Ryanodine binding sites measured in small skeletal muscle biopsies.
A method allowing measurement of the concentration of [3H]ryanodine binding sites in small skeletal muscle specimens (> 10-20 mg) was developed. A membrane fraction containing 87% of the [3H]ryanodine binding sites of the tissue and exhibiting one single KD of 18-27 nmol l-1 in rat and 8 nmol l-1 in human muscles (p < 0.05) was obtained. Maximum binding to rat EDL and soleus muscles equalled 59.1 and 16.2 pmol g-1 wet wt, whereas in human gluteus muscles binding was 12.3 pmol g-1 wet wt. The [3H]ryanodine binding showed a dependency on Mg2+ and pH similar to previously published results. As measured by Ca2+ selective mini-electrodes, the [Ca2+] causing 50% of maximum [3H]ryanodine binding (K0.5) was 200-400 nmol l-1 for different muscles. [Ca2+] higher than 1 mmol l-1 caused strong inhibition of the [3H]ryanodine binding, and both high and low [Ca2+] caused rapid dissociation of the complex. At ionic strength lower than 100 mmol l-1, more than 50% of the [3H]ryanodine was bound to particles with size less than 1.2 microns which were not retained by GF/C filters. Thus, we have obtained an almost complete quantitative recovery of functional RyRs from small muscle specimens exhibiting high affinity for Ca2+, which stimulated ligand binding.
Lunde
P K
PK
University of Oslo, Ullevaal Hospital, Norway.
Sejersted
O M
OM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D001707:Biopsy, Needle; D002118:Calcium / Q000494:pharmacology; D002793:Cholic Acids; D015925:Cryopreservation; D003902:Detergents; D004533:Egtazic Acid / Q000031:analogs & derivatives; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008274:Magnesium / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000473:pathology; D017830:Octoxynol; D011060:Poloxalene; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis* / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000737:chemistry; D013997:Time Factors; D014316:Tritium
D002793:Cholic Acids; D003902:Detergents; D007202:Indicators and Reagents; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D004533:Egtazic Acid; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D017830:Octoxynol; D011060:Poloxalene; D008274:Magnesium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium