Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake
| Autor: | Hideki Iwamoto, Jane Wright, Randy D. Blakely, Elizabeth A. Ennis, Shawn M. Ferguson, Ericka C. Holmstrand, C. David Weaver, Katie S. Emerson, Alicia M. Ruggiero, Michael T. Ivy, Michelle Lewis |
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| Rok vydání: | 2012 |
| Předmět: |
Physiology
Cognitive Neuroscience Allosteric regulation Presynaptic Terminals Biology Biochemistry Choline Xenopus laevis chemistry.chemical_compound Allosteric Regulation Hemicholinium-3 Chlorocebus aethiops medicine Animals Humans Staurosporine Membrane Transport Proteins Depolarization Cell Biology General Medicine Acetylcholinesterase High-Throughput Screening Assays Choline transporter Protein Transport HEK293 Cells chemistry COS Cells Biophysics Cholinergic Female Choline transport medicine.drug |
| Zdroj: | ACS Chemical Neuroscience. 3:767-781 |
| ISSN: | 1948-7193 |
| Popis: | Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling. |
| Databáze: | OpenAIRE |
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