A Cryo-EM Based Study of An Equivalent N Terminal Domain Mutation in Skeletal and Cardiac Ryanodine Receptor (RyR)

Abstract

Ca 2þ sparks constitute the fundamental units of sarcoplasmic reticulum (SR) Ca 2þ release in cardiomyocytes. However, despite more than 25 years of investigation, the precise nature by which ryanodine receptors (RyRs) collaborate to generate these release events remains unclear. This challenge is related to both technical limitations in imaging RyRs and the rapid time frame in which sparks occur. Unfortunately, various imaging techniques capable of resolving RyRs, including super-resolution microscopy (dSTORM, DNA-PAINT) and electron microscopy, require fixed samples. To circumvent this limitation, we developed a transgenic mouse with photo-activated (PA) tagRFP targeted to RyR2. This approach allows correlative pairing of RyR localization, determined by PA Localization Microscopy (PALM), and Ca 2þ sparks detected by high-speed imaging with a highly inclined light sheet (HILO). Ca 2þ spark recordings showed that a subset of events exhibited slow kinetics, with protracted rise times and durations. Subtracting estimated Ca 2þ diffusion revealed that prolonged Ca 2þ sparks exhibited multiple distinct releases, numbering between 2 and 8 events. Notably, consecutive releases were associated with displacement of the spark centroid. Paired imaging of RyRs confirmed that these ''travelling sparks'' moved between nearby RyR clusters, with some sparks exhibiting displacement as far as 500 nm along z-lines. Importantly, spark propagation often proceeded between clusters that were not within the closest proximity. Treatment with isoproterenol exaggerated this phenomenon, as a larger fraction of travelling sparks was observed which included as many as 12 distinct release sites. These data suggest that participation of discrete RyR clusters in Ca 2þ spark generation is dependent not only on RyR cluster position, but also other factors such as local post-translational modifications which are critically altered during badrenergic stimulation.

Defective ryanodine receptor N-terminus inter-subunit interaction is a common mechanism in neuromuscular and cardiac disorders

“…Our results are consistent with previous molecular dynamics simulations indicating that R169Q, as well as A165D and R176Q, increase the NTD-NTD inter-subunit distance (Xiong et al, 2018). Hence, the present study adds to the mounting evidence from biochemical, structural, and molecular dynamics analyses that RyR1 and RyR2 pathogenic mutations located within the β8-β9 loop disrupt NTD tetramerization (Table 1) (Iyer et al, 2020;Seidel et al, 2021;Xiong et al, 2018).…”

“…Here, we used chemical cross-linking to demonstrate that R163C impairs RyR1 NTD tetramerization (Figure 1). Our biochemical findings are in agreement with the 3D structure of RyR1-R163C, which was recently solved at near-atomic resolution under the same closed-state conditions as with RyR1-WT (Iyer et al, 2020). R163C was found to cause a shift and rotation in the NTD that resulted in increased NTD-NTD inter-subunit distance.…”

“…RyR1 R163C is one of the most studied MH mutations in heterologous (HEK293) and homologous expression systems (dyspedic myotubes) as well as in heterozygous knockin mice. Results obtained from single channel recordings, [ 3 H] ryanodine binding and Ca 2+ imaging indicate enhanced basal activity, increased sensitivity to Ca 2+ activation, increased sensitivity to pharmacological activators, and elevated resting cytosolic Ca 2+ concentration (Tong et al, 1997;Avila and Dirksen, 2001;Yang et al, 2003;Yang et al, 2006;Feng et al, 2011;Chen et al, 2018;Iyer et al, 2020). Here, we used chemical cross-linking to demonstrate that R163C impairs RyR1 NTD tetramerization (Figure 1).…”

“…Two different substitutions have been reported, R163C and R163L (Table 1). We chose to study the R163C variant because its gain of function characteristics have been studied extensively in vivo and in vitro (Tong et al, 1997;Avila and Dirksen, 2001;Yang et al, 2003;Yang et al, 2006;Feng et al, 2011;Chen et al, 2018;Iyer et al, 2020).…”

“…Neighboring NTDs from the four RyR subunits interact with each other to promote channel closure (Tung et al, 2010;Zissimopoulos et al, 2013;Zissimopoulos et al, 2014). Notably, pathogenic RyR1 and RyR2 mutations impair NTD-NTD inter-subunit interactions and result in hyperactive and leaky channels (Iyer et al, 2020;Seidel et al, 2021;Seidel et al, 2015;Xiong et al, 2018;Zheng and Liu, 2017).…”