Molecular cloning of a putative tetrodotoxin-resistant rat heart Na+ channel isoform.

Abstract

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...

Gene Duplications and Evolution of Vertebrate Voltage-Gated Sodium Channels

“…Both scn5Laa and scn5Lab were also expressed in nervous and cardiac tissues. In mammals, SCN5A is expressed in cardiac muscle and limited regions of the brain (Hartmann et al 1999;Rogart et al 1989), while SCN10A and SCN11A are expressed in peripheral nociceptive sensory neurons (Akopian et al 1996;Dib-Hajj et al 1999a;Sangameswaran et al 1996). However, mammalian expression of SCN5A, SCN10A or SCN11A has not been reported in spinal cord or eye.…”

The newborn rabbit sino‐atrial node expresses a neuronal type I‐like Na+ channel.

“…mRNA encoding type I was the most abundant, and this mRNA is also represented in cardiac cDNA libraries (Rogart, Cribbs, Muglia, Kephart & Kaiser, 1989). In these studies, the specific cell type(s) within the heart tissue responsible for the results was not determined.…”

Deduced amino acid sequence of a putative sodium channel from the scyphozoan jellyfish Cyanea capillata.

“…The high degree of sequence similarity between the putative jellyfish channel (CYNA1) and other Na+ channels is evident in Fig. 1, where CYNAl has been aligned with the eel Na+ channel (15), the mammalian TTX-resistant cardiac Na+ channel (16), and the rat brain II Na+ channel (17). The degree of similarity is greatest in the transmembrane segments and less so in the large cytoplasmic loops that connect individual domains.…”

“…The program PAUP (12) was used to find the most parsimonious tree for the relationship between the different domains of CYNAl and between CYNAl and a variety of Na+ channel and Ca2+ channel amino acid sequences (Fig. 4) (19), rat brain 11 (17), and cardiac muscle (16) of CYNAl shows that domains II and IV are more closely related to one another than they are to domains I and III (data not shown). This confirms the model of gene duplication recently proposed (1) and indicates that the two domains created by the initial round of gene duplication of the single domain precursor must have undergone considerable divergence prior to the second round of gene duplication that ultimately created domains III and IV.…”