Structural and functional characterisation of SLC45A4, a neuronal polyamine transporter

Thesis

Polyamines (PAs) are ubiquitous polycationic metabolites essential for diverse cellular processes, including proliferation, protein synthesis, and ion channel regulation, and have been linked to pain signalling through modulation of nociceptor activity. However, no plasma membrane transporter responsible for PA import has previously been identified in mammals. Here, SLC45A4, a previously orphan MFS transporter implicated in chronic pain, was identified as the principal neuronal plasma membrane PA transporter. Metabolomic analysis allowed for identification of PAs as substrates and subsequent cell-based uptake assays, demonstrate that SLC45A4 catalyses high-affinity, low-turnover transport of spermidine and agmatine, a PA-like metabolite. CryoEM structures revealed a novel autoinhibitory plug domain, suggesting a mechanism for regulated transport of PAs, metabolism of which is tightly controlled. Deletion of this domain enabled determination of substrate-bound, outward-facing structures that define key substrate-recognition interactions and, together with biochemical analysis, suggest a mechanism of substrate-induced uniport. Finally, identification of a toxin-derived inhibitor, and determination of inhibitor-bound structures, demonstrate the feasibility of pharmacological inhibition of SLC45A4, and may inform rational design of novel analgesics. By regulation of intracellular PA levels, SLC45A4 presents itself as a previously unknown regulator of neuronal physiology and highlights its link to chronic pain.

Authors: Markusson, S

• DOI: 10.5287/ora-qyoynpnd4
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: pain signalling; membrane transport; polyamine transport and metabolism; major facilitator superfamily; SLC transporters; cryo-em
• Subjects: Structural Biology; Biochemistry; Biophysics