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Highlights from the Literature

Edited by Christopher D. Verrico

Nominated by Amira Klip
The Hospital for Sick Children
amira{at}sickkids.ca

Question: Can photo-cross-linking be used in vivo to define protein interactions?

Background: Protein interactions and the dynamic assemblies they form are essential for many mechanical and chemical manipulations in a cell. Benzophenones (diphenyl ketones), which form a covalent linkage between two macromolecules, have been used extensively as photo-physical probes to identify peptide-protein interactions. In contrast to other photo-cross-linkers, they are stable and can be manipulated under ambient light. Emulating these interactions in vivo, however, has proven difficult because available techniques to define peptide-ligand complexes have been limited to in vitro studies.

Observations: Farrell et al. describe a two-plasmid protocol for studying protein interactions in vivo: one plasmid expresses an orthogonal aminoacyl-tRNA synthetase/tRNA pair, and a second plasmid contains an amber mutant of the gene of interest. This allows the in vivo incorporation of p-benzoyl-L-phenylalanine (pBpa) into the position encoded by the amber codon in any gene transformed into Escherichia coli.

Significance: This methodology allows for the visualization of site-specific incorporation of pBpa into proteins expressed in E. coli with high translational fidelity and efficiency. In principle, this can be applied to any protein that is expressed in E. coli and can help define a protein’s binding partners, allowing one to detect even weak or transient protein interactions by irreversibly trapping them, something that, until now, was impossible.

Nominated by Litsa Kranias
University of Cincinnati Medical College
Litsa.Kranias{at}uc.edu

Question: Do ß₁- and ß₂-adrenergic receptors (ß₁AR and ß₂AR, respectively) form functionally relevant dimers in a native cellular environment?

Background: G protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in signal transmission, are activated by peptide and nonpeptide neurotransmitters, hormones, growth factors, odorant molecules, and light. The cellular responses mediated by GPCR result from numerous intricate networks of intracellular signaling pathways. Although traditionally thought of as monomers, dimerization of the ß₁ARs and ß₂ARs has been shown to exist and to display unique ligand-binding properties and signaling cascades in vitro. However, the physiological relevance of these interactions in a native cellular environment has not been demonstrated.

Observations: By using ß₁AR and ß₂AR double-knockout mice and then expressing each subtype singly or simultaneously in cardiomyocytes, Zhu et al. determined that these receptors are able to form heterodimers with distinct functional and pharmacological profiles. Expression of the heterodimer increased ligand-binding affinity. Moreover, they found that in contrast to ß₁AR expression alone, ß₂AR expression alone increased baseline contractility, which was eliminated by the presence of ß₁AR.

Significance: This work indicates that intermolecular interactions between ß1ARs and ß2ARs exist, resulting in functionally distinct ßAR heterodimers. Interestingly, they also reported discrepancies between previous studies of this heterodimer in vitro when contrasted with their current results, highlighting the importance of using a native cellular environment. This ß1-ß2AR complex may be particularly relevant, as ßARs are targets of many therapeutic agents, including numerous top-selling drugs.

Nominated by Graham Lamb
La Trobe University
g.lamb{at}latrobe.edu.au

Question: Can a method to image free Ca²⁺ within organelles be developed?

Background: Intracellularly, Ca²⁺ regulates numerous physiological phenomena, including muscle contraction, cellular division, secretion of hormones and neurotransmitters, and apoptosis. Much of this Ca²⁺ is released into the intracellular space from intracellular stores such as the sarcoplasmic reticulum (SR). Although numerous methods have been developed to measure intracellular Ca²⁺, the effects of Ca²⁺ within the SR remain vague.

Observations: This study by Launikonis et al. describes a technique, termed shifted excitation and emission ratioing (SEER), for quantifying free Ca²⁺ in intracellular compartments that is suited to confocal microscopy, which has the advantages of high spatial and temporal resolution. They provide quantitative confocal images of free Ca²⁺ inside the SR of skeletal muscle fibers. Interestingly, SR Ca²⁺ concentrations are substantially higher than in the nearby mitochondria, even when cytoplasmic Ca²⁺ is raised above its normal resting level.

Significance: This paper describes a flexible confocal imaging and analysis technique with dynamic range and sensitivity and high spatial and temporal resolution, an exciting advancement that is likely to have widespread use. Using this novel methodology, the authors provide the first direct quantification of free Ca²⁺ inside the SR. Importantly, this approach is not likely limited to its current application, as it could potentially be applied to quantify other parameters, such as membrane potential.

