Ruggiero_2012_ACS.Chem.Neurosci_3_767

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.