A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation

Abstract

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca²⁺-mediated activation of skeletal muscle Ca²⁺ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca²⁺ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca²⁺ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca²⁺ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca²⁺-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca²⁺-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca²⁺ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca²⁺ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca²⁺ of the two mutants, whereas the interaction of Ca²⁺ with directly interacting RyR1 amino acid residues Glu³⁸⁹³, Glu³⁹⁶⁷, and Thr⁵⁰⁰¹ was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca²⁺-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

Keywords: central core disease; homology modeling; ryanodine receptor; sarcoplasmic reticulum; single-channel recording.

Fig. 1.

Location of Ca²⁺-, ATP-, and caffeine-binding sites of open type 1 ryanodine receptor-wild type (RyR1-WT; PDB code 5TAL). Protein structure is shown as a transparent surface. Ca²⁺-, ATP-, and caffeine-binding sites are shown in green, red, and blue, respectively. Inset: structure of Ca²⁺-binding site of open RyR1-Q3970K mutant in the presence of Ca²⁺, ATP, and caffeine. [Modified from Xu et al. (31) with permission.]

Fig. 2.

Immunoblot and caffeine-induced Ca²⁺ release by HEK293 cells expressing wild-type (WT) and mutant type 1 ryanodine receptors (RyR1s). A: immunoblots of HEK293 cells transfected with pCMV5 vector and RyR1-WT expression vector (left), and 565 kDa RyR1-Q3970E, RyR1-Q3970K, and RyR1-WT (right). The blots identified the RyR1 protein band by its absence from the pCMV5 vector-transfected sample (left). B: Ca²⁺ transients in HEK293 cells expressing RyR1-WT (top), RyR1-Q3970E (middle), and RyR1-Q3970K (bottom) as changes of Fluo-4 fluorescence before and following the addition of 8 mM caffeine (arrow) to the bath solution. AU, arbitrary units.

Fig. 3.

Effects of cytosolic Ca²⁺ on type 1 ryanodine receptor-wild type (RyR1-WT) and mutant channel open probability (P_o). A–C: representative single-channel currents of RyR1-WT (A), RyR1-Q3970E (B), and RyR1-Q3970K (C) were recorded as downward deflections from the closed state (c–) in 250 mM symmetrical KCl at −20 mV with 2 µM sarcoplasmic reticulum (SR) luminal Ca²⁺ and the indicated cytosolic Ca²⁺ concentrations. D: Ca²⁺ dependence of RyR1-WT, RyR1-Q3970E, and RyR1-Q3970K channel open probabilities. Data are the mean ± SE of 3–21 recordings. Inset: open probabilities of WT and Q3970K in a logarithmic scale. [WT data in D were obtained from Xu et al. (31) under recording conditions used in the present report.]

Fig. 4.

Effects of caffeine and ATP on type 1 ryanodine receptor- wild type (RyR1-WT) and mutant channel open probabilities (P_o). A–C: representative single-channel currents of RyR1-WT (A), RyR1-Q3970E (B), and RyR1-Q3970K (C) were recorded as downward deflections from the closed state (C–) in 250 mM symmetrical KCl at −20 mV. Luminal Ca²⁺ was 2 µM. Cytosolic solutions contained 30 µM Ca²⁺ (top traces), 30 µM Ca²⁺ and 5 mM caffeine (middle traces), and 30 µM Ca²⁺, 5 mM caffeine, and 2 mM ATP (bottom traces). Free Ca²⁺ was 5 µM in the presence of 5 mM caffeine and 2 mM ATP as described under materials and methods. D: channel open probability in the presence of 30 µM cytoplasmic Ca²⁺ (open symbols), 30 µM Ca²⁺ and 5 mM caffeine (shaded symbols), and 5 µM Ca²⁺ plus 5 mM caffeine and 2 mM ATP (closed symbols). Data are the mean ± SE of 3–6 recordings. *P < 0.05 compared with respective controls by one-way ANOVA followed by Tukey’s test. [WT data in D were obtained from Xu et al. (31) under recording conditions used in the present report.]

Fig. 5.

Interactions of Ca²⁺ with wild type (WT) and mutant type 1 ryanodine receptors (RyR1s). Shown are predicted interactions of Ca²⁺ with WT and mutant RyR1s in the presence of Ca²⁺, ATP, and caffeine. Residues displaying electrostatic interactions with Ca²⁺ in the Ca²⁺/ATP/caffeine closed (5TAQ) and open (5TAL) states are depicted in stick representation. Backbone of RyR1 is shown as a ribbon. Ca²⁺ is shown as a green sphere. Strong electrostatic interactions (distance <3.2 Å) are shown as blue dashed lines and weak electrostatic interactions (distance <4 Å) are shown as red dashed lines between Ca²⁺ and RyR1 residues. Attractive and repulsive amino acid interactions (distance <3.5 Å) are depicted as blue and green dashed lines, respectively.