Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle.

1. The characteristics of saxitoxin (STX) binding to the mammalian Na channel have been studied in purified sarcolemma isolated from rat skeletal muscle. 2. STX binds specifically to isolated sarcolemma with a Kd of 1.43 x 10(-9) M and Bmax of 7-8 p-mole STX bound/mg membrane protein at 0 degrees C in the presence of 140 mM-NaCl. In rat muscle homogenate under the same conditions the corresponding values are Kd = 1.53 x 10(-9) M and Bmax = 0.15-0.20 p-mole/mg protein (18-20 p-mole/g wet wt.). Membrane purification produced a fortyfold increase in STX binding site concentration per milligram protein. Calculated binding site density in isolated sarcolemma was about 30 sites/micron 2 of membrane surface. 3. Denervation (10-14 days) results in a 43% reduction in the density of high-affinity STX binding sites in purified sarcolemma, but the Kd for this class of sites is not changed. 4. In sarcolemma, the apparent Kd for STX binding is dependent on temperature pH and ionic strength. The Q10 for Kd between 0 and 40 degrees C is 1.3. Protonation of a group having a pK of 6.0 markedly raises Kd without affecting Bmax. Apparent Kd increases eightfold when ionic strength is raised from 20 to 600 mM. 5. Dissociation and association rate constants for STX binding are temperature dependent with Q10 of 2.6 and 1.9 respectively between 0 and 20 degrees C. 6. STX binding is competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ greater than Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+ is seen for the monovalent cation-binding site. 7. The STX binding site is relatively stable to heat and to enzymic degradation. A specific modifier of carboxyl residues inactivates subsequent STX binding. This process can be prevented by the presence of STX during the reaction. 8. Characteristics of the STX binding site in isolated sarcolemma are compared to those reported for other isolated excitable membranes and for studies of whole muscle and muscle homogenate. Sarcolemma provides a potential source of enriched Na channels for further purification efforts in a mammalian system.