Ligand sensitivity of type-1 inositol 1,4,5-trisphosphate receptor is enhanced by the D2594K mutation.

Inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP3R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP3 sensitivity. We hypothesized the IP3R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP3R1 regulation by IP3, cytosolic, and luminal Ca2+ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca2+ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP3 ligand sensitivity. Single-channel IP3R1 studies revealed that the conductance of IP3R1-WT and -D2594K channels is similar. However, IP3R1-D2594K channels exhibit higher IP3 sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP3R1-D2594K showed a bell-shape cytosolic Ca2+-dependency, but D2594K had greater activity at each tested cytosolic free Ca2+ concentration. The IP3R1-D2594K also had altered luminal Ca2+ sensitivity. Unlike IP3R1-WT, D2594K channel activity did not decrease at low luminal Ca2+ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels' pore cytosolic exit affects the channel's gating behavior thereby explaining the enhanced ligand-channel's sensitivity.The authors received the support of research grants from the National Institutes of Health grant/award numbers R01GM111397 to S. R. W. Chen, M. Fill, and J. Ramos‐Franco, R01HL057832 to M. Fill and by the Canadian Institutes of Health Research, grant/award number PJT‐173352. S. R. W. Chen holds the Heart and Stroke Foundation Chair in Cardiovascular Research (END611955). A. Tambeaux was supported by the Graduate College of Rush University Medical Center.S

Impaired Binding to Junctophilin-2 and Nanostructural Alteration in CPVT Mutation

RATIONALE: Catecholaminergic polymorphic ventricular tachycardia is a rare disease, manifested by syncope or sudden death in children or young adults under stress conditions. Mutations in the Ca2+ release channel/type 2 ryanodine receptor (RyR2) gene account for about 60% of the identified mutations. Recently, we found and described a mutation in RyR2 N-terminal domain, RyR2R420Q. OBJECTIVE: To determine the arrhythmogenic mechanisms of this mutation. METHODS AND RESULTS: Ventricular tachycardias under stress conditions were observed in both patients with catecholaminergic polymorphic ventricular tachycardia and knock-in mice. During action potential recording (by patch-clamp in knock-in mouse cardiomyocytes and by microelectrodes in mutant human induced pluripotent stem cell-derived cardiomyocytes), we observed an increased occurrence of delayed afterdepolarizations under isoproterenol stimulation, associated with increased Ca2+ waves during confocal Ca2+ recording in both mouse and human RyR2R420Q cardiomyocytes. In addition, Ca2+-induced Ca2+-release, as well as a rough indicator of fractional Ca2+ release, were higher and Ca2+ sparks longer in the RyR2R420Q-expressing cells. At the ultrastructural nanodomain level, we observed smaller RyR2 clusters and widened junctional sarcoplasmic reticulum measured by gated stimulated emission depletion super-resolution and electronic microscopy, respectively. The increase in junctional sarcoplasmic reticulum width might be due to the impairment of RyR2R420Q binding to junctophilin-2, as there were less junctophilin-2 coimmunoprecipitated with RyR2R420Q. At the single current level, the RyR2R420Q channel dwells longer in the open state at low intracellular Ca2+ ([Ca2+]i), but there is predominance of a subconductance state. The latter might be correlated with an enhanced interaction between the N terminus and the core solenoid, a RyR2 interdomain association that has not been previously implicated in the pathogenesis of arrhythmias and sudden cardiac death. CONCLUSIONS: The RyR2R420Q catecholaminergic polymorphic ventricular tachycardia mutation modifies the interdomain interaction of the channel and weakens its association with junctophillin-2. These defects may underlie both nanoscale disarrangement of the dyad and channel dysfunction. GRAPHIC ABSTRACT: An online graphic abstract is available for this article.This work was funded by Inserm and University Paris-Sud, and grants from ANR (ANR-19-CE14-0031-01 to A.M. Gómez), LabEx LERMIT (ANR-10-LABX-33), “Instituto de Salud Carlos III”; FEDER “Union Europea, Una forma de hacer Europa” (PI18/01582), La Fe Biobank (PT17/0015/0043) and Memorial Nacho Barberá to E. Zorio; Swiss National Science Foundation (SNSF grants no. 31003A 179325 and 310030 156375 to E. Niggli), British Heart Foundation (FS/15/30/31494) to S. Zissimopoulos; Italian Telethon ONLUS Foundation (GGP19231) and Italian MIUR (PRIN no. 2015ZZR4W3) to F. Protasi; National Institutes of Health (NIH) grants to J. Ramos-Franco (R01GM111397) and H.H. Valdivia–A.M. Gómez (2R01HL055438-22); and European Union H2020 (MSCA-RISE AMD-734931- 6) to A.M. Gómez. H.H. Valdivia was recipient of a Fullbright-Tockeville chair to work on A.M. Gómez laboratory. J.L. Álvarez and C.R. Valdivia were recipient of a visiting program Alambert from University Paris-Sud to work in A.M. Gómez laboratory. A. Zahradnikova was recipient of a postdoctoral position from University Paris-Sud. R. Rizzetto was recipient of a postdoctoral fellowship from CORDDIM (Région Ile de France). L. Yin was recipient of the CSC (Chinese Scholarship council)