Richard McGee

Markov, fractal, diffusion, and related models of ion channel gating. A comparison with experimental data from two ion channels

The gating kinetics of single-ion channels are generally modeled in terms of Markov processes with relatively small numbers of channel states. More recently, fractal (Liebovitch et al. 1987. Math. Biosci. 84:37-68) and diffusion (Millhauser et al. 1988. Proc. Natl. Acad. Sci. USA. 85:1502-1507) models of channel gating have been proposed. These models propose the existence of many similar conformational substrates of the channel protein, all of which contribute to the observed gating kinetics. It is important to determine whether or not Markov models provide the most accurate description of channel kinetics if progress is to be made in understanding the molecular events of channel gating. In this study six alternative classes of gating model are tested against experimental single-channel data. The single-channel data employed are from (a) delayed rectifier K+ channels of NG 108-15 cells and (b) locust muscle glutamate receptor channels. The models tested are (a) Markov, (b) fractal, (c) one-dimensional diffusion, (d) three-dimensional diffusion, (e) stretched exponential, and (f) expo-exponential. The models are compared by fitting the predicted distributions of channel open and closed times to those observed experimentally. The models are ranked in order of goodness-of-fit using a boot-strap resampling procedure. The results suggest that Markov models provide a markedly better description of the observed open and closed time distributions for both types of channel. This provides justification for the continued use of Markov models to explore channel gating mechanisms.

Determination of total (Na++K+)-ATPase activity of isolated or cultured cells

The aim of this work was to determine if the total (Na+ + K+)-ATPase of the plasma membrane of a cell population could be assayed without cell homogenization and partial purification of the enzyme. Several types of intact cells that were placed in an assay medium containing MgATP, Na+, and K+ hydrolyzed little or none of the added ATP. When the cells were pretreated with the ionophore alamethicin and then placed in the assay medium, they exhibited an ouabain-sensitive (Na+ + K+)-ATPase activity that increased and reached a limiting value with increasing alamethicin concentration. Since alamethicin did not increase the activity of the purified membrane-bound (Na+ + K+)-ATPase, its effects on the intact cells are probably due to the formation of large channels within the plasma membrane that allow the free access of the components of the assay medium to the intracellular domains of (Na+ + K+)-ATPase. Utilizing whole cells treated with alamethicin, total (Na+ + K+)-ATPase activity was determined in clonal pheochromocytoma cells (PC12), neuroblastoma x glioma hybrid cells (NG108-15), and myocytes isolated from adult and neonatal rat hearts. With the use of this whole-cell assay, the ouabain sensitivities of the enzymes in adult and neonatal rat heart myocytes were determined and found to be the same as those that have been determined with the use of partially purified enzymes.

Training Mentors of Clinical and Translational Research Scholars: A Randomized Controlled Trial

Purpose To determine whether a structured mentoring curriculum improves research mentoring skills. Method The authors conducted a randomized controlled trial (RCT) at 16 academic health centers (June 2010 to July 2011). Faculty mentors of trainees who were conducting clinical/translational research ≥50% of the time were eligible. The intervention was an eight-hour, case-based curriculum focused on six mentoring competencies. The primary outcome was the change in mentors’ self-reported pretest to posttest composite scores on the Mentoring Competency Assessment (MCA). Secondary outcomes included changes in the following: mentors’ awareness as measured by their self-reported retrospective change in MCA scores, mentees’ ratings of their mentors’ competency as measured by MCA scores, and mentoring behaviors as reported by mentors and their mentees. Results A total of 283 mentor–mentee pairs were enrolled: 144 mentors were randomized to the intervention; 139 to the control condition. Self-reported pre-/posttest change in MCA composite scores was higher for mentors in the intervention group compared with controls (P < .001). Retrospective changes in MCA composite scores between the two groups were even greater, and extended to all six subscale scores (P < .001). More intervention-group mentors reported changes in their mentoring practices than control mentors (P < .001). Mentees working with intervention-group mentors reported larger changes in retrospective MCA pre-/posttest scores (P = .003) and more changes in their mentors’ behavior (P = .002) than those paired with control mentors. Conclusions This RCT demonstrates that a competency-based research mentor training program can improve mentors’ skills.

