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

Abstract

The characteristics of saxitoxin (STX) binding to the mammalian Na channel were studied in purified sarcolemma isolated from rat skeletal muscle. STX bound specifically to isolated sarcolemma with a Kd of 1.43 .times. 10-9 M and Bmax [maximum number of binding sites] of 7-8 pmol STX bound/mg membrane protein at 0.degree. C in the presence of 140 mM-NaCl. In rat muscle homogenate under the same conditions the corresponding values were Kd = 1.53 .times. 10-9 M and Bmax = 0.15-0.20 pmol/mg protein (18-20 pmol/g wet wt). Membrane purification produced a 40-fold increase in STX binding site concentration/mg protein. Calculated binding site density in isolated sarcolemma was .apprx. 30 sites/.mu.m2 of membrane surface. Denervation (10-14 days) caused a 43% reduction in the density of high-affinity STX binding sites in purified sarcolemma, but the Kd for this class of sites was not changed. In sarcolemma, the apparent Kd for STX binding was dependent on temperature, pH and ionic strength. The Q10 for Kd between 0 and 40.degree. C was 1.3. Protonation of a group having a pK of 6.0 markedly raised Kd without affecting Bmax. Apparent Kd increased 8-fold when ionic strength was raised from 20-600 mM. Kd and association rate constants for STX binding were temperature dependent with Q10 of 2.6 and 1.9, respectively, between 0 and 20.degree. C. STX binding was competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ > Li+ > Na+ > K+ > Rb+ > Cs+ was seen for the monovalent cation-binding site. The STX binding site was relatively stable to heat and to enzymic degradation. A specific modifier of carboxyl residues inactivated subsequent STX binding. This process can be prevented by the presence of STX during the reaction. Characteristics of the STX binding site in isolated sarcolemma were 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.