The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Abstract

The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.

Modeling polymorphic ventricular tachycardia at rest using patient-specific induced pluripotent stem cell-derived cardiomyocytes

“…Our results contrast with those published by Xiao et al. describing no consequence of the recombinant RyR2-H29D mutant compared to RyR2-WT when expressed in HEK cells [ 31 ]. Using the 3D RyR2 and RyR1 available structures, we performed 3D in-silico protein modeling to investigate the impact of the RyR2-H29D mutation on the structure/function relationship.…”

“Ryanopathies” and RyR2 dysfunctions: can we further decipher them using in vitro human disease models?

“… [ 46 ] F2307L Genetic screening for long QT and CPVT syndrome patients in Norway. [ 47 ] V2113M, Y2156C, H2168Q, E2183V, D2216V, E2296Q, F2307L, V2321M, R2404T, R2420W, M2389L See findings of the L62F mutation. [ 31 ] H2217Y, C2402Y See findings of the D242V mutation.…”

“… [ 56 ] R4144C See findings of the F2307L mutation. [ 47 ] L3879P, Q3925E, G3946A, S3959L, M3972I, D3973H, L3974Q, K3997E, S4124G, Y4149s, R4157Q, Q4159P, N4178S, E4187Q See findings of the L62F mutation. [ 31 ] S3799P, G3946D, D3977Y, A4091V, A4091T See findings of the D242V mutation.…”

Tritium-labelled alkaloids: Synthesis and applications

“…The likely mechanism resulting in the observed multiple tritium resonances at locations close to the 21‐methyl group in the product has been explained as a direct catalyst‐mediated tritium exchange into the nearby positions of the olefin . As in the case of [ 3 H]muscimol, over 1000 published studies using [ 3 H]ryanodine testify to its value as a radioligand …”

“…115 As in the case of [ 3 H]muscimol, over 1000 published studies using [ 3 H]ryanodine testify to its value as a radioligand. 116,117 6 | TROPANE ALKALOIDS…”