Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major

Isaac N Pessah, Mary J. Schiedt

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Heavy sacroplasmic reticulum (SR membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystophic PM (0.80 vs 0.96% mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4 and 5.5 weeks ex ovo. [3H]ryanodine binding to normal SR from PM 2 days ex novo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax - 17 pmol /mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respetively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from distrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340 000 and 320 000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffein and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM. Since the [3H]ryanodine receptor is a specific marker for the SR Ca2+ release channel at the muscle triad and a key component of excitation-contraction coupling, abnormal expression of [3H]ryanodine receptors may be of a fundamental importance to the etiology of muscular dystrophy in the chicken.

Original languageEnglish (US)
Pages (from-to)98-106
Number of pages9
JournalBBA - Biomembranes
Volume1023
Issue number1
DOIs
StatePublished - Mar 30 1990

Keywords

  • Muscle development
  • Muscular dystrophy
  • Ryanodine receptor
  • Sarcoplasmic reticuluml
  • Skeletal muscle

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

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