Ryanodine binding sites measured in small skeletal muscle biopsies

Abstract

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 K_D of 18-27 nmol I^-1 in rat and 8 nmol I^-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 (K_0.5) was 200-400 nmol I^-1 for different muscles. [Ca²⁺] higher than 1 mmol I^-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 I^-1, more than 50% of the [³H]ryanodine was bound to particles with size less than 1.2 μn 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.

Key Words:

• EDL
• muscle biopsies
• ryanodine receptor
• Soleus