Dyspedic Mouse Skeletal Muscle Expresses Major Elements of the Triadic Junction but Lacks Detectable Ryanodine Receptor Protein and Function

doi:10.1074/jbc.272.11.7360

Dyspedic Mouse Skeletal Muscle Expresses Major Elements of the Triadic Junction but Lacks Detectable Ryanodine Receptor Protein and Function*

From the ^‡ Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616 the
^§Department of Cardiology, Children's Hospital, Boston, Massachusetts 02115, and
** Department of Anesthesia, Brigham and Women's Hospital, Boston, Massachusetts 02115

^∥ To whom correspondence should be addressed. Tel.: 916-752-6696; Fax: 916-752-4698.

Abstract

The ry1⁵³ dyspedic mouse contains two disrupted alleles for ryanodine receptor type 1 (skeletal isoform of ryanodine receptor; Ry₁R) resulting in perinatal death. In the present study, whole skeletal muscle homogenates and sucrose gradient-purified junctional sarcoplasmic reticulum from neonatal wild-type and dyspedic mice were assayed for biochemical and functional markers. Equilibrium binding experiments performed with 1-120 nM [³H]ryanodine reveal saturable high and low affinity binding to membrane preparations from wild-type mice, but not to preparations from dyspedic mice. Binding experiments performed with [³H]PN200 show a 2-fold reduction in [³H]PN200 binding capacity in dyspedic muscle, compared to age-matched wild-type muscle, with no change in receptor affinity. The presence or absence of proteins known to be critical for normal ryanodine receptor/Ca²⁺ channel complex function was assessed by Western blot analysis. Results indicate that FKBP-12, DHPRα1, triadin, calsequestrin, SERCA1 (sarco(endo)plasmic reticulum Ca²⁺ ATPase), and skeletal muscle myosin heavy chain are present in both dyspedic and wild-type muscle. Only wild-type membranes showed immunoreactivity toward Ry₁R antibody. Neither dyspedic nor wild-type mouse muscle showed detectable immunoreactivity toward Ry₂R or Ry₃R antibodies, even after sucrose gradient purification of sarcoplasmic reticulum. These results indicate that proteins critical for ryanodine receptor function are expressed in dyspedic skeletal muscle in the absence of Ry₁R. Ca²⁺ transport measurements show that membranes from wild-type controls, but not dyspedic mice, release Ca²⁺ upon exposure to ryanodine. Dyspedic mice and cells derived from them serve as excellent homologous expression systems in which to study how Ry₁R structure relates to function.

Footnotes

↵^¶ Supported by the Boston Heart Foundation.
↵* This work was supported in part by grants from the Muscular Dystrophy Association and the Brigham and Women's Hospital Anesthesia Foundation (to P. D. A.), the American Heart Association, California Affiliate (to I. N. P. and E. D. B.), and United States Public Health Service Grants 1RO1-AR43140 (to P. D. A. and I. N. P.) and 1RO1-E505002 (to I. N. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Ry₁R

ryanodine receptor, skeletal isoform

Ry₂R

ryanodine receptor, cardiac isoform

Ry₃R

ryanodine receptor, brain isoform

DHP

dihydropyridine

DHPR

dihydropyridine receptor

E-C

excitation-contraction

ECL

enhanced chemiluminescence

PVDF

polyvinylidene difluoride

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

SR

sarcoplasmic reticulum

t-tubule

transverse tubule

MOPS

4-morpholinepropanesulfonic acid.
↵² H. T. Nguyen, E. D. Buck, S. Mukherjee, S. Schieferl, P. C. Dolber, J. R. Sommer, I. N. Pessah, and P. D. Allen, submitted for publication.
↵³ C. Franzini-Armstrong, personal communication.
- Received July 23, 1996.
- Revision received November 18, 1996.