Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block.

Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers. Single-channel conductances were studied for [Na+] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states. Two other conductance states were observed: a subconductance state, usually seen at [NaCl] greater than or equal to 0.5 M, and a flickery state, usually seen at [NaCl] less than or equal to 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M. The K+/Na+ permeability ratio was approximately 0.16 in 0.5 and 2.5 M salt, independent of the Na+ mole fraction, which indicates that there are no interactions among permeant ions in the channels. Impermeant and permeant blocking ions (tetraethylammonium, Ca++, Zn++, and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs. [Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] greater than or equal to 1.0 M. At low [NaCl] (less than or equal to 0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge. Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.

Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures.

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.

Steady-state gating of batrachotoxin-modified sodium channels. Variability and electrolyte-dependent modulation.

The steady-state gating of individual batrachotoxin-modified sodium channels in neutral phospholipid bilayers exhibits spontaneous, reversible changes in channel activation, such that the midpoint potential (Va) for the gating curves may change, by 30 mV or more, with or without a change in the apparent gating valence (za). Consequently, estimates for Va and, in particular, za from ensemble-averaged gating curves differ from the average values for Va and za from single-channel gating curves. In addition to these spontaneous variations, the average Va shifts systematically as a function of [NaCl] (being -109, -88, and -75 mV at 0.1, 0.5, and 1.0 M NaCl), with no systematic variation in the average za (approximately 3.7). The [NaCl]-dependent shifts in Va were interpreted in terms of screening of fixed charges near the channels' gating machinery. Estimates for the extracellular and intracellular apparent charge densities (sigma e = -0.7 and sigma i = -0.08 e/nm2) were obtained from experiments in symmetrical and asymmetrical NaCl solutions using the Gouy-Chapman theory. In 0.1 M NaCl the extracellular and intracellular surface potentials are estimated to be -94 and -17 mV, respectively. The intrinsic midpoint potential, corrected for the surface potentials, is thus about -30 mV, and the standard free energy of activation is approximately -12 kJ/mol. In symmetrical 0.1 M NaCl, addition of 0.005 M Ba2+ to the extracellular solution produced a 17-mV depolarizing shift in Va and a slight reduction in za. The shift is consistent with predictions using the Gouy-Chapman theory and the above estimate for sigma e. Subsequent addition of 0.005 M Ba2+ to the intracellular solution produced a approximately 5-mV hyperpolarizing shift in the ensemble-averaged gating curve and reduced za by approximately 1. This Ba(2+)-induced shift is threefold larger than predicted, which together with the reduction in za implies that Ba2+ may bind at the intracellular channel surface.

Tools for integrating women's health into medical education: clinical cases and concept mapping.

The authors describe two teaching tools, case-based learning and concept mapping, and how they support cross-disciplinary, multidisciplinary, and interdisciplinary learning, use a biopsychosocial model, and promote the integration of sex- and gender-based science into the medical curriculum. The process of case development at MCP Hahnemann University (MCPHU) is outlined in detail for a specific case. That case, which integrates three different components of women's health, is then presented in full. The authors then provide an example of a concept map dealing with women and alcohol use; the map defines current knowledge and serves as a blueprint for developing curricular goals and learning objectives for the topic. Properly constructed concept maps and cases help teach patient-centered approaches to problem solving, address sex- and gender-based differences in disease as well as in pathophysiology and pharmacology, integrate psychosocial issues-such as family dynamics, environmental stressors, access to health care, effective gender-based communication between patient and provider, and cultural variations-along with biomedical ones, and encourage a multidisciplinary approach to patient care. The authors maintain that these tools might be used to transform medical education by making it more integrated and interdisciplinary.

Semisynthesis of linear gramicidins using diphenyl phosphorazidate (DPPA).

Sequential HPLC analysis has been used to optimize a synthetic scheme for the preparation of semisynthetic position 1 analogues of the channel-forming pentadecapeptide, gramicidin A. Diphenyl phosphorazidate (DPPA) was more efficient than dicyclohexylcarbodiimide (DCC) for the coupling of N-formyl amino acids to des(formyl-valyl) gramicidin A. The DPPA coupling proceeds rapidly, and with high yield, at 0 degree. The absence of significant (less than 0.1%) racemization is demonstrated by the lack of electrophysiologically active formyl-L-valine gramicidin A in preparations of formyl-D-valine gramicidin A. This scheme has proved useful for the preparation of microgram to gram amounts of position-1-substituted gramicidin analogues suitable for crystallography and electrophysiology. Details of the techniques for the preparation of these highly purified analogues are discussed.

Barriers and solutions to implementing a new curriculum: lessons from the women's health education program at MCP Hahnemann School of Medicine.

