Type 1 ryanodine receptor interactions

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dc.contributor.advisor Mackrill, John en
dc.contributor.author English, Kathleen Kelly
dc.date.accessioned 2016-02-02T11:20:37Z
dc.date.available 2016-02-02T11:20:37Z
dc.date.issued 2015
dc.date.submitted 2015
dc.identifier.citation English, K. K. 2015. Type 1 ryanodine receptor interactions. PhD Thesis, University College Cork. en
dc.identifier.endpage 285 en
dc.identifier.uri http://hdl.handle.net/10468/2238
dc.description.abstract Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research. en
dc.format.mimetype application/pdf en
dc.language.iso en en
dc.publisher University College Cork en
dc.rights © 2015, Kathleen K. English en
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/ en
dc.subject Calcium signalling en
dc.subject Sarcoplasmic reticulum en
dc.subject Junctophilin en
dc.subject Sarcopenia en
dc.subject Muscle en
dc.title Type 1 ryanodine receptor interactions en
dc.type Doctoral thesis en
dc.type.qualificationlevel Doctoral en
dc.type.qualificationname PhD (Medicine and Health) en
dc.internal.availability Full text available en
dc.check.info No embargo required en
dc.description.version Accepted Version
dc.contributor.funder Physiology, College of Medicine and Health, University College Cork en
dc.description.status Not peer reviewed en
dc.internal.school Physiology en
dc.check.type No Embargo Required
dc.check.reason No embargo required en
dc.check.opt-out Not applicable en
dc.thesis.opt-out false
dc.check.embargoformat Not applicable en
ucc.workflow.supervisor j.mackrill@ucc.ie
dc.internal.conferring Spring 2016 en

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