Nonoisotopic assay for the presynaptic choline transporter reveals capacity for allosteric modulation of choline uptake.
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.
Center for Molecular Neuroscience, Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, USA.
Alicia M
AM
Ruggiero
Jane
J
Wright
Shawn M
SM
Ferguson
Michelle
M
Lewis
Katie S
KS
Emerson
Hideki
H
Iwamoto
Michael T
MT
Ivy
Ericka C
EC
Holmstrand
Elizabeth A
EA
Ennis
C David
CD
Weaver
Randy D
RD
Blakely
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002794:Choline / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D017729:Presynaptic Terminals / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D014982:Xenopus laevis
D026901:Membrane Transport Proteins; C065637:choline transporter; D002794:Choline