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The Journal of Neuroscience, September 27, 2006, 26(39):9851-9859; doi:10.1523/JNEUROSCI.1862-06.2006
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Cellular/Molecular
Na+, Cl–, and pH Dependence of the Human Choline Transporter (hCHT) in Xenopus Oocytes: The Proton Inactivation Hypothesis of hCHT in Synaptic Vesicles
Hideki Iwamoto, Randy D. Blakely, andLouis J. De Felice Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville Tennessee 37232-8548
Correspondence should be addressed to Dr. Louis J. De Felice, Department of Pharmacology, Center for Molecular Neuroscience, 7130 Medical Research Building III, Vanderbilt University Medical Center, 465 21st Avenue South, Nashville, TN 37232-8548. Email: lou.defelice{at}vanderbilt.edu
The recent cloning of the human choline transporter (hCHT) has allowed its expression in Xenopus laevis oocytes and the simultaneous measurement of choline transport and choline-induced current under voltage clamp. hCHT currents and choline transport are evident in cRNA-injected oocytes and significantly enhanced by the hCHT trafficking mutant L530A/V531A. The charge/choline ratio of hCHT varies from 10e/choline at –80 mV to 3e/choline at –20 mV, in contrast with the reported fixed stoichiometry of the Na+-coupled glucose transporter in the same gene family. Ion substitution shows that the choline uptake and choline-induced current are Na+ and Cl– dependent; however, the reversal potential of the induced current suggests a Na+-selective mechanism, consigning Cl– to a regulatory role rather than a coupled, cotransported-ion role. The hCHT-specific inhibitor hemicholinium-3 (HC-3) blocks choline uptake and choline-induced current; in addition, HC-3 alone reveals a constitutive, depolarizing leak current through hCHT. We show that external protons reduce hCHT current, transport, and binding with a similar pKa of 7.4, suggesting proton titration of residue(s) that support choline binding and transport. Given the localization of the choline transporter to synaptic vesicles, we propose that proton inactivation of hCHT prevents acetylcholine and proton leakage from the acidic interior of cholinergic synaptic vesicles. This mechanism would allow cholinergic, activity-triggered delivery of silent choline transporters to the plasma membrane, in which normal pH would reactivate the transporters for choline uptake and subsequent acetylcholine synthesis.
Key words: choline transporter; Xenopus oocyte; choline-induced current; Na+; Cl–; pH
Received May 2, 2006; revised Aug. 11, 2006; accepted Aug. 11, 2006.
Correspondence should be addressed to Dr. Louis J. De Felice, Department of Pharmacology, Center for Molecular Neuroscience, 7130 Medical Research Building III, Vanderbilt University Medical Center, 465 21st Avenue South, Nashville, TN 37232-8548. Email: lou.defelice{at}vanderbilt.edu
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