Abstract
Despite its importance and abundance of experimental data, the molecular
mechanism of RyR2 activation by calcium is poorly understood. Recent
experimental studies involving coexpression of wild-type (WT) RyR2
together with a RyR2 mutant deficient in calcium-dependent activation
(Li, P., and S.R. Chen. 2001. J. Gen. Physiol. 118:33-44) revealed
large variations of calcium sensitivity of the RyR tetramers with
their monomer composition. Together with previous results on kinetics
of Ca activation (Zahradn�kov�, A., I. Zahradn�k, I. Gy�rke, and
S. Gy�rke. 1999. J. Gen. Physiol. 114:787-798), these data represent
benchmarks for construction and testing of RyR models that would
reproduce RyR behavior and be structurally realistic as well. Here
we present a theoretical study of the effects of RyR monomer substitution
by a calcium-insensitive mutant on the calcium dependence of RyR
activation. Three published models of tetrameric RyR channels were
used either directly or after adaptation to provide allosteric regulation.
Additionally, two alternative RyR models with Ca binding sites created
jointly by the monomers were developed. The models were modified
for description of channels composed of WT and mutant monomers. The
parameters of the models were optimized to provide the best approximation
of published experimental data. For reproducing the observed calcium
dependence of RyR tetramers containing mutant monomers (a) single,
independent Ca binding sites on each monomer were preferable to shared
binding sites; (b) allosteric models were preferable to linear models;
(c) in the WT channel, probability of opening to states containing
a Ca$^2+$-free monomer had to be extremely low; and (d) models
with fully Ca-bound closed states, additional to those of an Monod-Wyman-Changeaux
model, were preferable to models without such states. These results
provide support for the concept that RyR activation is possible (albeit
vanishingly small in WT channels) in the absence of Ca$^2+$ binding.
They also suggest further avenues toward understanding RyR gating.
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