5HT Fluxes Indicate Coupling Mechanism fin fout [5HT]in [5HT]out 5HT Gradient 5HT Flux

  15. The Human Serotonin Transporter Couples Like a Single-File Channel single file alternating access

  16. A Single-File Pore Supports Coupled Transport

  17. The Equilibrium 5HT Gradient Depends on [5HT]out

  18. The Flux Ratio Eliminates Binding and Gating Processes fout fin Binding/Gating Flux

  19. Flux Ratio Data Suggests Single-File Permeation Alternating Access Single-File

  20. A Single-File Pore Predicts Experimental Results

  21. Single file diffusion couples NE to Na - pore length replaces NE:Na stoichiometry NE Na NE NE NEv NE [NE]i / [NE]o = [Na]o / [Na]i

  22. Pore Length is Equivalent to Stoichiometry

  23. Manuscript M1:10783, Fig. 1 A B

  24. Manuscript M1:10783, Fig. 2

  25. Manuscript M1:10783, Fig. 6

  26. Bruns et al. Neuron 1993 Retzius-P cell leech synapse Ca flash to presynaptic neuron presynaptic response postsynaptic response

  27. Erice School of Biophysics 3-10 April 2002 NE, 5-HT, and DA Transporters: electrophysiology and micro-fluorometry Louis J. DeFelice Department of Pharmacology Center for Molecular Neuroscience Vanderbilt University Medical Center Nashville TN 37232-6600

  28. Synaptic Transmission neuron synapse (signals must turn off as well as on)

  29. ASP+ is structurally similar to MPP+ ASP+ MPP+

  30. PMT DIC Image ASP PMT Fluorescent Image Laser

  31. Light gathered from entire cell. n = 3

  32. Light gathered from a line drawn across cell.

  33. 1 hNET-293 Cell 2 ASP+ NET Trypan Blue

  34. ASP 800 nM [trypan blue]

  35. SCG Neurons Transport ASP+ - DS +DS

  36. Do NET results apply to other co-transporters? Glutamate transporters (EAAT) Cl channel Mike Kavanaugh Iron transporters (Nramp) proton coupled Differences between antiporters and symporters? Tapasree Goswami

More Related