Delivery of health care to the underserved: Potential contributions of telecommunications technology

OBJECTIVE To evaluate the potential uses of telecommunications in medicine (telemedicine), determine the most important principles in designing telemedicine applications, decide what research questions to address, and identify potential barriers to full use of telemedicine. DESIGN A consensus conference on telemedicine was convened in October 1993 to assemble a wide variety of participants with the assigned task of addressing the objective. RESULTS Consensus was achieved on several key principles for implementation of successful telemedicine. Two of the most important principles will be (1) to focus on the needs of the underserved people more than on the capabilities of the available technologies and the regional centers and (2) to use the least expensive but appropriate telecommunications technology for any specific application. Greater professional connectivity between providers of health care in underserved areas and colleagues in tertiary medical centers is expected to minimize professional isolation in underserved areas. CONCLUSION Telecommunications technologies have considerable potential for improving health care to the rural and underserved populations, but a systematic approach to implementation, which takes into account the identified key principles, is needed.

The effects of exposure to exogenous fatty acids and membrane fatty acid modification on the electrical properties of NG108-15 cells

Summary1.The role of membrane lipid composition in determining the electrical properties of neuronal cells was investigated by altering the available fatty acids in the growth medium of cultured neuroblastoma × glioma hybrid cells, clone NG108-15.2.Growth of the cells for several days in the presence of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic) caused a pronounced decrease in the Na+ action-potential rate of rise (dV/dt) and smaller decreases in the amplitude, measured by intracellular recording. Oleic acid had no effect on the action potentials generated by the cells. In contrast, a saturated fatty acid (palmitate) and atrans monounsaturated fatty acid (elaidate) caused increases in both the rate of rise and the amplitude.3.No changes in the resting membrane potentials or Ca2+ action potentials of fatty acid-treated cells were observed. The membrane capacitance and time constant were not altered by exposure to arachidonate, oleate, or elaidate, whereas arachidonate caused a small increase in membrane resistance.4.Examination of the membrane phospholipid fatty acid composition of cells grown with various fatty acids revealed no consistent alterations which could explain these results.5.To examine the mechanism for arachidonate-induced decreases indV/dt, the binding of3H-saxitoxin (known to interact with voltage-sensitive Na+) channels was measured. Membranes from cells grown with arachidonate contained fewer saxitoxin binding sites, suggesting fewer Na+ channels in these cells.6.We conclude that conditions which lead to major changes in the membrane fatty acid composition have no effect on the resting membrane potential, membrane capacitance, time constant, or Ca2+ action potentials in NG108-15 cells. Membrane resistance also does not appear to be very sensitive to membrane fatty acid composition. However, changes in the availability of fatty acids and/or changes in the subsequent membrane fatty acid composition lead to altered Na+ action potentials. The primary mechanism for this alteration appears to be through changes in the number of Na+ channels in the cells.

A Research Mentor Training Curriculum for Clinical and Translational Researchers

To design and evaluate a research mentor training curriculum for clinical and translational researchers. The resulting 8‐hour curriculum was implemented as part of a national mentor training trial.

The Mentoring Competency Assessment: validation of a new instrument to evaluate skills of research mentors.

Purpose To determine the psychometric properties of the Mentoring Competency Assessment (MCA), a 26-item skills inventory that enables research mentors and mentees to evaluate six competencies of mentors: maintaining effective communication, aligning expectations, assessing understanding, addressing diversity, fostering independence, and promoting professional development. Method In 2010, investigators administered the MCA to 283 mentor–mentee pairs from 16 universities participating in a trial of a mentoring curriculum for clinical and translational research mentors. The authors analyzed baseline MCA data to describe the instrument’s psychometric properties. Results Coefficient alpha scores for the MCA showed reliability (internal consistency). The hypothesized model with its six latent constructs (competencies) resulted in an acceptable fit to the data. For the instrument completed by mentors, chi-square = 663.20; df = 284; P < .001; root mean square error of approximation (RMSEA) = 0.069 (90% CI, 0.062–0.076); comparative fit index (CFI) = 0.85; and Tucker-Lewis index (TLI) = 0.83. For the instrument completed by mentees, chi-square = 840.62; df = 284; P < .001; RMSEA = 0.080 (90% CI, 0.063–0.077); CFI = 0.87; and TLI = 0.85. The correlations among the six competencies were high: 0.49–0.87 for mentors, 0.58–0.92 for mentees. All parameter estimates for the individual items were significant; standardized factor loadings ranged from 0.32 to 0.81 for mentors and 0.56 to 0.86 for mentees. Conclusions The findings demonstrate that the MCA has reliability and validity. In addition, this study provides preliminary norms derived from a national sample of mentors and mentees.

