␣-Scorpion toxins and sea anemone toxins bind to a common extracellular site on the Na ؉ channel and inhibit fast inactivation. Basic amino acids of the toxins and domains I and IV of the Na ؉ channel ␣ subunit have been previously implicated in toxin binding. To identify acidic residues required for toxin binding, extracellular acidic amino acids in domains I and IV of the type IIa Na ؉ channel ␣ subunit were converted to neutral or basic amino acids using site-directed mutagenesis, and altered channels were transiently expressed in tsA-201 cells and tested for 125 I-␣-scorpion toxin binding. Conversion of Glu 1613 at the extracellular end of transmembrane segment IVS3 to Arg or His blocked measurable ␣-scorpion toxin binding, but did not affect the level of expression or saxitoxin binding affinity. Conversion of individual residues in the IVS3-S4 extracellular loop to differently charged residues or to Ala identified seven additional residues whose mutation caused significant effects on binding of ␣-scorpion toxin or sea anemone toxin. Moreover, chimeric Na ؉ channels in which amino acid residues at the extracellular end of segment IVS3 of the ␣ subunit of cardiac Na ؉ channels were substituted into the type IIa channel sequence had reduced affinity for ␣-scorpion toxin characteristic of cardiac Na ؉ channels. Electrophysiological analysis showed that E1613R has 62-and 82-fold lower affinities for ␣-scorpion and sea anemone toxins, respectively. Dissociation of ␣-scorpion toxin is substantially accelerated at all potentials compared to wild-type channels. ␣-Scorpion toxin binding to wild type and E1613R had similar voltage dependence, which was slightly more positive and steeper than the voltage dependence of steady-state inactivation. These results indicate that nonidentical amino acids of the IVS3-S4 loop participate in ␣-scorpion toxin and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in ␣-scorpion toxin binding affinity between cardiac and neuronal Na ؉ channels. The results also support the hypothesis that this region of the Na ؉ channel is important for coupling channel activation to fast inactivation.Voltage-gated Na ϩ channels are responsible for the conduction of electrical impulses in most excitable tissues (1). The importance of their function is demonstrated by the effects of Na ϩ channel-specific neurotoxins that bind to at least six different receptor sites on the Na ϩ channel molecule and disrupt its normal behavior (reviewed in Refs. 2 and 3). These natural toxins are powerful tools for understanding and correlating ion channel structure and function, as exemplified by identification of molecular determinants for binding of the pore blocker tetrodotoxin, which has provided important information about the structure of the ion selectivity filter and pore (3, 4). Similarly, the identification of molecular determinants for binding of toxins that modify activation or inactivation will likely provide important ...

1 antagonists and agonists, while non-L-type Ca 2ϩ channels are insensitive to DHPs. Peptide segments which contribute to the DHP receptor site have been localized by photoaffinity labeling and antibody mapping to transmembrane segments IIIS6 and IVS6 and adjacent extracellular segments of the ␣ 1 subunit (1-3). Charged DHPs can only reach their receptor site from the extracellular side of the membrane (4). Access is optimal when the length of the alkyl spacer chain between the charged moiety and the binding center of the ligand is 10 methylene groups, suggesting that the DHP receptor site is approximately 11-14 Å into the lipid bilayer (5). Together, these results suggest the DHP receptor site is located within transmembrane segments IIIS6 and IVS6 about 25-35% of the distance across the lipid bilayer. In addition, a site in the intracellular carboxyl-terminal domain has also been photoaffinity-labeled by photoreactive DHPs (6). Like DHP antagonists, DHP agonists also act from the extracellular side of the membrane (7). Analysis of chimeric Ca 2ϩ channels (8) showed that the extracellular end of segment IVS6 is important for the action of DHP agonists. Here, we use site-directed mutagenesis and radioligand binding to identify individual amino acids in transmembrane segments IIIS6 and IVS6 that are critical determinants of DHP binding. EXPERIMENTAL PROCEDURESMaterials-tsA-201 cells were provided by Dr. Robert DuBridge (Cell Genesis, Foster City, CA). cDNA encoding the ␣ 1 (9, 10) and ␣ 2 ␦ (10) subunits cloned from rabbit skeletal muscle calcium channel was provided by Drs.

