STRUCTURE-FUNCTION ANALYSIS OF THE HUMAN EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 1

Site-directed mutagenesis was used to target residues within transmembrane domains 1 and 2 of the human equilibrative nucleoside transporter 1 (hENTl), in order to assess their contributions to transporter functionality. Through affinity changes in [ H]NBMPR binding, competitive inhibition profiles with ENT1 inhibitors and [ H]2- chloroadenosine uptake, these residues were found to make minor, but statistically significant, contributions to the inhibitor and/or substrate binding site of hENTl. An initial study was also undertaken to probe the potential oligomerization of hENTl. Typically considered to be a monomer to this date, under the native conditions of Blue Native Electrophoresis, hENTl was found to possess a molecular mass of 160 kDa. This is approximately three times the size of the known 55kDa hENTl monomer. Targeted site-directed mutagenesis of GxxxG motifs within the transporter, known to play a role in oligomerization, did not appear to affect the higher molecular mass hENTl complex observed

Name thesis:degree_name

M Sc

Discipline thesis:degree_discipline

Pharmacology and Toxicology

Year dc:date.issued

2011

Author dc:creator

• Cunningham, Frances Kay Marie

Advisor dc:contributor.advisor

• Hammond, James R.

• equilibrative nucleoside transporters
• site-directed mutagenesis
• transmembrane domains 1 and 2
• oligomerization
• blue native electrophoresis
• GxxxG motif

Handle dc:identifier.uri

https://hdl.handle.net/20.500.14721/18548

OAI identifier oai:identifier

oai:uwo.scholaris.ca:20.500.14721/18548