A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease

Abstract

Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals.

Figure 1

Pedigree of the Mexican family showing: CCD status, MHS status, haplotypes constructed with markers for loci D19S220 and D19S47, and the presence of the I4898T mutation. CCD-affected individuals are indicated by closed symbols and normal individuals are indicated by open symbols. The two individuals diagnosed as MHS are indicated by an asterisk next to the symbol. Diagonal bars through symbols denote deceased individuals. The haplotype segregating with the disease is indicated by the filled bar. The presence or absence of the I4898T mutation is indicated by + or −, respectively.

Figure 2

Sequence and detection of the I4898T mutation. (A) Nucleotide and deduced amino acid sequence of the RYR1 cDNA from individual III:4. The T14693C mutation changes the Ile-4898 codon, ATT, to a threonine codon, ACT. (B) Detection of the T14693C mutation by the GMPD method. The 33-nt digestion product represents the normal T14693 allele, while the 34-nt digestion product represents the mutant C14693 allele. Samples yielding only the 33-nt product are normal, while samples yielding both 33- and 34-nt products are heterozygous for the T14693C mutation.

Figure 3

Results of Ca²⁺ photometry and imaging assays and expression of the normal and I4897T mutant RYR1 cDNAs in HEK-293 cells. Traces of the responses in the Ca²⁺ photometry assay to incremental doses of caffeine in HEK-293 cells expressing: (A) the pcDNA3 vector, (B) normal RyR1, (C) I4897T mutant, (D) RyR1 plus SERCA1, (E) I4897T plus SERCA1, and (F) I4897T plus RyR1 plus SERCA1. Changes in cytosolic Ca²⁺ concentration are recorded as the ratio of fluorescence at 340/380 nm. The Ca²⁺ imaging assay demonstrates caffeine-induced Ca²⁺ release in a single HEK-293 cell expressing: (G) RyR1 plus SERCA1 or (H) I4897T plus RyR1 plus SERCA1. (I) Western blot of HEK-293 cell extracts 48 hr after transfection with pcDNA3 vector, normal RyR1, and I4897T mutant constructs.