An Approach to Successful Development of Clinician-Scientists in Neurology: The NINDS R25 Experience.

OBJECTIVE: Training clinician-scientists is a primary objective of many academic neurology departments, as these individuals are uniquely positioned to perform insightful clinical or laboratory-based research informed both by clinical knowledge and their own experiences caring for patients. Despite its importance, training clinician-scientists has perhaps never been so challenging. The National Institute of Neurologic Disorders and Stroke (NINDS) R25 program was designed in an attempt to support these individuals, decrease the time needed to obtain National Institutes of Health K awards, and to help educate a cohort of trainees preparing for a career in academic neurology. We endeavored to describe the structure and features of the program while examining its outcomes. METHODS: R25 outcome data from 2009 to 2024 were reviewed. Statistical comparisons were made using 2-sided Mann-Whitney U testing. RESULTS: A total of 67% of adult neurologists who received an R25 had a successful application for a National Institutes of Health K award compared with 45% of adult neurologists who had not received R25 support (p < 0.0001). Among child neurologists, 73% who applied went on to receive K funding after R25 support, compared with 45% who had not been part of the R25 program (p < 0.001). The average time between completion of residency and obtaining a K award for R25 participants was decreased by 26 months among those with an MD/PhD degree, and 32 months for those with an MD degree compared with non-R25 individuals. INTERPRETATION: The R25 program has been successful in achieving its training goals, but stands as only one component of support for aspiring clinician-scientists. Investments and commitments made by academic neurology departments are key to supporting this success. ANN NEUROL 2024.

The NIH "BEST" programs: Institutional programs, the program evaluation, and early data.

Biomedical research training has undergone considerable change over the past several years. At its core, the goal of graduate and postdoctoral training is to provide individuals with the skills and knowledge to become outstanding scientists and expand knowledge through the scientific method. Historically, graduate school training has focused on preparation for academic positions. Increasingly, however, a shift toward preparation for a wider range of career options has emerged. This is largely because most biomedical PhD graduates do not become Principal Investigators in academic laboratories. Here we describe an National Institutes of Health Common Fund program with the major goal of culture change for biomedical research training and training that prepares individuals for a broader expanse of careers in the biomedical research enterprise. These "Broadening Experiences in Scientific Training" (BEST) awards, issued in 2012 and 2013, provided support to institutions to develop innovative approaches to achieving these goals, as a complement to traditional training. Awardees were tasked with catalyzing change at their institutions and sharing best practices across the training community. Awardees were required to participate in a cross-site evaluation that assessed the impact of BEST activities on three main areas: (a) trainee confidence and knowledge to make career decisions, (b) influence of this added activity on time in training, and (c) ability of the institutions to sustain activities deemed to be beneficial. Here we present the fundamental approach to the BEST program and early evaluative data.

Myths and facts about getting an academic faculty position in neuroscience.

We at the National Institute of Neurological Disorders and Stroke routinely receive questions and statements from trainees and faculty that suggest widespread beliefs about the necessity of a National Institutes of Health K99/R00 award, other prior funding, and/or specific types of publications for obtaining one's first tenure-track position in neuroscience. To address these beliefs, we examined the funding and publication history of a cohort of investigators who began their first academic faculty position between 2009 and 2019, and we interviewed several senior academic leaders with extensive experience in hiring new faculty. Our data show that <11% of newly hired faculty had a K99/R00 award and that neither prior funding nor papers in prestigious journals were necessary to obtain a tenure-track faculty position. Interviews with academic leaders almost uniformly referred to critically important factors that were considered to be more important in the hiring process than funding or publishing in high-profile journals.

Creation of a comprehensive training and career development approach to increase the number of neurosurgeons supported by National Institutes of Health funding.

