RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

Abstract

In cardiomyocytes, Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca2+ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca2+-activated K+ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca2+ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca2+-activated K+ current (IK,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the IK,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on IK,Ca and Ca2+ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in IK,Ca (p<0.05) and [Ca2+]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca2+ release is responsible for the activation and modulation of SK channels in cardiac myocytes.

Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

“…10,38 We previously showed that RyR2 alters SK2 channel activity in cardiac myocytes due to changed in the intracellular Ca 2+ levels. 35 Our present results reveal that interfering RNA silencing of endogenous JP2 caused a significantly reduced density of I k,Ca. and reduced amplitude of the Ca 2+ transient in the mice cardiomyocytes.…”

“…In our previous study, we demonstrated co-immunoprecipitation of RyR2 and the SK2 channel in the heart. 35 Interestingly, we observed that JP2 was also immunoprecipitated with an antibody against RyR2 ( Figure 1C). Take together; these results indicate the specific interaction between JP2 and SK2 in native cardiac tissue.…”

“…Reciprocal co-immunoprecipitation (co-IP) was performed as previously reported. 35 In brief, 300 lg of solubilized proteins isolated from the adult mouse heart or from HEK293 cells was incubated with anti-JP2, anti-SK2 or anti-RyR2 antibodies overnight at 4°C, and control groups (Ctrl-N) were incubated with IgG antibodies of non-mouse and non-rabbit sources, followed by an additional incubation with protein G Sepharose (Santa Cruz Biotechnology, La Jolla, CA, USA) for 6 hour at 4°C. Protein-bead complexes were washed three times with a high-salt buffer (50 mmol L À1 Tris pH 8.0, 1 mol L À1 NaF, 500 mmol L À1 NaCl, 0.5% NP-40 and complete protease inhibitor mixture), then a low-salt buffer (50 mmol L À1 Tris pH 8.0, 1 mol L À1 NaF, 250 mmol L À1 NaCl, 0.5% NP-40, and complete protease inhibitor mixture) and finally a salt-free buffer (50 mmol L À1 Tris pH 8.0, 1 mol L À1 NaF, 0.5% NP-40, and complete protease inhibitor mixture) to remove non-specific binding.…”

Ca²⁺-Activated K⁺ Channels as Therapeutic Targets for Myocardial and Vascular Protection

“…29 In parallel, Mu et al showed that RyR2-mediated SR Ca 2+ release is at least in part responsible for activation of atrial plasmalemmal SKs. 30 Colocalization of SKCa channels with voltage-dependent Ca 2+ channels points to their involvement in repolarization of the action potential. However, responsiveness of SKs to SR Ca 2+ release suggests that they can play an additional important role counteracting the depolarizing force of the Na + /Ca 2+ exchanger.…”

Small-conductance Ca2+-activated K+ channels and cardiac arrhythmias

“…The co-localization of SK and Ca 2+ channels suggests the possibility that local subsarcolemmal Ca 2+ resulting from opening of Ca 2+ channels is sufficient to activate SK channels as was demonstrated for hippocampal neurons 46 . However, two recent studies suggested that sarcoplasmic reticulum (SR) Ca 2+ release is required for the activation of cardiac SK channels 47,48 . One of these studies utilized over-expressed SK channels and the regulation of the over-expressed SK channels maybe distinct compared to native cardiomyocytes.…”