The human cardiac ryanodine receptor (hRyR2) is a Ca²⁺ ion channel found on the sarcoplasmic reticulum of cardiomyocytes that plays a central role in excitation-contraction coupling. This channel is tightly regulated to prevent dysfunction, though mutation of hRyR2 is associated with arrhythmia and cardiomyopathy.  Inter-channel clustering of hRyR2 is proposed to regulate its function¹, preventing uncontrolled Ca²⁺ release and possibly promoting concerted gating². This research aims to functionally characterise arrhythmia / cardiomyopathy-linked mutations (R1051P, R1051C, and R1051H) found in the P1 domain – a region likely involved in inter-channel clustering³ – to determine whether any Ca²⁺ release dysfunction is influenced by functional changes at the clustering level.

HEK293 cells recombinantly expressing either wild-type (WT) or mutant hRyR2 were loaded with Calbryte 520 AM Ca²⁺ sensitive dye and imaged using confocal microscopy to assess whole cell Ca²⁺ release. Ca²⁺ release from populations of human recombinant RyR2 channels incorporated into droplet interface bilayers (DIBs)⁴ was analysed optically (with Cal590) using total internal reflection fluorescence microscopy (TIRF). Purified hRyR2 was incorporated into artificial bilayers for single channel recording and analysis of gating mechanisms⁵.

Spontaneous whole cell Ca²⁺ release from R1051-mutants was significantly different from WT hRYR2 (p<0.05, one-way ANOVA with Tukey post-hoc tests) and displayed longer duration of oscillations (WT = 17.7±0.18, R1051P = 21.4±0.51, R1051C = 21.3±0.34, R1051H = 20.5±0.30, seconds), and lower amplitude (WT = 1.15±0.2, R1051P = 0.88±0.03, R1051C = 0.76±0.02, R1051H = 0.71±0.02, ΔF/F₀) (n = number of cells, WT n=645, R1051P n=204, R1051C n=357, R1051H n=447, results expressed as mean±SEM), indicating Ca²⁺ release dysfunction. Populations of R1051P hRyR2 exhibited additional Ca²⁺ flux behaviours not observed for the WT indicative of rapid switching between stable periods of low and high Ca²⁺ flux (n=42-63 channel populations). Preliminary analysis estimating cluster size did not indicate any difference in cluster size between R1051P and WT hRyR2, although results suggested these behaviours manifested more frequently in larger populations. Single channel experiments showed comparable open probabilities (P_o) for mutant and WT channels (n=3-4 channels), although mathematical modelling of higher P_o traces indicated R1051P visited longer open states more frequently (WT τ = 36.5ms, amp = 54%, R1051P τ = 60.4ms. amp = 68%) with fewer flicker closings than WT hRyR2.

This research demonstrates that mutation of R1051 affects hRyR2 function at multiple organisational levels. Effects at the single channel level seem of minor impact, with dysfunction becoming more pronounced and distinct from the WT in populations of channels, suggesting that the mutational effect largely manifests at the cluster level. This has downstream effects on global Ca²⁺ release dynamics that may act as a substrate for arrhythmia. Future work aims to better quantify cluster size and correlate this to Ca²⁺ release behaviour from populations, and consideration of hRyR2 distribution within clusters will be important in consolidating the mechanism of dysfunction.