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The Journal of Neuroscience, January 23, 2008, 28(4):976-989; doi:10.1523/JNEUROSCI.2796-07.2008

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Cellular/Molecular
Currents in Response to Rapid Concentration Jumps of Amphetamine Uncover Novel Aspects of Human Dopamine Transporter Function

Kevin Erreger,^1,2,3 Christof Grewer,⁴ Jonathan A. Javitch,^5,6 and Aurelio Galli^1,2,3

¹Department of Molecular Physiology and Biophysics, ²Center for Molecular Neuroscience, and ³Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, Tennessee 37232, ⁴Department of Physiology and Biophysics, University of Miami, Miami, Florida 33101, and Departments of ⁵Pharmacology and ⁶Psychiatry, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Correspondence should be addressed to Aurelio Galli, Department of Molecular Physiology and Biophysics, Vanderbilt University, 7124 MRBIII, 465 21st Avenue South, Nashville, TN 37232. Email: aurelio.galli{at}vanderbilt.edu

Amphetamine (AMPH) is a widely abused psychostimulant that acts as a substrate for the human dopamine transporter (hDAT). Using a piezoelectric rapid application system, we measured AMPH-induced currents mediated by hDAT. Whole-cell patch-clamp recordings in a heterologous expression system reveal that AMPH induces a rapidly activating and subsequently decaying inward current mediated by hDAT. We hypothesize that this transient inward current reflects a conformational change associated with substrate translocation. The AMPH-induced current strictly depends on extracellular Na⁺. Elevated intracellular Na⁺ has no effect on the peak AMPH-induced current amplitude but inhibits the steady-state current. In addition, elevated intracellular Na⁺ causes an overshoot outward current upon washout of AMPH that reflects hDAT locked in a Na⁺-exchange mode. Furthermore, elevated intracellular Na⁺ dramatically accelerates the recovery time from desensitization of the AMPH-induced current, revealing a new role for intracellular Na⁺ in promoting the transition to the hDAT "outward-facing" conformation. Ion substitution suggests that both extracellular and intracellular Cl^– facilitate transporter turnover in contrast to the classical model of Cl^– as a cotransported ion. We present an alternating-access model of hDAT function that accurately fits the main features of the experimental data. The model predicts that translocation of substrate occurs within milliseconds of substrate binding but that slow reorientation of the empty transporter is the rate-limiting factor for turnover. The model provides a framework for interpreting perturbations of hDAT activity.

Key words: transporter; amphetamine; dopamine; drug abuse; monoamine; patch clamp

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Received March 6, 2007; revised Nov. 28, 2007; accepted Nov. 28, 2007.

Correspondence should be addressed to Aurelio Galli, Department of Molecular Physiology and Biophysics, Vanderbilt University, 7124 MRBIII, 465 21st Avenue South, Nashville, TN 37232. Email: aurelio.galli{at}vanderbilt.edu

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