Abstract
Triadin 1 (TRD) is an integral membrane protein that associates with
the ryanodine receptor (RyR2), calsequestrin (CASQ2) and junctin
to form a macromolecular Ca signaling complex in the cardiac junctional
sarcoplasmic reticulum (SR). To define the functional role of TRD,
we examined the effects of adenoviral-mediated overexpression of
the wild-type protein (TRD(WT)) or a TRD mutant lacking the putative
CASQ2 interaction domain residues 200 to 224 (TRD(Del.200-224)) on
intracellular Ca signaling in adult rat ventricular myocytes. Overexpression
of TRD(WT) reduced the amplitude of I(Ca)- induced Ca transients
(at 0 mV) but voltage dependency of the Ca transients was markedly
widened and flattened, such that even small I(Ca) at low and high
depolarizations triggered maximal Ca transients. The frequency of
spontaneous Ca sparks was significantly increased in TRD(WT) myocytes,
whereas the amplitude of individual sparks was reduced. Consistent
with these changes in Ca release signals, SR Ca content was decreased
in TRD(WT) myocytes. Periodic electrical stimulation of TRD(WT) myocytes
resulted in irregular, spontaneous Ca transients and arrhythmic oscillations
of the membrane potential. Expression of TRD(Del.200-224) failed
to produce any of the effects of the wild-type protein. The lipid
bilayer technique was used to record the activity of single RyR2
channels using microsome samples obtained from control, TRD(WT) and
TRD(Del.200-224) myocytes. Elevation of TRD(WT) levels increased
the open probability of RyR2 channels, whereas expression of the
mutant protein did not affect RyR2 activity. We conclude that TRD
enhances cardiac excitation-contraction coupling by directly stimulating
the RyR2. Interaction of TRD with RyR2 may involve amino acids 200
to 224 in C-terminal domain of TRD.
- adenoviridae,
- animals;
- arrhythmias,
- bilayers;
- biosynthesis/chemistry/genetics/physiology;
- calcium
- calcium,
- calcium-binding
- cardiac,
- cardiovascular;
- carrier
- channel
- channel,
- chemistry/physiology;
- contraction,
- dogs;
- electric
- expression;
- function
- fusion
- gating,
- gene
- genetic
- genetics/physiopathology;
- genetics;
- ion
- lipid
- macromolecular
- male;
- membrane
- metabolism;
- microsomes,
- mixed
- models,
- muscle
- myocardial
- myocytes,
- oxygenases,
- physiology/ultrastructure;
- physiology;
- potentials;
- protein
- proteins,
- rats,
- rats;
- receptor
- recombinant
- release
- reticulum,
- ryanodine
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