Abstract
Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.
Highlights
Ryanodine receptor type 2 (RyR2) is an intracellular Ca21 release channel that is expressed predominantly in the heart and brain and plays an essential role in many cellular processes, including muscle contraction, learning, and memory, by governing the release of Ca21 from intracellular Ca21 stores [1,2,3,4,5,6,7,8,9]
Of or mutants. [3H]ryanodine binding to RyR2 WT, E3848A, E3922A, or E3848A/E3922A mutant (E) and to RyR2 WT, H3850A, Q3925A, or T4931A mutant (F)
To assess the role of these Ca21-coordinating residues in RyR2 function, we mutated each of these residues to alanine (i.e. E3848A, E3922A, Q3925A, H3850A, and T4931A) in the mouse RyR2 and determined the Ca21-dependent [3H]ryanodine binding to each of these RyR2 mutants with a wide range of Ca21 concentrations
Summary
Recent 3D structural analyses revealed the RyR2 Ca21 activation site that is formed by residues Glu-3848, Glu-3922, Gln-. There was little or no [3H]ryanodine binding to RyR2 WT in the near absence of Ca21 (;0.1 nM) This indicates that there is little or no Ca21-independent basal activity of RyR2 WT. In the near absence of Ca21, the E3848A mutant exhibited a substantially higher level of [3H]ryanodine binding than WT (p , 0.0001) (Fig. 1, C and E). This indicates that the E3848A mutation markedly increases the Ca21-independent basal activity of RyR2. Different from WT, elevating Ca21 concentration from ;1 mM to ;10 mM decreased (rather than increased) [3H]ryanodine binding to the E3848A mutant This suggests the existence of a putative Ca21 inactivation site(s) in. At Ca21 concentrations .10 mM Ca21, [3H]ryanodine binding to the E3848A mutant increased but did not saturate even at 100
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