Expression and functional analysis of murine ryanodine receptor type 3.
Ryanodine receptors (RyRs) are intracellular homotetrameric Ca2+-release
channels constituting a family of three different isoforms, named RyRl, RyR2
and RyR3. RyRl and RyR2 are highly expressed in skeletal and cardiac
muscles respectively, where they localize in the terminal cisternae of the
sarcoplasmic reticulum (SR). Although RyRl and RyR2 have been found to be
expressed in several other tissues at much lower level than in striated muscles,
their major functional role is related to Ca2+-release from the SR following
electrical depolarization of the plasma membrane, a process referred to as
excitation-contraction (e-c) coupling and known to regulate striated muscle
The third isoform, RyR3, is characterized by a wide pattern of expression,
without any specific association to a tissue or a cell-type. The finding that
RyR3 is also expressed in mammalian skeletal muscles parallels the presence of
two distinct isoforms, o- and P-RyR, in non-mammalian vertebrate skeletal
muscles, and suggests that two functionally distinct RyRs may be involved in
the regulation of skeletal muscle contraction.
The expression of RyR3 was analyzed in murine skeletal muscle from late
foetal stages to adult, throughout neonatal phases of development. RyR3 was
expressed widely during skeletal muscle post-natal development, disappearing
in all muscles analyzed except diaphragm and soleus.
RyR3 knockout mice were generated, and contractile properties of skeletal
muscles were analyzed. Skeletal muscle contraction in RyR3-/- mice was
impaired during the neonatal phase of development. In skeletal muscles isolated
from RyR3-1- mice, the twitch elicited by electrical stimulation was strongly
depressed. A significant reduction of the contractile activity was also elicited
after stimulation with caffeine, an activator of Ca2+-release through RyRs. In the
adults, no differences were detected between wild-type and mutant mice.
These results are the first demonstrations of a physiological role of RyR3 in
excitation-contraction coupling mechanisms of skeletal muscle, and support the
model of a two-channel system regulating skeletal muscle contraction.
In order to further characterize the RyR3-1- mouse, [3H]ryanodine binding
experiments were performed on diaphragm and total hindlimb skeletal muscles
from RyR3+/+ and RyR3-1- mice. Preliminary results will be presented and