The KATP channel is a key regulator of the pancreatic β-cell, effectively linking metabolic status to electrical activity. Syntaxin-1A has been previously reported by our lab to both bind and inhibit the KATP channel via the nucleotide-binding folds (NBFs). The purpose of this thesis project was to elucidate the precise regions within the NBFs responsible for the Syn-1A- KATP interaction. In vitro binding assays revealed that Syn-1A associates with the Walker domains of both NBF1 and NBF2. Furthermore, site directed mutagenesis of the conserved lysine in Walker A of both NBFs abolishes Syn-1A affinity for this region. Electrophysiological recordings indicate that channel inhibition was mediated primarily through interaction with NBF1-Walker B and both Walkers of NBF2. Based on these results, we propose a model by which Syn-1A acts as an inhibitory clamp on the KATP channel, effectively buffering minor fluctuations in ATP/ADP concentration to prevent unnecessary channel activity.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/18249 |
| Date | 13 January 2010 |
| Creators | Chang, Nathan |
| Contributors | Gaisano, Herbert Young, Feng, Zhong-Ping |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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