Erice School of Biophysics 3-10 April 2002

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

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

7. 4 mM NE -80 mV cell-detached patch with few transporters

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

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

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

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

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

13. 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

14. Experimental Protocols

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

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

17. A Single-File Pore Supports Coupled Transport

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

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

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

21. A Single-File Pore Predicts Experimental Results

22. 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

23. Pore Length is Equivalent to Stoichiometry

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

26. Manuscript M1:10783, Fig. 2

27. Manuscript M1:10783, Fig. 6

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

30. 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

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

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

34. PMT DIC Image ASP PMT Fluorescent Image Laser

39. Light gathered from entire cell. n = 3

40. Light gathered from a line drawn across cell.

43. 1 hNET-293 Cell 2 ASP+ NET Trypan Blue

45. ASP 800 nM [trypan blue]

47. SCG Neurons Transport ASP+ - DS +DS

50. 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