Voltage-gated Na؉ channels consist of a large ␣ subunit of 260 kDa associated with ␤1 and/or ␤2 subunits of 36 and 33 kDa, respectively. ␣ subunits of rat cardiac Na ؉ channels (rH1) are functional when expressed alone in Xenopus oocytes or mammalian cells. ␤1 subunits are present in the heart, and localization of ␤1 subunit mRNA by in situ hybridization shows expression in the perinuclear cytoplasm of cardiac myocytes. Coexpression of ␤1 subunits with rH1 ␣ subunits in Xenopus oocytes increases Na ؉ currents up to 6-fold in a concentration-dependent manner. However, no effects of ␤1 subunit coexpression on the kinetics or voltage dependence of the rH1 Na ؉ current were detected. Increased expression of Na ؉ currents is not observed when an equivalent mRNA encoding a nonfunctional mutant ␤1 subunit is coexpressed. Our results show that ␤1 subunits are expressed in cardiac muscle cells and that they interact with ␣ subunits to increase the expression of cardiac Na ؉ channels in Xenopus oocytes, suggesting that ␤1 subunits are important determinants of the level of excitability of cardiac myocytes in vivo.

The distinct roles of the two magnesium ions essential to the activity of D-xylose isomerase from Streptomyces olivochromogenes were examined. The enzyme-magnesium complex was isolated, and the stoichiometry of cation binding determined by neutron activation analysis to be 2 mol of magnesium per mole of enzyme. A plot of Mg2+ added versus Mg2+ bound to enzyme is consistent with apparent KD values of < or = 0.5-1.0 mM for one Mg2+ and < or = 2-5 mM for the second. A site-directed mutant of D-xylose isomerase was designed to remove the tighter, tetracoordinated magnesium binding site (site 1, Mg-1); Glu180 was replaced with Lys180. The stoichiometry of metal binding to this mutant, E180K, is 1 mol of magnesium per mole of enzyme. Ring-opening assays with 1-thioglucose (H2S released upon ring opening) show E180K catalyzes the opening of the sugar ring at 20% the rate of the wild-type, but E180K does not catalyze isomerization of glucose to fructose. Thus, the magnesium bound to Glu180 is essential for isomerization but not essential for ring opening. The X-ray crystallographic structures of E180K in the absence of magnesium and in the presence and absence of 250 mM glucose were obtained to 1.8-A resolution and refined to R factors of 17.7% and 19.7%, respectively. The wild-type and both E180K structures show no significant structural differences, except the epsilon-amino group of Lys180, which occupies the position usually occupied by the Mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac rH1 Na+ channel a subunits were expressed in cells of the Chinese hamster lung 1610 cell line by transfection, and a stable cell line exprsing cardiac Na+ channels (SNa-rHl) was isolated. Mean Na+ currents of 2.2 ± 1.0 nA were recorded, which corresponds to a cell surface density of approximately 1-2 channels active at the peak of the Na+ current per pam2. The expressed cardiac Na+ current was tetrodotoxin resistant (Kd = 1.8 FM) and had voltagedependent properties similar to those of the Na+ current in neonatal ventricular myocytes. Activation of protein kinase C by 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10 saM) decreased this current -33% at a holding potential of -114 mV and 56% at -94 mV. This reduction in peak current was caused in part by an 8-to 14-mV shift of steady-state inactivation in the hyperpolarized direction. Na+ channel activation was unchanged. Effects of OAG in SNa-rH1 cells and in neonatal rat cardiac myocytes were similar, except that the time course of inactivation was slowed either transiently or persistently when protein kinase C was activated in myocytes bathed in low-Ca2+ (1 M) or Ca2+-free solution but was unaffected in SNa-rH1 cells. The effects of OAG on cardiac Na+ current were blocked in cells that had been previously microlijected with a peptide inhibitor of protein kinase C but not with a peptide inhibitor of cAMP-dependent protein kinase, indicating that protein kinase C is responsible for the effects of OAG. Single-channel recordings from SNa-rHi cells showed that the probability of channel opening was reduced by OAG, but the conductance was unaffected. OAG did not induce the late Na+ channel openings observed with PKC modulation ofneuronal and skeletal muscle Na+ channels. Thus, the substantial reduction in Na+ current at normal diastolic depolarizations with 10 pM OAG is due to failure of channel opening in response to depolarization. Such Na+ current reductions may have profound effects on cardiac cell excitability.The rapid upstroke of the cardiac action potential due to the Na+ current through voltage-dependent Na+ channels is responsible for the rapid spread of activation through the atria and ventricles that initiates and coordinates contraction of the heart. Cardiac function depends critically on the amplitude, timing, and voltage dependence of the Na+ current, and interventions that modulate Na+ current have potent physiological effects. The functional properties of mammalian cardiac Na+ channels are distinctive. They are less sensitive to tetrodotoxin (TTX) than most Na+ channels, with a Kd of approximately 1-10 ,uM (1), and their kinetics of activation and inactivation are slower and more complex (2). These properties are mediated by a distinct Na+ channel a subunit (rH1) that was originally characterized by cloning from newborn rat heart (3) and denervated rat skeletal muscle (4). A closely related Na+ channel has been described in human cardiac muscle (5). Expression of these a subunit cDNAs in Xenopus oocytes (5-7) yields Na+ currents with functiona...