Abstract
Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-β-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.
Highlights
Functional coupling between large-conductance Ca2؉-activated K؉ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues
It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2؉ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2؉ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SM cells (SMCs)
The frequency and area at which Ca2ϩ sparks spread (FWHM) were significantly increased, and the decay (t1⁄2 and FDHM) was prolonged by specific for JP2 (siJP2) (Fig. S5D). These results are consistent with previous findings that demonstrated enhanced RyR activity and reduced Naϩ/Ca2ϩ-exchanger activity in JP2 knockdown cardiac myocytes [31,32,33]. These results suggest that the knockdown of JP2 attenuates proximal coupling between BKCa channels and RyR, thereby decreasing the efficiency with which localized Ca2ϩ spark signals are converted to cellular spontaneous transient outward currents (STOCs) signals, i.e. Ca2ϩ derived from Ca2ϩ sparks did not sufficiently reach nearby BKCa channels
Summary
Functional coupling between large-conductance Ca2؉-activated K؉ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. The JP1 and JP2 isoforms have obligatory roles in the junctional membrane complexes (JMCs) formed under the transverse- (T-) tubular system in striated muscle cells They enable skeletal and cardiac myocytes to translate conformational changes in voltage-gated Ca2ϩ channels (VDCCs) in PM and Ca2ϩ influx through VDCC, respectively, to a marked elevation as a Ca2ϩ microdomain via highly effective Ca2ϩ release from SR through ryanodine receptors (RyR) during excitation-contraction (E-C) coupling (4 –6).
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