Nominated by Angus Nairn
Associate Editor, Journal of General Physiology
Yale University School of Medicine
angus.nairn{at}yale.edu

Question: What advantages can be gained by monitoring ion channels via "optical patch-clamping"?

Background: Ion channels are fundamental for regulating the activity of cells. The patch-clamp technique revolutionized our ability to monitor the activity of these channels, allowing currents to be recorded through single channels with subpicoampere and submillisecond resolution. Nevertheless, patch-clamping is limited by an inability to spatially map the channels, to independently record from more than one channel at a time, and by the disruption of the cytoskeleton caused by the micropipettes. Although optical imaging techniques have held some promise in circumventing these issues, the temporal resolution (~30 ms) and signal-to-noise ratio has hindered their use.

Observations: Technical enhancements allowed Demuro and Parker to use total internal reflection fluorescence microscopy (TIRFM) to examine the behavior of Ca²⁺-permeable ion channels in Xenopus oocytes. They imaged the activity of nicotinic acetylcholine receptors/channels, which notably have only partial permeability to Ca²⁺, by employing an ultrafast, high-sensitivity camera that improved the temporal resolution to ~2 ms. Thus, although this approach is restricted to imaging Ca²⁺ flux, it will be applicable to diverse channels that show appreciable Ca²⁺ permeability.

Significance: This technique marks an important step in the use of noninvasive optical techniques to examine ion channel kinetics of numerous individual ion channels. The major advantages of this technique include the ability to monitor channel motility over time, spatial resolution of channel locations, and simultaneous comparisons between channel-gating kinetics. As noted by the authors, single-channel imaging may also hold promise as a more approachable high-throughput screening process to determine drug effects on ion channel activities.

Nominated by Lawrence Palmer
Associate Editor, Journal of General Physiology
Cornell University
lgpalm{at}med.cornell.edu

Question: What is the pore architecture and gating mechanism of the P/Q-type Ca²⁺ channel?

Background: Voltage-gated Ca²⁺ channels (VGCCs) are essential for muscle contraction, neurotransmitter release, neurodevelopment, and gene expression. VGCCs are multimeric complexes of several distinct subunits, the most functionally defining of which are the ₁-subunits, which form the ion-conductance pore, voltage sensor, gating apparatus and carry many sites for regulation. ₁-Subunits consist of four homologous repeats with six transmembrane domains (S1–S6) and a pore loop between S5 and S6, which forms the ion-selectivity filter. In contrast to voltage-gated K⁺ channels, little is known about the architecture of VGCC inner pores and the molecular movements associated with gating.

Observations: In the first report by Yang and colleagues, they use the substituted-cysteine accessibility method (SCAM), which has been previously used in other channels to identify architecture and gating mechanisms, to identify the residues that line the pore region of P/Q-type Ca²⁺ channel. As anticipated, the inner pore region is in fact formed by S6, although the loose packing of the S6 and S5 segments is different from what is observed in K⁺ channels. In the accompanying report, they determined that the S6 helices of the P/Q-type Ca²⁺ channel undergo conformational changes during gating and that the location of the activation gate is at the intracellular end of the pore.

Significance: Although the VGCC described here shares some properties with that of K⁺ channels, it also appears to possess some unique features. Nonetheless, these results provide a framework for future studies of Ca²⁺ channel ion permeation, mechanisms of gating, and drug-binding sites. Of particular importance may be the loose packing of the S5 and S6 transmembrane segments, which would theoretically allow binding of large molecules.

Nominated by Pontus Persson
Editor, The American Journal of Physiology-Regulatory, Integrative, and Comparative Physiology
Humboldt University
pontus.persson{at}charite.de

Question: By what mechanism does vitamin E supplementation affect the aging process in mice?

Background: Held for decades, one theory of aging contends that oxygen free radicals induce damage to cell walls, metabolic machinery, and DNA, which induces the aging process. As such, mitochondria are a focal point in the study of aging, as they provide ATP for cellular processes via the citric acid cycle and the electron transport chain, which produces free radicals. Mitochondria possess their own DNA, which is susceptible to free radical-induced damage, and unlike nuclear DNA, mitochondria do not have as many repair mechanisms. This damage then can cause mitochondrial dysfunction leading to impaired energy production and cellular aging.

Observations: Using the CD-1/UCadiz strain of mice, which is a senescence-accelerated strain, Navarro et al. examined the affects of -tocopherol (vitamin E) supplementation (5 g/kg of food). Male mice showed a 40% increase in median and a 17% increase in maximal lifespan, with increased neuromuscular performance and cognitive exploratory activity. Additionally, they found a negative correlation between oxidative damage and mitochondrial enzymatic activities in the brain and liver. There was also a linear correlation described between brain mitochondrial enzymatic activities and neurological performance.