OBJECTIVE: To increase the number of independent National Institutes of Health (NIH)-funded neurosurgeons and to enhance neurosurgery research, the National Institute of Neurological Disorders and Stroke (NINDS) developed two national comprehensive programs (R25 [established 2009] for residents/fellows and K12 [2013] for early-career neurosurgical faculty) in consultation with neurosurgical leaders and academic departments to support in-training and early-career neurosurgeons. The authors assessed the effectiveness of these NINDS-initiated programs to increase the number of independent NIH-funded neurosurgeon-scientists and grow NIH neurosurgery research funding. METHODS: NIH funding data for faculty and clinical department funding were derived from the NIH, academic departments, and Blue Ridge Institute of Medical Research databases from 2006 to 2019. RESULTS: Between 2009 and 2019, the NINDS R25 funded 87 neurosurgical residents. Fifty-three (61%) have completed the award and training, and 39 (74%) are in academic practice. Compared to neurosurgeons who did not receive R25 funding, R25 awardees were twice as successful (64% vs 31%) in obtaining K-series awards and received the K-series award in a significantly shorter period of time after training (25.2 ± 10.1 months vs 53.9 ± 23.0 months; p < 0.004). Between 2013 and 2019, the NINDS K12 has supported 19 neurosurgeons. Thirteen (68%) have finished their K12 support and all (100%) have applied for federal funding. Eleven (85%) have obtained major individual NIH grant support. Since the establishment of these two programs, the number of unique neurosurgeons supported by either individual (R01 or DP-series) or collaborative (U- or P-series) NIH grants increased from 36 to 82 (a 2.3-fold increase). Overall, NIH funding to clinical neurological surgery departments between 2006 and 2019 increased from $66.9 million to $157.3 million (a 2.2-fold increase). CONCLUSIONS: Targeted research education and career development programs initiated by the NINDS led to a rapid and dramatic increase in the number of NIH-funded neurosurgeon-scientists and total NIH neurosurgery department funding.

Control of single channel conductance in the outer vestibule of the Kv2.1 potassium channel.

Current magnitude in Kv2.1 potassium channels is modulated by external [K+]. In contrast to behavior expected from the change in electrochemical driving force, outward current through Kv2.1 channels becomes larger when extracellular [K+] is increased within the physiological range. The mechanism that underlies this unusual property involves the opening of Kv2.1 channels into one of two different outer vestibule conformations, which are defined by their sensitivity to TEA. Channels that open into a TEA-sensitive conformation generate larger macroscopic currents, whereas channels that open into a TEA-insensitive conformation generate smaller macroscopic currents. At higher [K+], more channels open into the TEA-sensitive conformation. In this manuscript, we examined the mechanism by which the conformational change produced a change in current magnitude. We started by testing the simplest hypothesis: that each pharmacologically defined channel conformation produces a different single channel conductance, one smaller and one larger, and that the [K+]-dependent change in current magnitude reflects the [K+]-dependent change in the percentage of channels that open into each of the two conformations. Using single channel and macroscopic recordings, as well as hidden Markov modeling, we were able to quantitatively account for [K+]-dependent regulation of macroscopic current with this model. Combined with previously published work, these results support a model whereby an outer vestibule lysine interferes with K+ flux through the channel, and that the [K+]-dependent change in orientation of this lysine alters single channel conductance by changing the level of this interference. Moreover, these results provide an experimental example of single channel conductance being modulated at the outer end of the conduction pathway by a mechanism that involves channel activation into open states with different outer vestibule conformations.

Influence of permeant ions on voltage sensor function in the Kv2.1 potassium channel.

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K(+) occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K(+)]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K(+)] modulate the rate of channel activation. Elevation of symmetrical [K(+)] or [Rb(+)] from 0 to 3 mM doubled the rate of on-gating charge movement (Q(on)), measured at 0 mV. Cs(+) produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03-3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Q(on). However, for each of these permeant ions, the speeding of Q(on) occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K(+)-dependent reorientation of the outer vestibule, also abolished the K(+)-dependence of Q(on). Together, the data indicate that the K(+)-dependent reorientation in the outer vestibule was responsible for the change in Q(on). Moreover, similar [K(+)]-dependence and effects of mutagenesis indicate that the K(+)-dependent change in rate of Q(on) can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K(+) reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.

Control of outer vestibule dynamics and current magnitude in the Kv2.1 potassium channel.

