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Facile visualization of biomolecules is an essential component of the undergraduate biochemistry curriculum. In the past, a number of tools have been used to display biomolecules. More recently, the advent of greater accessibility to virtual reality (VR) and augmented reality (AR) programs has created a new mechanism to visualize biomolecules. However, creation of VR and AR programs often requires extensive coding experience. In order to expand the utility of AR programs in visualization of biomolecules, we enabled undergraduate students to create their own AR protein visualization tool using an off-the-shelf program running in multiplatform smartphones or tablets to enhance student inclusivity. Assessment of this program shows that students can create their own biomolecule models, were enthusiastic about doing so, and found that their learning was enhanced when this AR module was inserted into their upper level biochemistry class. This activity could be readily adapted for remote and online learning.

Cation conducting channelrhodopsins (ChRs) are a popular tool used in optogenetics to control the activity of excitable cells and tissues using light. ChRs with altered ion selectivity are in high demand for use in different cell types and for other specialized applications. However, a detailed mechanism of ion permeation in ChRs is not fully resolved. Here, we use complementary experimental and computational methods to uncover the mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2, in the high- and low-conducting open states. Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca2+ permeability vs. Na+ by nearly two-fold at peak current, but less so at stationary current. We then developed molecular models of dimeric wild-type C1C2 and N297D mutant channels in both open states and calculated the PMF profiles for Na+ and Ca2+ permeation through each protein using well-tempered/multiple-walker metadynamics. Results of these studies agree well with experimental measurements and demonstrate that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by sidechain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and sidechain swinging. We also found that altered cation binding affinities at both the extracellular entrance and central binding sites underly the distinct transport properties of the low-conducting open state. This work significantly advances our understanding of ion selectivity and permeation in cation channelrhodopsins and provides the insights needed for successful development of new ion-selective optogenetic tools. [ABSTRACT FROM AUTHOR]

According to the most recent data from the federal Chemical Safety and Hazard Investigation Board, between 2001 and 2011, more than 120 university laboratory accidents have caused injuries (including one death) and millions of dollars in damages. Laboratory-safety lessons normally comprise lecture slides alongside occasional movies and tours of laboratory facilities. This approach limits the realism of safety instruction within the laboratory. In addition, enthusiasm for laboratory-safety lectures is often low for both instructors and participants. To address these issues, we have developed an augmented-reality (AR) program to increase enthusiasm and enhance the learning experience for laboratory safety using Microsoft HoloLens. AR is an emerging field that uses computer technologies to generate realistic images, sounds, and other sensations that replicate a real environment. When compared with static or one-sided laboratory lectures, our approach creates an interactive learning environment where students must physically move around the laboratory to learn about laboratory safety. As each of the images and holograms for this open-source program can be placed in any location for any laboratory configuration, our program is designed to be used in any laboratory around the globe.

The [Na.sup.+]/[K.sup.+]-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of [Na.sup.+] and [K.sup.+]. It is comprised of at least two subunits, a large catalytic subunit that mediates ATP hydrolysis and ion transport, and an ancillary [beta] subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the a subunit have been extensively studied, little is known about the participation of the [beta] subunit in conformational changes of the enzyme. To elucidate the role of the [beta] subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep [[beta].sub.1] subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the [[beta].sub.1] subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the [Na.sub.+]/[K.sub.+]-ATPase through a fluorophore-labeled [beta] subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the [beta] subunit to certain reaction steps of the [Na.sup.+]/[K.sup.+]-ATPase, which involve changes in the occupancy of the two principle conformational states, [E.sub.1]P and [E.sub.2]P. From these experiments, evidence is provided that the [[beta].sub.1]-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in [[beta].sub.1] isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release. KEY WORDS: voltage-clamp fluorometry * conformational change * kinetics * electrogenic steps * subunit interaction

The conformational dynamics of the [gamma] subunit during ion transport and its position in relation to the [alpha] and the [beta] subunits are examined by using fluorescence resonance energy transfer under voltage clamp conditions. The labeling of the [gamma] subunit at specific residues with fluorophores has induced a decrease in [K.sup.+]-induced stationary current, which is attributed to the perturbation in the [K.sup.+] branch of the reaction cycle of the pump representing a new way to inhibit the pump.

The light-gated cation channel channelrhodopsin-2 (ChR2) has rapidly become an important tool in neuroscience, and its use is being considered in therapeutic interventions. Although wild-type and known variant ChR2s are able to drive light-activated spike trains, their use in potential clinical applications is limited by either low light sensitivity or slow channel kinetics. We present a new variant, calcium translocating channelrhodopsin (CatCh), which mediates an accelerated response time and a voltage response that is ∼70-fold more light sensitive than that of wild-type ChR2. CatCh's superior properties stem from its enhanced Ca2+ permeability. An increase in [Ca2+]i elevates the internal surface potential, facilitating activation of voltage-gated Na+ channels and indirectly increasing light sensitivity. Repolarization following light-stimulation is markedly accelerated by Ca2+-dependent BK channel activation. Our results demonstrate a previously unknown principle: shifting permeability from monovalent to divalent cations to increase sensitivity without compromising fast kinetics of neuronal activation. This paves the way for clinical use of light-gated channels. [ABSTRACT FROM AUTHOR]

Abstract: This paper summarizes our recent work investigating the conformational dynamics and structural arrangement of the Na+/K+-ATPase using voltage clamp fluorometry as well as the latest biochemical, biophysical and structural results from other laboratories. Our research has been focused on combining site-specific fluorophore labeling on the alpha, beta and/or gamma subunit with electrophysiological studies to investigate partial reactions of the ion pump by monitoring changes in fluorescence intensity following voltage pulses and/or solution exchange. As a consequence of these studies, we have been able to identify a residue on the beta subunit, which following labeling with tetramethylrhodamine-6-maleimide can be used as a reporter group to monitor the conformational state of the holoenzyme. Furthermore, we have been able to delineate distance constraints between the alpha, beta and gamma subunits and to examine the relative movements of these proteins during ion transport. Concurrent to this research, significant advancements have been made in understanding the molecular mechanism of the Na+/K+-ATPase. Thus, our research will be compared with the results from other groups and future experimental directions will be proposed. [Copyright &y& Elsevier]

The Na+/K+-ATPase couples the chemical energy in ATP to transport Na+ and K+ across the plasma membrane against a concentration gradient. The ion pump is composed of two mandatory subunits: the α subunit, which is the major catalytic subunit, and the β subunit, which is required for proper trafficking of the complex to the plasma membrane. In some tissues, the ion pump also contains an optional third subunit, γ, which modulates the pump activity. To examine the conformational dynamics of the γ subunit during ion transport and its position in relation to the α and the β subunits, we have used fluorescence resonance energy transfer under voltage clamp conditions. From these experiments, evidence is provided that the γ subunit is located adjacent to the M2-M6-M9 pocket of the ct subunit at the transmembrane-extracellular interface. We have also used fluorescence resonance energy transfer to investigate the relative movement of the three subunits as the ion pump shuttles between the two main conformational states, E1 and E2, as described by the Albers-Post scheme. The results from this study suggest that there is no relative change in distance between the α and γ subunits but there is a relative change in distance between the β and γ subunits during the E2 to E1 transition. It was also observed that labeling the γ subunit at specific residues with fluorophores induces a decrease in K+-induced stationary current. This result could be due to a pertu/bation in the K+ branch of the reaction cycle of the pump, representing a new way to inhibit the pump. [ABSTRACT FROM AUTHOR]