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.
Highlights
Small-conductance Ca2+-activated K+ (SK or KCa2) channels are a subfamily of Ca2+-activated K+ channels (KCa) observed in neuronal and non-neuronal tissues [1,2,3]
We investigated whether the SK2 channel and ryanodine receptors (RyRs) functionally interact in cardiac myocytes
The current density-voltage relations are summarized in Fig. 1B, which shows a significant decrease in the current density in the presence of a ryanodine receptor type 2 (RyR2) inhibitor with 20 mM ryanodine (Alexis, n = 8 cells)
Summary
Small-conductance Ca2+-activated K+ (SK or KCa2) channels are a subfamily of Ca2+-activated K+ channels (KCa) observed in neuronal and non-neuronal tissues [1,2,3]. The selective knockout of SK2 channels in the mouse revealed multifaceted functions of this channel in cardiac myocytes [6,7]. These channels link the intracellular calcium concentration to a wide variety of cellular processes [1,9]. The calcium sensitivity of SK channels depends on calmodulin, which is constitutively bound to the C-terminal domain of the channel. The binding of calcium to calmodulin results in a conformational change of the channel, which leads to the opening of the channel pore [10,11]. Intracellular Ca2+ ions are derived from the influx of Ca2+ into the cell through voltage-dependent Ca2+ channels (VDCCs) and by the release of Ca2+ from internal Ca2+ stores [12]
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