Membrane fatty acid modification of the neuroblastoma X glioma hybrid, NG108-15

As a first step in studying the effects of membrane lipid modification on complex cellular functions we have modified the membrane fatty acid composition of the neuroblastoma X glioma hybrid clone, NG108-15. These cultured cells were chosen because they exhibit many complex neuronal functions in vitro. Unsaturated fatty acids (oleate, linoleate, linolenate and arachidonate) were accumulated, metabolized and esterified by the cells. These unsaturated fatty acids stimulated cell growth, whereas saturated fatty acids were toxic to the cells. Changes as large as 40-fold in the ratio of monounsaturated/polyunsaturated fatty acids in the membrane phospholipids were produced by addition of fatty acids directly to serum-containing culture medium. As a result of the exposure of NG108-15 cells to unsaturated fatty acids the amount of phosphatidylethanolamine in the cells was increased by as much as 60%. Polyunsaturated fatty acids also caused a small decrease in the membrane cholesterol/phospholipid molar ratio. These experiments demonstrate that large changes in membrane fatty acid composition can be created in clonal cells capable of differentiated neuronal activities. Additional changes in membrane lipid composition also appear to be induced by these manipulations. The question of the importance of specific membrane lipid composition to neuronal cellular function now can be addressed.

Regulation of Depolarization‐Dependent Release of Neurotransmitters by Adenosine: Cyclic AMP‐Dependent Enhancement of Release from PC12 Cells

Abstract: We have used pheochromocytoma cells, clone PC12, as a model system for studying the effects of adenosine on neurosecretion. Exposure of the cells to adenosine or 2‐chloroadenosine caused immediate activation of adenylate cyclase, increases in cellular cyclic AMP content, and inhibition of SAM‐dependent phospholipid N‐methylation and protein carboxymethylation. However, the effects on methylation were only observed with concentrations of adenosine 100 times greater than those that elevated cyclic AMP. Exposure of the cells to adenosine and 2‐chloroadenosine did not alter the release of [3H]norepinephrine ([3H]NE) in the absence of depolarization. However, depolarization‐dependent release of [3H]NE was markedly elevated by short (1–20 min) pretreatments with adenosine or 2‐chloroadenosine. The enhancement of release was observed irrespective of the nature of the depolarizing stimulus (elevated K+, carbamylcholine, or veratridine). Release of [3H]acetylcholine in response to elevated K+ also was increased by adenosine pretreatment. These effects of adenosine and 2‐chloroadenosine on neurotransmitter release closely paralleled elevation of cellular cyclic AMP but not inhibition of methylation. Taken together, the results show that adenosine, probably acting through adenosine receptors coupled to stimulation of adenylate cyclase, is able to modulate the neurosecretory process in PC12 cells. Furthermore, the enhancement of release occurred even though the extent of depolarization (measured as 86Rb+ flux through the acetylcholine receptor channel) and the amount of 45Ca2+ which entered upon depolarization were unchanged. Therefore, the enhancement of release produced by elevated cyclic AMP appeared to reflect increased efficiency of the stimulus‐secretion coupling process.

Deriving Competencies for Mentors of Clinical and Translational Scholars

Although the importance of research mentorship has been well established, the role of mentors of junior clinical and translational science investigators is not clearly defined. The authors attempt to derive a list of actionable competencies for mentors from a series of complementary methods. We examined focus groups, the literature, competencies derived for clinical and translational scholars, mentor training curricula, mentor evaluation forms and finally conducted an expert panel process in order to compose this list. These efforts resulted in a set of competencies that include generic competencies expected of all mentors, competencies specific to scientists, and competencies that are clinical and translational research specific. They are divided into six thematic areas: (1) Communication and managing the relationship, (2) Psychosocial support, (3) Career and professional development, (4) Professional enculturation and scientific integrity, (5) Research development, and (6) Clinical and translational investigator development. For each thematic area, we have listed associated competencies, 19 in total. For each competency, we list examples that are actionable and measurable. Although a comprehensive approach was used to derive this list of competencies, further work will be required to parse out how to apply and adapt them, as well future research directions and evaluation processes. Clin Trans Sci 2012; Volume 5: 273–280