The cardiac ryanodine receptor (RyR2) contains structural elements within the channel pore that function as gates to regulate the release of intracellular calcium, initiating cardiac muscle contraction. The precise regulation of these gates is critical in maintaining normal cardiac function, and channel dysfunction, resulting in altered calcium handling, underlies the mechanisms of arrhythmia and sudden cardiac death. The enormous size of RyR2 has impeded the gathering of detailed structural information, hence the structural determinants for channel gating remain unknown. Structural modelling studies have revealed similarities between the RyR2 pore and the K+ channel, KcsA, providing a framework in which to test channel gating mechanisms. A region termed the selectivity filter is a gating component in K+ channels, involved in inactivation and flicker closings, and its conformation is maintained by a transient hydrogen-bonding network. This project examined the role of the RyR2 selectivity filter in channel gating by generating mutants at analogous positions to KcsA that either disrupted (Y4813A, D4829A and Y4839A) or maintained (Y4813W, D4829E and Y4839W) a proposed hydrogen-bonding network, and assessed their intracellular Ca2+ release, ryanodine modification and biophysical properties. Y4813A and D4829A had drastic effects on channel function, whereas retaining physicochemical properties of conservative mutations, Y4813W and Y4839W, maintained the functional characteristics of WT RyR2. Flicker closings were affected by Y4839A mutation however, in general, single-channel gating for Y4813W, Y4839A and Y4839W was comparable to WT RyR2. Interestingly, monitoring single-channels for prolonged periods revealed novel insights into channel behaviour, characterised by inherent fluctuations in channel activity under steady-state conditions. This thesis reveals that the selectivity filter region is an important component for RyR2 channel function. However, it remains unclear whether the selectivity filter regulates channel gating, as the proposed hydrogen-bonding network would not be possible due to altered residue distances revealed from recent high-resolution RyR structural models.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:683654 |
| Date | January 2015 |
| Creators | Firth, Jahn Michael |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/89098/ |
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