Significance: These results suggest that vitamin E supplementation has positive effects on several parameters of aging, which importantly includes neurological functions. This is in accord with the prophylactic effects of vitamin E on age-associated cardiovascular disease, chronic inflammation, and neurological disorders. Although studies are still needed to determine the threshold dose, this work suggests that the mechanism by which vitamin E mitigates the damaging effects of oxidative stress is by increasing mitochondrial enzymatic activities.

Nominated by Michael Welsh
University of Iowa
michael-welsh{at}uiowa.edu

Question: What role does apoptosis have in the aging process?

Background: Mitochondrial dysfunction, which can be defined as the progressive decline in metabolic and physiological processes, may be due to damage of mitochondrial DNA (mtDNA) and may underlie the aging process. However, the role of mtDNA damage in the aging process is unclear. It has been posited that free radical-mediated mitochondrial damage accumulates over time and accounts for the induction of apoptosis, which in turn contributes to the aging process. Conversely, apoptosis could contribute to the aging process independently of free radical-mediated mtDNA damage.

Observations: Prolla and colleagues created mice with impaired DNA proofreading capacity, which resulted in a severalfold increase in mtDNA mutations. Intriguingly, the mutated mice developed symptoms of old age more commonly seen in humans than in mice, such as gray hair and hair loss. More importantly, they found that the accelerated aging phenotype was correlated with the induction of apoptotic markers but not several markers of oxidative stress, including oxidative damage to DNA and RNA. Notably, there was also an increase in apoptotic markers as normal mice aged.

Significance: These findings suggest that apoptosis, not oxidative stress, may be a key mechanism of aging. This is supported by studies that retard aging by restricting calories, which causes an attenuation of mtDNA mutations and the accumulation of apoptotic pathways. However, what needs to be determined is whether the mutant mice in this study died via a similar pathology to that seen in normal mice and are thus truly representative of an aging model. If true then pharmaceutical interventions could potentially mitigate these genetic defects and increase life expectancy.

Nominated by Jeff Sands
Editor, The American Journal of Physiology-Renal
Physiology
Emory University
jsands{at}emory.edu

Question: Can cGMP phosphodiesterase inhibitors induce aquaporin 2 (AQP2) membrane insertions?

Background: AQP2 is a water channel expressed in collecting duct principal cells of the kidneys, where it contributes to fluid homeostasis. One widely known mechanism of AQP2 membrane accumulation is via stimulation of the antidiuretic hormone vasopressin (VP) type 2 receptor (V2R), which is activated through cAMP. More recently, Bouley, Brown, and colleagues demonstrated that AQP2 membrane insertion can also be induced via a cGMP-dependent mechanism and can thus stimulate water permeability.

Observations: The current investigations were focused on determining whether inhibition of the cGMP phosphodiesterases (specifically PDE5, which is expressed in renal collecting ducts) would produce AQP2 membrane insertion. In vitro tests illustrated that inhibition of PDE5 resulted in increased levels of cGMP. Administration of Viagra, a PDE5 inhibitor, to rat kidney collecting duct principal cells demonstrated that the rapid rise in cGMP levels resulted because of attenuated cGMP breakdown. In vivo, Viagra stimulated cell surface accumulation of AQP2 in Brattleboro rats (which possess a genetic mutation that impairs vasopressin synthesis).

Significance: Normally the kidneys filter the blood, removing salts and minerals and concentrating them into urine. People with nephrogenic diabetes insipidus (NDI) have problems with AQP2 protein trafficking, which results in an inability to maximally concentrate urine, severe dehydration, and electrolyte imbalance. Drugs that target the V2Rs could have therapeutic efficacy, but the widespread distribution of V2Rs presents a dilemma. As such, this work provides important preclinical, pharmacological evidence that PDE5 inhibitors could represent an alternative pathway to induce AQP2 plasma membrane insertion in NDI patients, effectively circumventing the V2R pathway.

Akt1/protein kinase B is critical for ischemic and VEGF-mediated angiogenesis.

Nominated by William Sessa
Yale University School of Medicine
william.sessa{at}yale.edu

Question: What is the role of Akt1 in the regulation of angiogenesis and the adaptive/maladaptive consequences following chronic Akt activation in the cardiovascular system?

Background: Akt, also known as protein kinase B (PKB), is an important signaling molecule involved in many cellular functions, including growth, survival, and metabolism. In mammalian cells Akt exists in three isoforms (Akt1, Akt2, and Akt3) that have some degree of functional overlap, but their precise roles are not clear. In the heart and vasculature Akt regulates angiogenesis, apoptosis, and cardiac hypertrophy. In fact, Akt overexpression has been shown to have a protective role in the cardiovascular system. However, long-term Akt overexpression appears to have detrimental consequences on cardiac function. The mechanism by which Akt becomes deleterious is unknown, as are the specific roles of the Akt isoforms in this effect.

