Louis J. DeFelice Department of Pharmacology Center for Molecular Neuroscience Vanderbilt University

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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
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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
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Synaptic Transmission
neuron
synapse
(signals must turn off as well as on)
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alternating access of co-transport

Na
NEi / NEo Nao / Nai
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cell-attached patch with many transporters
4 mM NE
-80 mV
hNET-transfected HEK cell
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4 mM NE
-80 mV
cell-detached patch with few transporters
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alternating access model modified to include a
channel mode of conduction
Na
NEi / NEo Nao / Nai
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By the AA model, one million transporters working
at once would generate only 0.16 pA.
But which ions carry the measured current?
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To answer that question, we turn to amperometry.
inside-out patch
4 mM NE
66,000 NE
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NE-induced current and NE uptake are correlated
6000 NE
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channel mode of conduction modified to include
NE
NE
Na
NE
NE
NE
?
NE
NEi / NEo Nao / Nai
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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 IoutSout
• gate complicates kinetic measurements
• Experiments must
• measure equilibrium gradients, over wide So
• eliminate gating

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Experimental Protocols
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Unidirectional 5HT Fluxes Indicate Coupling
Mechanism
fin
fout
5HTin
5HTout
5HT Gradient
5HT Flux
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The Human Serotonin Transporter Couples Like a
Single-File Channel
single file
alternating access
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A Single-File Pore Supports Coupled Transport
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The Equilibrium 5HT Gradient Depends on 5HTout
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The Flux Ratio Eliminates Binding and Gating
Processes
fout
fin
Binding/Gating
Flux
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Flux Ratio Data Suggests Single-File Permeation
Alternating Access
Single-File
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A Single-File Pore Predicts Experimental Results
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Single file diffusion couples NE to Na
- pore length replaces NENa stoichiometry
NE
Na
NE
NE
NEv
NE
NEi / NEo Nao / Nai
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Pore Length is Equivalent to Stoichiometry
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Manuscript M110783, Fig. 1
A                    
 
B
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Manuscript M110783, Fig. 2
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Manuscript M110783, Fig. 6
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Bruns et al. Neuron 1993 Retzius-P cell leech
synapse
Ca flash to presynaptic neuron
presynaptic response
postsynaptic response
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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
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Synaptic Transmission
neuron
synapse
(signals must turn off as well as on)
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ASP is structurally similar to MPP
ASP
MPP
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Laser
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Light gathered from entire cell. n 3
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Light gathered from a line drawn across cell.
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1
hNET-293 Cell
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ASP
NET
Trypan Blue
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ASP 800 nM
trypan blue
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SCG Neurons Transport ASP
- DS
DS
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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
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Thanks! lady in red ...
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