Interactions of key charged residues contributing to selective block of neuronal sodium channels by µ-conotoxin KIIIA.

Voltage-gated sodium channels are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias (Na(v)1.5), myotonias (Na(v)1.4), epilepsies (Na(v)1.2), and pain (Na(v)1.7). Thus, there is strong motivation to identify isoform-specific blockers and the molecular determinants underlying their selectivity among these channels. µ-Conotoxin KIIIA blocks rNa(v)1.2 (IC(50), 5 nM), rNa(v)1.4 (37 nM), and hNa(v)1.7 (97 nM), expressed in mammalian cells, with high affinity and a maximal block at saturating concentrations of 90 to 95%. Mutations of charged residues on both the toxin and channel modulate the maximal block and/or affinity of KIIIA. Two toxin substitutions, K7A and R10A, modulate the maximal block (52-70%). KIIIA-H12A and R14A were the only derivatives tested that altered Na(v) isoform specificity. KIIIA-R14A showed the highest affinity for Na(v)1.7, a channel involved in pain signaling. Wild-type KIIIA has a 2-fold higher affinity for Na(v)1.4 than for Na(v)1.7, which can be attributed to a missing outer vestibule charge in domain III of Na(v)1.7. Reciprocal mutations Na(v)1.4 D1241I and Na(v)1.7 I1410D remove the affinity differences between these two channels for wild-type KIIIA without affecting their affinities for KIIIA-R14A. KIIIA is the first µ-conotoxin to show enhanced activity as pH is lowered, apparently resulting from titration of the free N terminus. Removal of this free amino group reduced the pH sensitivity by 10-fold. Recognition of these molecular determinants of KIIIA block may facilitate further development of subtype-specific, sodium channel blockers to treat hyperexcitability disorders.

Orientation of µ-conotoxin PIIIA in a sodium channel vestibule, based on voltage dependence of its binding.

Mutant cycle analysis has been used in previous studies to constrain possible docking orientations for various toxins. As an independent test of the bound orientation of µ-conotoxin PIIIA, a selectively targeted sodium channel pore blocker, we determined the contributions to binding voltage dependence of specific residues on the surface of the toxin. A change in the "apparent valence" (zδ) of the block, which is associated with a change of a specific toxin charge, reflects a change in the charge movement within the transmembrane electric field as the toxin binds. Toxin derivatives with charge-conserving mutations (R12K, R14K, and K17R) showed zδ values similar to those of wild type (0.61 ± 0.01, mean ± S.E.M.). Charge-changing mutations produced a range of responses. Neutralizing substitutions for Arg14 and Lys17 showed the largest reductions in zδ values, to 0.18 ± 0.06 and 0.20 ± 0.06, respectively, whereas unit charge-changing substitutions for Arg12, Ser13, and Arg20 gave intermediate values (0.24 ± 0.07, 0.33 ± 0.04, and 0.32 ± 0.05), which suggests that each of these residues contributes to the dependence of binding on the transmembrane voltage. Two mutations, R2A and G6K, yielded no significant change in zδ. These observations suggest that the toxin binds with Arg2 and Gly6 facing the extracellular solution, and Arg14 and Lys17 positioned most deeply in the pore. In this study, we used molecular dynamics to simulate toxin docking and performed Poisson-Boltzmann calculations to estimate the changes in local electrostatic potential when individual charges were substituted on the toxin's surface. Consideration of two limiting possibilities suggests that most of the charge movement associated with toxin binding reflects sodium redistribution within the narrow part of the pore.

A survey of videodisc and interactive videodisc projects in North America: Part I.

Selected videodisc (VD) and interactive videodisc (IVD) programs, projects and topics are presented in two articles in this and a subsequent issue of the journal. Part I reviews the impact of Information Science developments on image management. The American Society of Hematology Slide Bank and other specific applications in urology, paediatric neurology, obstetrical nursing, medical decision making, dental diagnosis and treatment (DDT), and paediatric cardiology, are reviewed as educational and informatics research projects. This is followed by a section on three-dimensional reconstructions of the brain which stresses digital images. Multi-purposing and repurposing are reviewed in two prototype programs. A discussion of the multidisciplinary Slice of Life projects completes this first article.

Interactive computer-video modules for health sciences education.

Advances in electronic image recording and computer technology have resulted in a remarkable increase in the power and flexibility of interactive computer-video teaching systems. The University of Washington Health Science Videodisc Development Group first demonstrated a laser videodisc controlled by a remote central computer in 1980. Even this rudimentary unit highlighted basic medical informatics principles including: rapid accessibility; a "generic" or multi-purposed format; ease of computer control; and large collections of valid, rigorously reviewed images. Advances in medical informatics have led to the development of the following previously undescribed series of teaching units: 1. The hypertext programs Hypercard, Linkway, and Guide have been used with videodiscs to develop easy-to-use instructional and reference materials. These materials demonstrate the ease with which a computer-naive instructor may develop new programs and the advantages that the intuitive nature of these programs brings to student users. 2. Patient simulations using single and double screens plus pre-defined knowledge structures; 3. Interactive single topic tutorials using preset knowledge structures; 4. A key-word-based disc searching system; 5. Electronic video microscopy; 6. A series of programs developed independently by health science faculty who have purchased multi-purpose videodiscs that demonstrate the flexibility of the multi-purpose or "generic": collection concept.

The effect of an educational program on transfusion practices in a regional blood program.

With the increased complexity of blood component therapy, it is important to be able to modify physician behavior with reliable educational programs. A standardized educational program on the use of red blood cells and whole blood was tested in 22 hospitals in a regional blood program using medical audit and computer monitoring to evaluate effectiveness. Most hospitals were eager to take advantage of the education program but were unwilling to use the audit-education cycle. At the same time, computer monitoring of indivisual hospital ordering and transfusion practices demonstrated an increased utilization of red blood cells in 64 per cent of hospitals with an overall improvement of 8 per cent. The improved use of red blood cells was appreciated within the month of the program and then sustained for six to twelve months at the new level.