Observations: The studies of Shiojima et al. and Nagoshi et al. provide some mechanistic insights into the cardiac dysfunction that follows long-term Akt activation. Shiojima et al. found that coordinated angiogenesis and cellular growth was required to maintain the benefits observed following acute Akt1 activation. This is evidenced by the fact that inhibiting angiogenesis in the acute phase of Akt1 activation led to dysfunction and that angiogenesis diminished as hearts underwent pathological remodeling following long-term Akt1 activation. Nagoshi et al. found that the adverse effects of chronic Akt1 activation on cardiac function are likely caused by an inhibition of upstream feedback signaling pathways. This is demonstrated by the fact that when the signaling pathway is restored they were able to rescue function and prevent dysfunction. The work of Ackah et al. explored which Akt isoform is responsible for mediating adaptive postnatal angiogenesis. Their work suggests that the Akt1 isoform, but not Akt2 or Akt3, regulates adaptive angiogenesis and arteriogenesis.

Significance: Collectively, these findings are of considerable medical and pharmaceutical interest due to the connection of Akt with diabetes, cell survival, and cancer. These papers demonstrate definitive in vivo functions of this kinase and provide important new insights into the central role of Akt1 in the regulation of angiogenesis and the maladaptive consequences of chronic unregulated Akt activation in the heart.

Nominated by Kevin Strange
Vanderbilt University
kevin.strange{at}vanderbilt.edu

Question: Can a mathematical model accurately predict osmoregulatory responses in yeast?

Background: Osmoregulation is a homeostatic mechanism by which organisms actively maintain their internal solute concentration. The yeast Saccharomyces cerevisiae has been used extensively to study the phenomenon of osmoregulation by monitoring its responses to osmotic shock. Yeasts possess a membrane sensor (Sln 1) that when triggered transiently activates the high-osmolarity glycerol (HOG) signaling system, which in turn affects gene expression and ultimately stimulates glycerol production to increase intracellular osmotic pressure.

Observations: Klipp et al. describe a mathematical model that integrates the response of yeast cells to hyperosmotic shock including the membrane osmosensor, the HOG signaling system, gene expression, glycerol accumulation, and the feedback effect of internal glycerol levels on osmotic pressure and cellular volume. Using a combination of experimental studies and simulations, they found that the model was predictive since it identified previously unknown aspects of the feedback system that were empirically confirmed.

Significance: The model accurately provides novel and unexpected insights concerning the role of phosphotases in shutting down the HOG pathway and ending the osmotic shock response. The model suggests that the mechanism responsible is not enhanced expression of genes encoding phosphatases but the rapid closure of the Fps1 glycerol efflux channel. Both experiments and simulations demonstrate that the phosphotase mechanism of feedback control is too slow and weak but that closure of Fps 1 channels is essential; otherwise the stimulated glycerol produced would leak into the extracellular environment. Perhaps more importantly, this model could potentially be used to model osmoregulation in other organisms.

Nominated by Michael Welsh
University of Iowa
michael-welsh{at}uiowa.edu

Question: Is there a feedback mechanism from postsynaptic cells that modifies the functional properties of its presynaptic connections?

Background: There are billions of synapses in the brain. These heterogeneous connections form between neurons with varying efficacy, the strength of which depends on the number of contacts and the number of postsynaptic receptors. It is also dependent on the probability of neurotransmitter being released, which is a function of the action potential-induced increase in the Ca²⁺ concentration. However, measuring release probability has only been achieved in tissue culture.

Observations: Koester and Johnston, using optical quantal analysis of whole-cell recordings from pairs of monosynaptically coupled neurons in rat somatosensory cortical slices, describe a correlation between the amount of stimulated presynaptic Ca²⁺ released and the probability of neurotransmitter release. Surprisingly, however, they determined that the size of the Ca²⁺ signal from a presynaptic terminal that induces neurotransmitter release depends on its postsynaptic target.

Significance: These results suggest that there is communication between the presynaptic cells with their postsynaptic targets and that this interaction functions to normalize the Ca²⁺ transients and release probabilities. This was surely unexpected, and the mechanism of communication will undoubtedly be the focus of future research. Additionally, the authors noted that the pyramidal-to-pyramidal connections possessed the greatest variation in release probability, a fact that may endow them with the ability to change more readily as a function of usage.

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