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Title: Function and pharmacology of TRPM3 ion channel
Author: Naylor, Jacqueline
ISNI:       0000 0001 3440 2022
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2008
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For many ion channels there are few, if any, pharmacological agents, and even fewer showing specificity. In this study, a set of pharmacological tools were developed to investigate TRPM3, a widely expressed transient receptor potential (TRP) channel for which no functional role has yet been identified. Human TRPM3 was first expressed in HEK 293 cells and shown to be activated by hypo-osmotic challenge or sphingosine, consistent with previous reports. In addition, TRPM3 was activated by pregnenolone sulphate. Hydrophobicity analysis of the TRPM3 amino acid sequence revealed a short and reasonably unique peptide in the 3rd extracellular loop (E3) region, to which polyclonal antiserum (TM3E3) was produced. Extracellular application of TM3E3 inhibited TRPM3 function with a high degree of specificity, having no effect on TRPM2 or example members of other sub-types of mammalian TRP, TRPC5 or TRPV4. The data validate E3-targeting as an approach for production of isoform-specific channel blockers and reveal a specific agent for blocking TRPM3. The cellular and tissue functions of TRPM3 were also investigated. RT-PCR and immunocytochemistry demonstrated TRPM3 expression in human saphenous vein smooth muscle cells, where sphingosine- and pregnenolone sulphate-induced calcium responses were also apparent. These calcium responses could be selectively blocked by TM3E3. Furthermore, TRPM3 activators inhibited matrix metalloproteinase and interleukin-6 secretion, indicating a protective function for TRPM3 in vascular smooth muscle cells. Medium throughput screening systems were employed to screen a library of compounds for further TRPM3 modulators with vascular relevance. Cholesterol, antidepressants, antipsychotics, calmodulin inhibitors, and PIP2 all inhibited TRPM3, whereas nifedipine and elevated temperature activated the channel. TRPM3 appears to be regulated by a large number of different chemicals and mechanisms. In summary, TRPM3 has constitutive, protective, activity which can be suppressed by a multitude of compounds, including known vascular disease factors such as cholesterol.
Supervisor: Beech, D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available