The voltage-sensitive sodium channel is responsible for the increase in sodium conductance resulting from depolarization of cell membranes during the action potential in nerve, skeletal muscle, cardiac muscle, and neuroendocrine cells (Catterall, 1992). Sodium-based action potentials in different excitable tissues are very similar (Hille, 1992). Sodium channels that have been purified from many tissues show extensive homology (Trimmer & Agnew, 1989; Patlak, 1991), which is probably the basis of this functional similarity .
Molecular studies of eel (Electricus electrophorus) sodium channels revealed the first primary structure of a voltage-sensitive ion channel (Noda
et al. 1984). Since then, a multigene family has been identified in different tissues and species (Mandel, 1992).
The voltage-sensitive sodium channel exists either as a single protein or as a complex of proteins (Trimmer & Agnew, 1989). Irrespective of the source, all of the channels contain a large a subunit of approximately 260 kDa. The basic primary structure of the alpha subunit in the human heart shows a large polypeptide of 2016 amino acids with calculated molecular weight of 227,159 daltons. It contains four repeated domains (I -IV ) of 226 to 288 amino acids (Gellens et al. 1992). According to the single pore hypothesis (Hille, 1992), each domain is thought to form part of the wall around a central pore. Each domain contains 6 hydrophobic segments (Sl- S6) with the potential for formation of transmembrane a helices including a
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positively charged amphipathic segment (S4), which is comparable to other sodium channels (Catterall, 1986). Sodium channels in mammalian brain and skeletal muscle, in addition to the alpha subunit, contain one or two smaller beta subunits of 37 and 39 kDa (Catterall, 1986; Barchi, 1988). The primary structures of ßl subunit have been determined recently (Isom et al. 1992). Although the function of ß subunits is not clear yet, it may be anticipated that they contribute to the diversity of sodium channel structure and function (Catterall, 1992). It is seems however, that the cardiac sodium channel a-subunit alone can express the pharmacological and kinetic properties of native cardiac sodium channels (Satin et al. 1992a).
1.4.2. Structures responsible for activation and inactivation
The change in sodium conductance is biphasic, since it is controlled by two processes: activation, which increases the conductance in response to depolarization and inactivation, which subsequently decreases the conductance to the resting level even if depolarization is maintained.
Mutagenesis experiments provided direct evidence about the origin of voltage sensitivity. The S4 segments in sodium channels are thought to be the voltage sensors responsible for voltage-dependence of activation (Kra-Oz
et al. 1992). The S4 segment is one of the putative membrane-spanning
segments and it has a striking pattern of positive charged amino acids every third residue (Patlak, 1991). This structure is also typical of all voltage gated cation channels (Tanabe et al. 1987; Papazian et al. 1987). Neutralization of the one to three positively charged amino acid residues in the S4 segment in domain I of sodium channel a-subunit causes a progressive reduction in the steepness of the voltage-dependent activation of sodium channels as expected if these positive charged amino acid residues serve as gating charges (Stuhmer et al. 1989). In addition, mutation of a hydrophobic residue in the S4 segment in domain II from leucine to
phenylalanine causes a 20 mV shift in the voltage dependence of gating to more positive membrane potentials (Auld et al. 1990).
The primary structure responsible for inactivation has been identified in the short intracellular loop connecting domains III and IV by application of site specific antibodies in combination with voltage patch clamp techniques. In both whole cell and inside out patches, an antibody directed against this short intracellular segment inhibited sodium channel inactivation (Vassilev, Scheuer & Catterall, 1988; 1989). Mutagenesis experiments have also shown that expression of the sodium channel «-subunit in Xenopus
oocytes as two pieces corresponding to the first three domains (I-III) and the fourth domain (IV) results in channels that activate normally but have slowed inactivation (Stuhmer et al. 1989). It is believed that the single phenylalanine in the centre of a three-residue hydrophobic cluster is the critical residue, since its conversion to glutamine is sufficient by itself to nearly completely prevent fast channel inactivation (West et al. 1992).
1.4.3. Structures responsible f o r TTX sensitivity
Although the structure and function of sodium channels in different tissues are very similar, the presence of multiple sodium channel isoforms is clearly established (Trimmer & Agnew, 1989; Mandel, 1992). These isoforms of sodium channels can be distinguished by their electrophysiology and pharmacology.
A significant difference between sodium channels in cardiac tissues and other excitable tissues is their sensitivity to TTX. One class of sodium channels, present in brain and adult skeletal muscle, is blocked by application of TTX in the nanomolar range. On the other hand, sodium channels in cardiac muscle and in developing or denervated skeletal muscle, are blocked only by higher TTX concentrations, in the 1-10 micromolar range (Mandel, 1992). Direct binding studies also indicate the presence of
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approximately four times more low affinity binding sites than high affinity sites for the TTX analog, saxitoxin (STX) in cardiac tissue (Catterall & Coppersmith, 1981). This raises the possibility that there exist different types of sodium channel even in cardiac tissues themselves.
Three TTX insensitive cDNAs: rSkm2 from rat skeletal muscle (Kallen et al. 1990), RH1 from rat cardiac tissue (Rogart et al. 1989) and hHl from human cardiac tissue (Gellens et al. 1992) have been identified. The degree of primary structure identity that exists between hHl and rSkM2 (93.8%) is significantly greater than that between the two rat isoforms rSkMl (brain) and rSkM2 (59%), while the RH1 appears to be identical to rSkM2 (Rogart et al. 1989).
The SS2 domain has been postulated to contribute to the binding site for TTX. SS2 is a seven-amino acid segment that forms part of the external loop that connects membrane-spanning segments S5 and S6. Two of the seven amino acids in SS2 of the first NH2-terminal repeat or domain I differ between the TTX-resistant and TTX-sensitive Na channels (Guy & Conti, 1990; Satin et al. 1992a). In SS2 of TTX-resistant Na channels, asparagine is replaced by arginine and phenylalanine or tyrosine is replaced by cysteine.
Because the asparagine residue that is present in this region in all TTX-sensitive sodium channel isoforms is replaced by arginine in both hHl and rSkM2, it has been suggested that the charge difference could contribute to the decreased affinity for TTX exhibited by these channels (Gellens et al. 1992; White et al. 1991). However, mutation experiments have shown recently that an increase of negative charge by substitution of the positively charged Arg377 for a neutral Ans to RH1 Na channel caused a decrease in TTX affinity rather than an increase.
When Cys374 in SS2 region was replaced by Tyr, however, it resulted in an 730-fold increase in TTX binding affinity (Satin et al. 1992a), whereas replacement of phenylalanine (385) of brain sodium channel by
cysteine reduces sensitivity to TTX (Heinemann, Terlau & Imoto, 1992). Therefore it is believed that the cysteine rather than arginine in SS2 of cardiac Na channel is critical to TTX binding. These obsevations also suggest that TTX binding in the Na channel may not be a simple electrostatic attraction between the positively charged toxin guanidinium groups and negatively charged acidic group in Na channel binding site, perhaps involving an ionized hydrogen bond (Satin et al. 1992a)
1.5. Other ion currents involved in the action potential of cardiac