In Kv2.1 potassium channels, changes in external [K+] modulate current magnitude as a result of a K+-dependent interconversion between two outer vestibule conformations. Previous evidence indicated that outer vestibule conformation (and thus current magnitude) is regulated by the occupancy of a selectivity filter binding site by K+. In this paper, we used the change in current magnitude as an assay to study how the interconversion between outer vestibule conformations is controlled. With 100 mM internal K+, rapid elevation of external [K+] from 0 to 10 mM while channels were activated produced no change in current magnitude (outer vestibule conformation did not change). When channels were subsequently closed and reopened in the presence of elevated [K+], current magnitude was increased (outer vestibule conformation had changed). When channels were activated in the presence of low internal [K+], or when K+ flow into conducting channels was transiently interrupted by an internal channel blocker, increasing external [K+] during activation did increase current magnitude (channel conformation did change). These data indicate that, when channels are in the activated state under physiological conditions, the outer vestibule conformation remains fixed despite changes in external [K+]. In contrast, when channel occupancy is lowered, (by channel closing, an internal blocker or low internal [K+]), the outer vestibule can interconvert between the two conformations. We discuss evidence that the ability of the outer vestibule conformation to change is regulated by the occupancy of a nonselectivity filter site by K+. Independent of the outer vestibule-based potentiation mechanism, Kv2.1 was remarkably insensitive to K+-dependent processes that influence current magnitude (current magnitude changed by <7% at membrane potentials between -20 and 30 mV). Replacement of two outer vestibule lysines in Kv2.1 by smaller neutral amino acids made current magnitude dramatically more sensitive to the reduction in K+ driving force (current magnitude changed by as much as 40%). When combined, these outer vestibule properties (fixed conformation during activation and the presence of lysines) all but prevent variation in Kv2.1 current magnitude when [K+] changes during activation. Moreover, the insensitivity of Kv2.1 current magnitude to changes in K+ driving force promotes a more uniform modulation of current over a wide range of membrane potentials by the K+-dependent regulation of outer vestibule conformation.

Influence of pore residues on permeation properties in the Kv2.1 potassium channel. Evidence for a selective functional interaction of K+ with the outer vestibule.

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K(+)]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K(+) and Na(+) permeation. Permeability ratio measurements, measurements of the lowest [K(+)] required for interaction with the selectivity filter, and measurements of macroscopic K(+) and Na(+) conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K(+) to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K(+) permeation properties, Lys 356 appeared to be without effect on Na(+) permeation. This suggests that Lys 356 limited K(+) flux by interfering with a selective K(+) binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K(+) permeation properties. Together, these results indicate that a selective, functional K(+) binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K(+) channels. We suggest that this site is the location of K(+) hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K(+) current magnitude, but did not influence the ability of internal K(+) to enter the pore. These data indicate that in Kv2.1, exit of K(+) from the selectivity filter, rather than entry of internal K(+) into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K(+) channels.

Potassium channels.

Potassium channels are integral membrane proteins that selectively transport K+ across the cell membrane. They are present in all mammalian cells and have a wide variety of roles in both excitable and nonexcitable cells. The phenotypic diversity required to accomplish their various roles is created by differences in conductance, the timecourse and mechanisms of different gating events, and the interaction of channels with a variety of accessory proteins. Through the integration of biophysical, molecular, structural, and theoretical studies, significant progress has been made toward understanding the structural basis of K+ channel function, and diseases associated with K+ channel dysfunction.

Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines.

