Expression and functional analysis of murine ryanodine receptor type 3

Bertocchini, Federica (2000). Expression and functional analysis of murine ryanodine receptor type 3. PhD thesis The Open University.



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 contraction.

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 discussed.

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