Date of Award

1-1-2012

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (viii, 109 pages) : illustrations (some color)

Dissertation/Thesis Chair

Terence Wagenknecht

Committee Members

Rajendra Agrawal, Mark Fleck, Joachim Jaeger, Michael Koonce

Keywords

calmodulin, cryo-electron microscopy, ryanodine recepter, Ryanodine, Cryomicroscopy, Calmodulin, Binding sites (Biochemistry)

Subject Categories

Molecular Biology

Abstract

Ryanodine receptor (RyR) is a key player in excitation-contraction coupling (E-C coupling). Calmodulin (CaM) is one of the important regulatory factors of RyR. Two mammalian RyR isoforms, RyR1 and RyR2, are highly enriched in skeletal and cardiac muscle, respectively. Apo-calmodulin weakly activates RyR1 but inhibits RyR2, whereas Ca2+-calmodulin inhibits both the isoforms. Previous cryo-electron microscopy studies showed distinctly different binding locations on RyR1 for the two states of calmodulin. However, recent studies employing fluorescence resonance energy transfer appeared to challenge these findings. In chapter 1, using cryo-electron microscopy, we have determined that a mutant calmodulin, which is incapable of binding calcium, binds to RyR1 at the "apo" site, regardless of the calcium concentration. We have also re-determined the location of RyR1-bound Ca2+-calmodulin using uniform experimental conditions. Our results show the existence of the two overlapping but distinct binding sites for calmodulin in RyR1, and imply that the binding location switch is due to Ca2+ binding to calmodulin, as opposed to direct effects of Ca2+ on RyR1. We also discuss explanations that could resolve the apparent conflict between the cryo-electron microscopy results and fluorescence resonance energy transfer results. Interestingly, apo-calmodulin binds to RyR2 at a similar binding location to that of Ca2+-calmodulin on RyR1, in seeming agreement with the inhibitory effects of these two forms of CaM towards their respective receptors. In chapter 2, using site-specific GFP insertion coupled with cryo-EM techniques, we localize three amino-acid sequences of RyR1 that were hypothesized to be important in CaM regulatory effects on the intact, full-length RyR2. These are residues 1975-1999, 3614-3643 and 4064-4210 of RyR1 (1942-1966, 3581-3612 and 4261-4286 of RyR2). By comparing the locations of these sequences with the CaM-binding sites determined in chapter 1, we find that 3614-3643 and 4064-4210 are likely to involve in CaM binding, while 1975-1999 might be an internal buried sequence that does not directly bind to CaM. The results gained from these studies help uncover how RyR is regulated by CaM and exclude some incorrect hypotheses, or even provide hints about the mechanisms of other regulatory factors of RyR. Eventually, this knowledge will contribute to methods for controlling RyR and might help develop therapies for RyR related diseases.

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