RyR1-related myopathy mutations in ATP and calcium binding sites impair channel regulation

Abstract

The type 1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel on the sarcoplasmic/endoplasmic reticulum that is required for skeletal muscle contraction. RyR1 channel activity is modulated by ligands, including the activators Ca2+ and ATP. Patients with inherited mutations in RyR1 may exhibit muscle weakness as part of a heterogeneous, complex disorder known as RYR1-related myopathy (RYR1-RM) or more recently termed RYR1-related disorders (RYR1-RD). Guided by high-resolution structures of skeletal muscle RyR1, obtained using cryogenic electron microscopy, we introduced mutations into putative Ca2+ and ATP binding sites and studied the function of the resulting mutant channels. These mutations confirmed the functional significance of the Ca2+ and ATP binding sites identified by structural studies based on the effects on channel regulation. Under normal conditions, Ca2+ activates RyR1 at low concentrations (µM) and inhibits it at high concentrations (mM). Mutations in the Ca2+-binding site impaired both activating and inhibitory regulation of the channel, suggesting a single site for both high and low affinity Ca2+-dependent regulation of RyR1 function. Mutation of residues that interact with the adenine ring of ATP abrogated ATP binding to the channel, whereas mutating residues that interact with the triphosphate tail only affected the degree of activation. In addition, patients with mutations at the Ca2+ or ATP binding sites suffer from muscle weakness, therefore impaired RyR1 channel regulation by either Ca2+ or ATP may contribute to the pathophysiology of RYR1-RM in some patients.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics

“…The first mode represents the initiation of activation, where the adenine group of ATP formed a hydrogen bond with H4983 and the ribose formed a hydrogen bond with T4979 in the closed state without strong interactions with the S6c segment. Mutation T4979 results in the failure of this binding mode of ATP . After RyR1 activates, inducing a structural rearrangement in the ATP binding site, ATP switches to the second binding mode.…”

“…Mutation T4979 results in the failure of this binding mode of ATP. 56 After RyR1 activates, inducing a structural rearrangement in the ATP binding site, ATP switches to the second binding mode. In this mode, ATP maintains an open conformation, enabling adenine to switch its interaction to the hydrophobic cleft, and ribose changes the location of hydrogen bonding to M4954, preserving the direction of the S6c segment in the open state.…”

Mutations in proteins involved in E-C coupling and SOCE and congenital myopathies

“…RYR1 loss-of-function mutations result in reduction of Ca 2+ release due to either an impairment of RYR1 and DHPR functional coupling, through the so called “uncoupling” mechanism ( Avila et al, 2001 ; Avila et al, 2003 ), or a reduced channel opening resulting from alterations in sites of interaction with endogenous activators ( Yuan et al, 2021 ). The majority of uncoupling mutations are localized in the C-terminal region of the channel containing the pore domain, within the 4895 GGGIGDE 4901 amino acid sequence in humans ( Gillespie et al, 2014 ).…”

“…Indeed, the RYR1 mutation T4980M, associated with a recessive form of congenital myopathy with cores, is located within the RYR1 ATP-binding site ( Klein et al, 2011 ; Maggi et al, 2013 ). Functional studies showed that this mutation results in reduced channel activation, which may explain the muscle weakness observed in humans ( Yuan et al, 2021 ). Similarly, the Q3969K mutation, linked to a form of CCD, is located close to the Ca 2+ binding site of RYR1 and results in a reduction in Ca 2+ -dependent channel activation ( Yuan et al, 2021 ).…”

“…Functional studies showed that this mutation results in reduced channel activation, which may explain the muscle weakness observed in humans ( Yuan et al, 2021 ). Similarly, the Q3969K mutation, linked to a form of CCD, is located close to the Ca 2+ binding site of RYR1 and results in a reduction in Ca 2+ -dependent channel activation ( Yuan et al, 2021 ).…”