Distinct Properties of CPVT Mutations Located in the Central Domain of Human RyR2

Abstract

The cardiac ryanodine receptor (RyR2) Ca2+ release channel plays a central role in the rapid calcium release from the sarcoplasmic reticulum that is essential for muscle excitation-contraction coupling. Discrete mutations that have been discovered within the RyR2 associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) are found to cluster in distinct regions of RyR2. These regions may represent important regulatory domains of the molecule that either directly or indirectly can affect channel gating. In response to a physiological trigger, RyR2 mutations are believed to cause diastolic Ca2+ leak which results in arrhythmia, making them an important potential therapeutic target.We have expressed the RyR2 central domain CPVT-associated region and examined parameters which contribute to the structural and functional stability. A wild-type construct was compared with three constructs each containing a different CPVT mutation (P2328S, N2368I or A2387P). Circular dichroism spectroscopy revealed that none of the mutations significantly altered the percentage ellipticity of the protein, indicating that the overall conformation of the polypeptide backbone is only slightly affected by the mutations. Chemical denaturation using guanidine hydrochloride and monitoring tryptophan fluorescence suggested that the P2328S mutation was less stable than the wild-type or the other two mutations, indicated by a lower free energy change upon unfolding. A predicted ATP binding motif has previously been proposed in this domain and using fluorescence spectroscopy for the wild-type construct, ATP binding was observed with a Kd ∼0.03mM.