BACKGROUND: Many molecular studies of ion channel function rely on the ability to obtain high quality voltage clamp recordings using the patch clamp technique. For a variety of channel types studied in mammalian cell heterologous expression systems, the lack of experimenter control over expression levels severely hinders the ability to obtain a high percentage of cells with an expression level suitable for high quality recordings. Moreover, it has been nearly impossible to obtain expression levels in mammalian cells well suited for single channel recordings. We describe here the use of an inducible promoter system in a stably transfected mammalian cell line that produces nearly 100% success in obtaining ion channel expression levels suitable for either whole cell or single ion channel studies. RESULTS: We used a tetracycline-regulated expression system to control K+ channel expression in a CHO (Chinese hamster ovary) cell line. Current magnitudes within a reasonably narrow range could be easily and reliably obtained for either macroscopic or single channel recordings. Macroscopic currents of 1-2 nA could be obtained in nearly 100% of cells tested. The desired expression level could be obtained within just 2 to 3 hours, and remained stable at room temperature. Very low expression levels of transfected channels could also be obtained, which resulted in a >70% success rate in the ability to record single channel currents from a patch. Moreover, at these low expression levels, it appeared that endogenous channels produced little or no contamination. CONCLUSION: This approach to controlling ion channel expression is relatively simple, greatly enhances the speed and efficiency with which high quality macroscopic current data can be collected, and makes it possible to easily and reliably record single channel currents in a mammalian cell heterologous expression system. Whereas we demonstrate the ability of this system to control expression levels of voltage-gated K+ channels, it should be applicable to all other channel types that express well in mammalian expression systems.

Effect of external pH on activation of the Kv1.5 potassium channel.

We studied the mechanism by which external acidification from pH 7.3 to 6.8 reduced current magnitude in the Kv1.5 potassium channel. At physiological external [K(+)], a shift in the voltage-dependence of activation was entirely responsible for the acidification-induced decrease in Kv1.5 current magnitude (pK = 7.15). Elevation of external [Ca(2+)] or [Mg(2+)] identically shifted activation curves to the right and identically shifted the pH-sensitivity of the activation curves to more acidic values. Similar observations were made with the Kv2.1 K(+) channel, except that the pK for the activation shift was out of the physiological range. These data are consistent with a mechanism by which acidification shifted activation via modification of a local surface potential. Elimination of eight positive charges within the outer vestibule of the conduction pathway had no effect on the voltage-dependence of activation at pH 7.3 or higher, which suggested that sites exposed to the conduction pathway within the outer vestibule did not directly contribute to the relevant local surface potential. However, mutations at position 487 (within the conduction pathway) displaced the pK of the pH-sensitive shift in activation, such that the sensitivity of Kv1.5 current to physiologically relevant changes in pH was reduced or eliminated. These results suggest that, among voltage-gated K(+) channels, activation in Kv1.5 is uniquely sensitive to physiologically relevant changes in pH because the pK for the sites that contribute to the local surface potential effect is near pH 7. Moreover, the pK for the activation shift depends not only on the nature of the sites involved but also on structural orientation conferred, in part, by at least one residue within the conduction pathway.

The external TEA binding site and C-type inactivation in voltage-gated potassium channels.

The location of the tetraethylammonium (TEA) binding site in the outer vestibule of K+ channels, and the mechanism by which external TEA slows C-type inactivation, have been considered well-understood. The prevailing model has been that TEA is coordinated by four amino acid side chains at the position equivalent to Shaker T449, and that TEA prevents a constriction that underlies inactivation via a foot-in-the-door mechanism at this same position. However, a growing body of evidence has suggested that this picture may not be entirely correct. In this study, we reexamined these two issues, using both the Kv2.1 and Shaker potassium channels. In contrast to results previously obtained with Shaker, substitution of the tyrosine at Kv2.1 position 380 (equivalent to Shaker 449) with a threonine or cysteine had a relatively minor effect on TEA potency. In both Kv2.1 and Shaker, modification of cysteines at position 380/449 by 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET) proceeded at identical rates in the absence and presence of TEA. Additional experiments in Shaker demonstrated that TEA bound well to C-type inactivated channels, but did not interfere with MTSET modification of C449 in inactivated channels. Together, these findings rule out the possibility that TEA binding involves an intimate interaction with the four side chains at the position equivalent to Shaker 449. Moreover, these results argue against the model whereby TEA slows inactivation via a foot-in-the-door mechanism at position 449, and also argue against the hypothesis that the position 449 side chains move toward the center of the conduction pathway during inactivation. Occupancy by TEA completely prevented MTSET modification of a cysteine in the outer-vestibule turret (Kv2.1 position 356/Shaker position 425), which has been shown to interfere with both TEA binding and the interaction of K+ with an external binding site. Together, these data suggest that TEA is stabilized in a more external position in the outer vestibule, and does not bind via direct coordination with any specific outer-vestibule residues.