Single-channel properties of skeletal muscle ryanodine receptor pore Δ⁴⁹²³FF⁴⁹²⁴ in two brothers with a lethal form of fetal akinesia

Highlights

• De novo RYR1 c.14767_14772del p.(Phe4923_Phe4924del) pore variant in FADS.

• Homotetrameric channel has an unstable pore structure.

• Heterotetramercic channel complexes composed of WT and mutant subunits conduct Ca²⁺.

• RYR1 p.(Phe4923_Phe4924del) contributed to but was not sufficient to cause FADS.

Abstract

Ryanodine receptor ion channels (RyR1s) release Ca²⁺ ions from the sarcoplasmic reticulum to regulate skeletal muscle contraction. By whole-exome sequencing, we identified the heterozygous RYR1 variant c.14767_14772del resulting in the in-frame deletion p.(Phe4923_Phe4924del) in two brothers with a lethal form of the fetal akinesia deformation syndrome (FADS). The two deleted phenylalanines (RyR1-Δ⁴⁹²³FF⁴⁹²⁴) are located in the S6 pore-lining helix of RyR1. Clinical features in one of the two siblings included severe hypotonia, thin ribs, swallowing inability, and respiratory insufficiency that caused early death. Functional consequences of the RyR1-Δ⁴⁹²³FF⁴⁹²⁴ variant were determined using recombinant 2,200-kDa homotetrameric and heterotetrameric RyR1 channel complexes that were expressed in HEK293 cells and characterized by cellular, electrophysiological, and computational methods. Cellular Ca²⁺ release in response to caffeine indicated that the homotetrameric variant formed caffeine-sensitive Ca²⁺ conducting channels in HEK293 cells. In contrast, the homotetrameric channel complex was not activated by Ca²⁺ and did not conduct Ca²⁺ based on single-channel measurements. The computational analysis suggested decreased protein stability and loss of salt bridge interactions between RyR1-R4944 and RyR1-D4938, increasing the electrostatic interaction energy of Ca²⁺ in a region 20 Å from the mutant site. Co-expression of wild-type and mutant RyR1s resulted in Ca²⁺-dependent channel activities that displayed intermediate Ca²⁺ conductances and suggested maintenance of a reduced Ca²⁺ release in the two patients. Our findings reveal that the RYR1 pore variant p.(Phe4923_Phe4924del) attenuates the flow of Ca²⁺ through heterotetrameric channels, but alone was not sufficient to cause FADS, indicating additional genetic factors to be involved.

Introduction

Skeletal muscle type 1 ryanodine receptor ion channels (RyR1s) rapidly release Ca²⁺ from the sarcoplasmic reticulum (SR) into the myoplasm to initiate muscle contraction. The 2200 kDa RyR1s are comprised of four RyR1 subunits of ∼5000 residues and four FK506 binding proteins (FKBP) of ∼110 residues [[1], [2], [3]]. RyR1 is controlled in skeletal muscle by Ca_v1.1 voltage-gated channel and Ca²⁺ by a not well-understood mechanism. Studies with membrane isolates and purified preparations show that micromolar Ca²⁺ activates and millimolar Ca²⁺ inhibits RyR1. Exogenous ligands include the plant alkaloid ryanodine and caffeine. Ryanodine modifies the gating and ion conductance properties by binding with nanomolar affinity and high specificity to the open RyRs. Caffeine activates the RyRs at millimolar concentrations without altering their ion conductance properties.

A large number of RYR1 variants have been linked to autosomal dominantly and recessively inherited skeletal muscle myopathies, including central core disease (CCD), multi minicore disease, core-rod myopathy, and congenital neuromuscular disease [[4], [5], [6]]. Core diseases result in weakening of skeletal muscle function and manifest as cores that lack mitochondria and oxidative enzymes [7]. Dominantly acting CCD-associated RYR1 variants tend to be located in the pore region [[8], [9], [10], [11]], whereas recessive variants widely spread throughout the RYR1 gene [[4], [5], [6]]. The spectrum of ryanodinopathies was broadened by the identification of biallelic RYR1 variants associated with fetal akinesia deformation sequence (FADS)/arthrogryposis multiplex congenita and lethal multiple pterygium syndrome (LMPS) [7,[12], [13], [14], [15], [16], [17], [18]]. These recessive RYR1 variants likely cause complete protein loss or loss-of-function of the encoded protein, which can lead to lethal outcomes.

We performed whole-exome sequencing and identified a heterozygous deletion of 6 bp (c.14767_14772del), resulting in the in-frame deletion of two phenylalanines [p.(Phe4923_Phe4924del)] in the carboxyl-terminal pore region of RyR1 in two brothers with FADS. The index patient, patient 2, died at the age of 10 weeks, while the first affected child, patient 1, died on the first day of life. The two affected siblings inherited the heterozygous RYR1 variant from their healthy father, who is a somatic mosaic. No second variant on the maternal RYR1 allele was identified that could explain the severe, autosomal recessive FADS in the two affected boys. The deletion of the two phenylalanines was arbitrarily assigned to the most 3′ position, according to the sequence variant nomenclature [19], and deletes the third and fourth phenylalanine (RYR1-Δ⁴⁹²³FF⁴⁹²⁴) in a stretch of four successive phenylalanines located in the human S6 pore-lining helix of the RyR1 C-terminus.

To determine the functional effects of the pore deletion mutant, HEK293 cells were transfected with rabbit wild-type RyR1 (RyR1-WT) and RyR1-Δ⁴⁹²²FF⁴⁹²³ (which is analogous to Δ⁴⁹²³FF⁴⁹²⁴ in human RYR1) expression vectors. Homotetrameric and heterotetrameric channel complexes were characterized by cellular Ca²⁺ release measurements, single-channel recordings, and computational methods. The results indicated that homotetrameric RyR1-Δ⁴⁹²²FF⁴⁹²³ channels were not activated by Ca²⁺ and did not conduct Ca²⁺ in single-channel measurements, whereas heterotetrameric channels composed of wild-type and mutant subunits maintained a Ca²⁺-dependent channel activity but exhibited reduced Ca²⁺ conductances compared to wild type. The findings suggest that the RYR1 pore variant p.(Phe4923_Phe4924del) contributed to but alone was not sufficient to cause the severe FADS in the two brothers.