The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.

1. Voltage‐clamp studies were carried out on single rabbit myelinated nerve fibres at 14 degrees C with the method of Dodge & Frankenhaeuser (1958). 2. A method was developed to allow the ionic currents through the modal membrane to be calibrated exactly under voltage‐clamp conditions by measuring the resistance of the internode through which the current was injected. 3. The ionic currents in a rabbit node of Ranvier can be resolved into two components, a sodium current and a leak current. Potassium current is almost entirely absent. 4. The sodium currents in rabbit nodes were fitted by the Hodgkin‐Huxley model using m2h kinetics. The kinetics of sodium currents in a rabbit node differ from that in a frog node under similar experimental conditions in two respects: (a) inactivation is faster, tau h for rabbit being 2‐3 times smaller around ‐50 mV; (b) the P(Na) (E) curve for mammal is shifted 10‐15 mV in the hyperpolarizing direction. 5. From the kinetics of sodium current, the non‐propagating rabbit action potential was reconstructed at 14 degrees C. The transient inward sodium current is responsible for the fast initial depolarization phase of the action potential, while the repolarizing phase is accounted for by leak alone. The computed shape of the action potential was in good agreement with the experimentally obtained action potential. 6. At 14 degrees C, frog and rabbit nodes with similar diameters have similar measured gNa values.

Voltage-gated Na' channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na' channels are blocked by 1-10 IAM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity PH]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire a subunit of a cardiacspecific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles.Voltage-gated Na+ channels are transmembrane proteins that mediate the early increase in Na+ flux underlying the initial depolarization of the action potential in many excitable cells (1). It is now clear that mammalian Na+ channels are encoded by a multigene family. Isolation of separate cDNA clones has led to the identification of three structurally distinct Na+ channel isoforms in rat brain (2,3,16) and at least one other distinct isoform in rat skeletal muscle (4). The relationship of cardiac Na+ channels to other members of this multigene family is still uncertain.Na+ channels in mammalian cardiac membrane have recently been shown to have functional properties quite distinct from Na+ channels in nerve and skeletal muscle. The most widely known difference is that tetrodotoxin-sensitive (TTX-S) Na+ channels in nerve and skeletal muscle are blocked by nanomolar concentrations of the neurotoxins, saxitoxin (STX), and tetrodotoxin (TTX), whereas TTX-resistant (TTX-R) Na+ channels in cardiac membrane require 1-10 ,uM TTX to block them (5). Cardiac Na+ channels are also 1000 times more sensitive to inhibition by the antiarrhythmic agent lidocaine (6), which is significant clinically in that most lethal arrhythmias involve cardiac Na+ channels and cause 15-20% of U.S. deaths (7). Differences between Na+ channels in heart and other excitable tissues are relevant for a molecular understanding ofthe basic structure of these channels and the mode of action of antiarrhythmic drugs.Knowing that an alternative form of Na+ channel is expressed in heart, we undertook this study to determine the molecular basis by which this cardiac isoform arises. We show here that in newborn rat heart, where only low-affinity...

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Density of sodium channels in mammalian myelinated nerve fibers and nature of the axonal membrane under the myelin sheath Communicated by Alfred Gilman, October 26, 1976 ABSTRACT The density of sodium channels in mammalian myelinated fibers has been estimated from measurements of the binding of [3Hjsaxitoxin to rabbit sciatic nerve. Binding both to intact and to homogenized nerve consists of a linear, nonspecific, component and a saturable component that represents binding to the sodium channel. The maxim um saturable binding capacity in intact nerve is 19.9 ± 1.9 fmolPmg wetl; the equilibrium dissociation constant, K,, is 3.4 + 2.0 nM. Homogenization makes little difference, the maximum binding capacity being 19.9 i 1.5 fmol-mg wet-l with Kt = 1.3 + 0.7 nM. These values correspond to a density of about 700,000 sodium channels per node-i.e., about 12,000 per tm2 of nodal membrane. From