The integration of new knowledge into the medical school curriculum is a difficult process. This article proposes effective strategies for overcoming obstacles to curricular integration of women's health and sex and gender topics. Some techniques developed to overcome barriers to the integration of new material into an existing curriculum include faculty development, faculty rewards, development of competencies and assessment tools, interdisciplinary team teaching, standardized patients, and reference resources. An interdisciplinary approach to implementing women's health education, as with most new curricular material, is endorsed for integration of the new field into the medical school curriculum. This proposed model results in general institutional participation and support, especially from senior level leadership. The outlined process enables students and faculty to learn techniques for incorporating emerging information in all disciplines, helping them to become "life-long learners."

Integrating a domestic violence education program into a medical school curriculum: challenges and strategies.

BACKGROUND: Domestic violence affects millions of women in the United States each year. Physicians are in a unique position to screen for violence in their patients' homes and refer individuals before they are injured or killed. All U.S. medical schools are expected to incorporate partner violence into their curricula. DESCRIPTION: The MCP Hahnemann School of Medicine has developed and integrated a domestic violence educational intervention program into the existing problem-based medical school curriculum. This educational intervention includes modification of an existing teaching case to include signs and symptoms specific to domestic violence, written learning objectives on domestic violence in the health care setting, relevant resource material, and a 3-hr training program conducted by an interdisciplinary teaching team. EVALUATION: Based on quantitative and qualitative data over a 3-year period, this domestic violence education program has been successfully integrated into an existing problem-based curriculum. Overall, the high student exam scores indicate the effectiveness of the teaching program. In addition, the students' evaluation of the program reflects a high level of student satisfaction with the curriculum presented on domestic violence. CONCLUSION: This article describes both the content of a domestic violence teaching program and the process used to integrate such a program into a medical school curriculum. Although medical schools and their teaching programs are uniquely structured, with slight modifications they are able to accommodate domestic violence teaching programs.

Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position 1 in gramicidin A.

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.

A model for integrating women's health issues into a problem-based curriculum.

The process of incorporating new material into an existing medical curriculum frequently produces lengthy debate, political maneuvering, and competition for curricular time. The faculty of the Women's Health Education Program at MCP-Hahnemann School of Medicine, developed a stepwise process, or framework, for including women's health teaching for students in the problem-based curriculum. This process can be applied to the integration of any body of information. The key elements of the process are to define the full scope of what needs to be taught, develop teaching objectives, identify opportunities to introduce the information into the curriculum, develop strategies that capitalize on existing curricula, enlist the collaboration of a broad range of key faculty, develop evaluation tools, and assess whether students have achieved the expected competencies.

Improving the regulation of carcinogens by expediting cancer potency estimation.

The statutory language of the Safe Drinking Water and Toxic Enforcement Act of 1986 (Proposition 65; California Health and Safety Code 25249.5 et seq.) encourages rapid adoption of "no significant risk levels" (NSRLs), intakes associated with estimated cancer risks of no more than 1 in 100,000. Derivation of an NSRL for a carcinogen listed under Proposition 65 requires the development of a cancer potency value. This paper discusses the methodology for the derivation of cancer potencies using an expedited procedure, and provides potency estimates for a number of agents listed as carcinogens under Proposition 65. To derive expedited potency values, default risk assessment methods are applied to data sets selected from an extensive tabulation of animal cancer bioassays according to criteria used by regulatory agencies. A subset of these expedited values is compared to values previously developed by regulatory agencies using conventional quantitative risk assessment and found to be in good agreement. Specific regulatory activities which could be facilitated by adopting similar expedited procedures are identified.

STM imaging of molecular collagen and phospholipid membranes.

The application of STM to biological materials has been limited by poor conductivity, sample geometry and stability of biological materials. In this paper we describe an STM study of the monomeric helical forms of collagen, a stable, conductive and widely prevalent structural protein. We have also used STM to image artificial Langmuir DPE (dipalmitoyl phosphatidyl ethanolamine) phospholipid membranes. Both molecular collagen and the phospholipid membranes were dried in air on highly oriented pyrolytic graphite (HOPG). Our STM images of collagen dried on HOPG reveal strands 15 A in diameter with a periodicity of about 30 A which correlates with that known to occur in collagen. Spikes which periodically protrude from strands in our STM images of collagen appear to represent pyrrolidine ring structures in the amino acids proline and hydroxyproline. Thus, we report the first STM imaging of native biomolecules revealing intramolecular details and what appear to be specific amino acids. STM imaging of phospholipid membranes show a lattice pattern with densities spaced approximately 4.5 A apart. These are thought to represent individual phospholipid molecules in an artificial membrane formed on the HOPG. We believe STM and its related technologies will have great future utility in biomolecular studies.