An investigation into the effects of single point mutations found in patients with hypertrophic cardiomyopathy upon Junctophilin-2 structure and stability

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Excitation contraction coupling in cardiac muscle requires a precise spatial organisation of the L-type voltage-gated calcium channel (LTCC), on the t-tubule (t-t) membrane, and the ryanodine receptor (RyR2) localised to the junctional sarcoplasmic reticulum (jSR)1. In the heart, Junctophilin-2 (JP2) is thought to act as a scaffold to bridge, and stabilise, the gap between the two membranes forming the dyadic cleft. JP2 is believed to be tethered to the SR, via a C-terminal transmembrane domain, span the cytosol, and bind to the inner leaflet of the t-t via the N-terminal region. Genetic defects in JP2 have been associated with hypertrophic cardiomyopathy (HCM), which is the most common cause of sudden cardiac death in young people2. Several single amino acid mutations have been discovered within JP2 in HCM patients (S101R, S165F, Y141H, G505S) significantly, these carriers did not have any mutations of the Z-disc or contractile proteins typically associated with HCM3. The mechanisms by which these mutations lead to disease are still not understood. In order to investigate how the introduction of single point mutations influence the function of JP2 we have expressed and purified wild-type and mutant JP2 proteins from E. coli. Using fluorescence spectroscopy, exploiting the presence of instrinsic tryptophan residues within the JP2 amino acid sequence, we examined the thermal denaturation profiles of each protein. Slit widths were set at 5 nm, the excitation wavelength at 280 nm and the emission collected at 280 nm, 330 nm and 350 nm. The temperature was increased at a rate of 0.5oC min-1 (20 – 80oC). The transition temperature (Tm), the temperature at which 50% of the protein is denatured, was determined for wild-type and mutant JP2 by plotting the ratio of the emission intensity F330/F350. Guanidine hydrochloride was employed to study the effects of chemical denaturation and secondary structure analysis was conducted using circular dichroism (CD). Thermal and chemical denaturation experiments revealed the mutations had no overall effect on protein stability and the Tm for each protein was 52oC. CD revealed that JP2 is comprised of 38% α-helix, 16% β-sheet, 18% turns and 28% random coil, correlating with predicted secondary structure analysis. However, the S101R and Y141H mutants exhibited changes to the secondary structure profile with a reduction to the overall helical content and increase in random coil. These results indicate the JP2 mutations do not appear to effect the stability of the protein but lead to a modification of the structure which may alter the t-t binding properties and/or disrupt potential protein interactions between JP2 and dyadic cleft proteins involved in calcium homeostasis.
Original languageEnglish
Publication statusPublished - 2013
Event37th Congress of the International Union of Physiological Sciences - Birmingham, United Kingdom
Duration: 21 Jul 201326 Jul 2013


Conference37th Congress of the International Union of Physiological Sciences
Country/TerritoryUnited Kingdom


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