Abstract
In cardiac muscle, intracellular Ca$^2+$ release is controlled
by a number of proteins including the ryanodine receptor (RyR2),
calsequestrin (CASQ2), triadin-1 (Trd) and junctin (Jn) which form
a complex in the junctional sarcoplasmic reticulum (SR) membrane.
Within this complex, Trd appears to link CASQ2 to RyR2 although the
functional significance of this interaction is unclear. In this study,
we explored the functional importance of Trd-CASQ2 interactions for
intracellular Ca$^2+$ handling in rat ventricular myocytes. A
peptide encompassing the homologous CASQ2 binding domain of Trd (residues
206-230 in the rat; TrdPt) was expressed in the lumen of the SR to
disrupt Trd-CASQ2 interactions. Myocytes expressing TrdPt exhibited
increased responsiveness of SR Ca$^2+$ release to activation
by ICa as manifested by flattened and broadened voltage dependency
of the amplitude of cytosolic Ca$^2+$ transients. Rhythmically
paced, TrdPt-expressing myocytes exhibited spontaneous arrhythmogenic
oscillations of intracellular Ca$^2+$ and membrane potential
that was not seen in control cells. In addition, the frequency of
spontaneous Ca$^2+$ sparks and Ca$^2+$ waves was significantly
increased in TrdPt-expressing, permeabilized myocytes. These alterations
in SR Ca$^2+$ release were accompanied by a significant decrease
in basal free intra-SRCa$^2+$ and total SR Ca$^2+$ content
in TrdPt-expressing cells. At the same time a synthetic peptide corresponding
to the CASQ2 binding domain of Trd produced no direct effects on
the activity of single RyR2 channels incorporated into lipid bilayers
while interfering with the ability of CASQ2 to inhibit the RyR2 channel.
These results suggest that CASQ2 stabilizes SR Ca$^2+$ release
by inhibiting the RyR2 channel through interaction with Trd. They
also show that intracellular Ca$^2+$ cycling in the heart relies
on coordinated interactions between proteins of the RyR2 channel
complex and that disruption of these interactions may represent a
molecular mechanism for cardiac disease.
- action
- animals;
- atpases,
- calcium
- calcium,
- calcium-transporting
- calsequestrin,
- cardiac,
- carrier
- channel,
- electrophysiology;
- male;
- metabolism
- metabolism;
- muscle
- myocytes,
- patch-clamp
- physiology;
- potentials,
- proteins,
- rats,
- rats;
- receptor
- release
- reticulum
- reticulum,
- ryanodine
- sarcoplasmic
- sprague-dawley;
- techniques;
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