Background:
The cardiac ryanodine receptor-calcium release channel (RyR2) is an integral membrane protein that governs the rapid release of calcium ions from the intracellular sarcoplasmic reticulum (SR) into the cytoplasm, thereby initiating muscle contraction. Cardiac myosin binding protein-C (cMyBP-C) is a sarcomeric thick filament component that regulates sarcomere structure and function. The proposed study will biochemically and functionally characterise the potential retrograde regulation of calcium release by the sarcomere, mediated by the cMyBP-C association with the RyR2. We have recently obtained evidence that the RyR2 can physically interact with the cMyBP-C protein.
We will identify the minimal-interacting regions using human RyR2 and cMyBP-C deletion constructs in yeast two-hybrid interaction, co-immunoprecipitation and GST pull-down assays. Involvement of cMyBP-C residues/motifs specific for RyR2 and/or myosin binding will be investigated by site-directed mutagenesis.
Second, the functional implications of this protein interaction at the molecular and cellular level will employ two complementary techniques: single channel electrophysiological recordings and cardiomyocyte Ca2+ imaging using confocal laser microscopy.
Also, we will assess whether cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM), and whether RyR2 mutations associated with cardiac arrhythmias, result in aberrant RyR2:cMyBP-C interaction, as this may provide a potential mechanism explaining dysfunctional SR calcium release leading to cardiac disease.
Aims:
Investigate the association between the sarcomeric protein cMyBP-C and the RyR2. Our hypothesis is that cMyBP-C is in a dynamic equilibrium between the sarcomere and SR acting both as a regulator of sarcomere-mediated contraction and RyR2-mediated calcium release. Thus, cMyBP-C provides a retrograde link between contraction and its trigger, i.e. SR Ca2+ release. In particular, the following points will be addressed:
• Identify minimal interacting regions/critical residues involved in RyR2:cMyBP-C association
• Identification of cMyBP-C residues/motifs specific for RyR2 and/or myosin binding
• Role of cMyBP-C in RyR2 channel regulation
• Involvement of arrhythmia-linked RyR2 mutations and cardiac hypertrophy-linked cMyBP-C mutations in RyR2:cMyBP-C association
This project involves several well-established research teams with excellent track records within the Cardiff Institute for Molecular & Experimental Medicine.
Training:
The student will face the stimulating challenge of correlating biological function of the cardiac muscle ryanodine receptor (RyR2) complex with the molecular interactions of associated protein components. The project involves training in the acquisition and analysis of biochemical, biophysical and structural data. Proteins of interest will be cloned, sequenced, expressed, purified, characterised, and molecular interactions determined using biochemical and biophysical approaches. The requisite molecular biology, biochemistry, biophysical and computational facilities are already established within the Wales Heart Research Institute. The experienced supervisory team provides a creative learning environment that is complimented with expert staff for training the student in all relevant techniques. The biophysical component of the studentship may also utilise the world-leading synchrotron facilities at Diamond Light Source, enabling high-level exposure to state-of-the-art methods in biomedical research. This should encourage the student to develop the expertise to use new methods for further biomolecular interaction studies. The student will have the opportunity to attend international conferences to discuss their results with leading scientists and generate new ideas and research avenues.
Funding Notes:
The PhD studentship stipend is funded at the standard Medical Research Council rate (£13-14K/annum, tax-free) for 3 years.
Please forward applications, comprising a brief cover letter and full Curriculum Vitae, by email to Professor Tony Lai; lait a cf.ac.uk
References:
Disparate Ryanodine Receptor Association with the FK506-binding Proteins in Mammalian Heart.
Zissimopoulos S, Seifan S, Maxwell C, Williams AJ, Lai FA.
J Cell Sci. 2012 Apr 1;125(Pt 7):1759-69.
PMID: 22328519
Spatial organization of RYRs and BK channels underlying the activation of STOCs by Ca(2+) sparks in airway myocytes.
Lifshitz LM, Carmichael JD, Lai FA, Sorrentino V, Bellvé K, Fogarty KE, ZhuGe R.
J Gen Physiol. 2011 Aug;138(2):195-209
PMID: 21746845
Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation.
Sedej S, Heinzel FR, Walther S, Dybkova N, Wakula P, Groborz J, Gronau P, Maier LS, Vos MA, Lai FA, Napolitano C, Priori SG, Kockskämper J, Pieske B.
Cardiovasc Res. 2010 Jul 1;87(1):50-9.
PMID: 20080988
A mechanism of ryanodine receptor modulation by FKBP12/12.6, protein kinase A, and K201.
Blayney LM, Jones JL, Griffiths J, Lai FA.
Cardiovasc Res. 2010 Jan 1;85(1):68-78.
PMID: 19661110
Mineralocorticoid modulation of cardiac ryanodine receptor activity is associated with downregulation of FK506-binding proteins.
Gómez AM, Rueda A, Sainte-Marie Y, Pereira L, Zissimopoulos S, Zhu X, Schaub R, Perrier E, Perrier R, Latouche C, Richard S, Picot MC, Jaisser F, Lai FA, Valdivia HH, Benitah JP.
Circulation. 2009 Apr 28;119(16):2179-87.
PMID:19364981
Ryanodine receptor-mediated arrhythmias and sudden cardiac death.
Blayney LM, Lai FA.
Pharmacol Ther. 2009 Aug;123(2):151-77.
PMID:19345240
FKBP12.6 binding of ryanodine receptors carrying mutations associated with arrhythmogenic cardiac disease.
Zissimopoulos S, Thomas NL, Jamaluddin WW, Lai FA.
Biochem J. 2009 Apr 15;419(2):273-8.
PMID: 19226252
Ryanodine receptor arrays: not just a pretty pattern?
Yin CC, D'Cruz LG, Lai FA.
Trends Cell Biol. 2008 Apr;18(4):149-56.
PMID: 18329877