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

Abstract

SUMMARY1. 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 0C in the presence of 140 mM-NaCl. 3. Denervation (10-14 days) results in a 43 % reduction in the density of highaffinity STX binding sites in purified sarcolemma, but the 1Kd 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 0C is 1P3. Protonation of a group having a pK of 6*0 markedly raises Kd without affecting Bmasx. 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 'C.6. STX binding is competitively inhibited by monovalent and divalent cations under conditions of constant total ionic strength. An affinity sequence of Tl+ > Li+ > Na+ > K+ > Rb+ > 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.

Sodium Channel and Sodium Pump in Normal and Pathological Muscles from Patients with Myotonic Muscular Dystrophy and Lower Motor Neuron Impairment

“…Measurements of the amount of Na+ were carried out in a choline chloride medium as previously Fig. 4A, B (19), synaptosomes (24), and rat muscle (25).…”

“…Their number expressed per milligram of protein (Table III) The affinity of saxitoxin for its receptor was the same in innervated and denervated muscle (25,46). In contrast with the observations made after experimental denervation of rat muscle, Gruener et al (7,33) reported that TTX-resistant action potentials are not observed in pathologically nerve-impaired human muscle.…”

Purification from rat sarcolemma of the saxitoxin-binding component of the excitable membrane sodium channel.

“…for further purification by using an immobilized lectin specific for this carbohydrate. The lower limit of molecular weight for the sodium channel has been estimated to be 230,000-250,000, although an upper limit cannot yet be set (16). If this lower limit is assumed and one molecule of toxin is bound per 250,000-dalton unit, a theoretical maximal specific activity of 4000 pmol/mg of protein is expected.…”

Developmental Biology of Neoplastic Growth

“…One of these (trimethyloxonium ion) revealed that acidic amino acids were important in the binding of TTX and STX. Pretreatment with this reagent abolished the channel's ability to bind the toxin, whereas pre-exposure of the channel to the toxin protected it from protein modification (Barchi and Weigele 1979;Doyle et al 1993;Spalding 1980;Worley et al 1986). Later, after Na channel genes were sequenced and successfully expressed in recombinant systems, mutagenesis studies pinpointed a series of carboxylate sidechain-containing amino acids in homologous positions within each of the four domains of the channel involved in binding the toxin (Terlau et al 1991) and were hypothesized to form two predominantly negatively charged rings near the ion pore.…”

“…Increasing temperature decreases affinity of both STX and TTX for the toxin sensitive isoforms of the Na channel (Barchi and Weigele 1979;Bay and Strichartz 1980;Weigele and Barchi 1978). Warming 20°C can reduce affinity of the toxins 10-to 20-fold.…”