Dissertation committee:
Μαίρη Μαυρή-Βαβαγιάννη Αναπλ. Καθηγήτρια, (Επιβλέπουσα), Ντία Γαλανοπούλου Καθηγήτρια, Ευαγγελία Λιβανίου Ερευνήτρια Α΄ Ε.Κ.Ε.Φ.Ε. Δημόκριτος
Summary:
Calmodulin (CaM) is an important regulator (inhibitor) of the cardiac ryanodine
receptor type-2 (RyR2) function as a calcium release channel of SR, playing a
pivotal role in excitation-contraction coupling. Although the mechanism of
CaM/RyR2 interaction is not fully understood, it is widely accepted that the
main CaM binding domain is located within residues 3583–3603 of RyR2, while a
series of data suggest that additional regions in RyR2 can act as potential CaM
binding domains. On the other hand, anumber of CaM mutants, with possibly
impaired RyR2-regulatory capacity, have been recently characterized in patients
with heart dysfunction. The goal of the present thesis was to study the
mechanism of the interaction between wild type CaM (CaMWT) and RyR2 as well as
to study the interaction between certain CaM mutants and RyR2. In order to
study the CaMWT/RyR2 interaction, eight peptide fragments of RyR2 and
derivatives thereof were synthesized. Potential binding of the above synthetic
peptides to CaMWT was studied by means of pull-down, ELISA-type and isothermal
titration calorimetry experiments. Moreover, some of the RyR2-peptides were
used as antigens for the development of polyclonal antibodies, while
anti-peptide polyclonal antibodies for full-length CaMWT were also developed
and used in the ELISA-type experiments. According to the results obtained, the
C-terminus of RyR2 seems to contain an additional CaMWT binding domain
(4255-4277). For studying the interaction between CaM mutants and RyR2, six CaM
mutants were produced and heavy SR vehicles from pig hearts (as a source of
RyR2) were prepared. Consequently, the effect of the CaM mutants on the RyR2
activity was tested, in comparison with CaMWT, with the [3H]ryanodine binding
assay, while binding of the CaM mutants to RyR2 was also evaluated, in
comparison with CaMWT too, with pull-down experiments. As revealed by the
results obtained, specific CaM mutations (N54I, F90L, D96V, D130G) affect both,
binding capacity to RyR2 and the channel activity. Further exploitation of the
molecular tools and the techniques developed in the framework of this thesis
will help us better understand and elucidate the mechanisms underlying cardiac
function.
Keywords:
Ryanodine receptor type 2 (RyR2), Calmodulin (CaM), Ca2+ ions, CaM mutants, CaM binding domains in RyR2 (CaMBDs)
