Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains.

The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel alpha subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III-IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. 89:10910-10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.

Sodium channel inactivation is altered by substitution of voltage sensor positive charges.

The role of the voltage sensor positive charges in fast and slow inactivation of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing mutations (by glutamine substitution) and charge-conserving mutations were constructed in a cDNA encoding the sodium channel alpha subunit. To determine if fast inactivation altered the effects of the mutations on slow inactivation, the mutations were also constructed in a channel that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III-IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. 89:10910- 10914). Most of the mutations shifted the vof fast inactivation in the negative direction, with the largest effects resulting from mutations in domains I and II. These shifts were in the opposite direction compared with those observed for activation. The effects of the mutations on slow inactivation depended on whether fast inactivation was intact or not. When fast inactivation was eliminated, most of the mutations resulted in positive shifts in the v of slow inactivation. The largest effects again resulted from mutations in domains I and II. When fast inactivation was intact, the mutations in domains II and III resulted in negative shifts in the v of slow inactivation. Neutralization of the fourth charge in domain I or II resulted in the appearance of a second component in the voltage dependence of slow inactivation that was only observable when fast inactivation was intact. These results suggest the S4 regions of all four domains of the sodium channel are involved in the voltage dependence of inactivation, but to varying extents. Fast inactivation is not strictly coupled to activation, but it derives some independent voltage sensitivity from the charges in the S4 domains. Finally, there is an interaction between the fast and slow inactivation processes.

Performance of a fully automated in vitro allergy testing system.

BACKGROUND: Since the development of the radioallergosorbent test (RAST) for quantification of allergen-specific IgE, numerous non-radoisotopic methods have been devised which combine the proven cellulose disc technology with enzyme-linked immunoassay methods. The HY.TEC EIA (Hycor Biomedical, Inc. Irvine, CA) was compared with Pharmacia CAP with respect to overall system features and assay performance characteristics. METHODS: The HY.TEC EIA and Pharmacia CAP were compared with respect to calibrator range, sensitivity, type of detection, type of solid phase, throughput, and mode of operation. To determine the assay sensitivity and specificity for a variety of allergens, a total of 2,447 tests were performed on both CAP and HY.TEC EIA. The samples were scored positive in both cases using a cutoff of 0.35 IU/mL. RESULTS: The general features of the HY.TEC EIA system are comparable to Pharmacia UniCAP, with the added advantage of higher throughput. Intra-assay precision was 7% and inter-assay precision was 9-15%. Using CAP as a comparative method, HY.TEC EIA has a sensitivity of 94.0% and a specificity of 94.4%. CONCLUSIONS: The HY.TEC EIA demonstrates excellent agreement with the Pharmacia CAP system in the determination of allergen-specific IgE. With the automation necessary in today's clinical laboratory, we conclude that the HY.TEC EIA is a state-of-the-art tool for the diagnosis of allergic disease.

Site-directed mutagenesis of the putative pore region of the rat IIA sodium channel.

We have used site-directed mutagenesis to examine the functional role of each of the eight acidic amino acid residues in the region between proposed transmembrane segments 5 and 6 (S5-S6) of domain II of the rat brain IIA sodium channel alpha subunit. The mutant sodium channels were expressed in Xenopus oocytes and analyzed by two-microelectrode voltage clamping with respect to voltage-dependent activation, inactivation, ion selectivity, and sensitivity to the pore-blocking neurotoxins tetrodotoxin (TTX) and saxitoxin (STX). None of the mutations had significant effects on voltage-dependent gating, ion selectivity, or block by protons or calcium. Three of the mutations had significant effects on the sensitivity of the channel to block by TTX and STX. Neutralization of negative charges at positions 942 and 945 greatly reduced the block by TTX and STX, suggesting that these two residues interact directly with the toxins. Substitution of a nearby negative charge at position 949 resulted in a smaller decrease in TTX and STX block, although analysis of TTX block of this mutant at low ionic strength suggests that the interaction is not simply by an electrostatic through-space mechanism. None of the other five mutations had any effects on block by either TTX or STX. The two acidic residues that had dramatic effects on toxin binding had significantly smaller effects at a depolarized membrane potential. The sodium channel interacts with TTX and STX with higher affinity at depolarized potentials, so these two residues must make a greater contribution to toxin binding in the low affinity state. These results define a small segment of the sodium channel alpha subunit domain II S5-S6 region that interacts with TTX and STX and therefore must lie near the mouth of the channel pore.

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines.

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.