ThermoTRP channels as modular proteins with allosteric gating

Ion channels activate by sensing stimuli such as membrane voltage, ligand binding or temperature and transduce this information into conformational changes that open the channel pore. Thus, a key question in understanding ion channel function is how do the protein domains involved in sensing stimuli (sensors) and opening the pore (gates) communicate. In this regard, transient receptor potential (TRP) channels that confer thermosensation [A. Dhaka, V. Viswanath, A. Patapoutian, TRP ion channels and temperature sensation, Annu. Rev. Neurosci. 29 (2006) 135-161; I.S. Ramsey, M. Delling, D.E. Clapham, An introduction to TRP channels, Annu. Rev. Physiol. 68 (2006) 619-647] (thermoTRP; Q(10)>10) are unique to the extent that they integrate a variety of physical and chemical stimuli. In some cases such as, for example, the vanilloid receptor TRPV1 [M.J. Caterina, M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, D. Julius, The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature 389 (1997) 816-824] and TRPA1 [G.M. Story, A.M. Peier, A.J. Reeve, S.R. Eid, J. Mosbacher, T.R. Hricik, T.J. Earley, A.C. Hergarden, D.A. Andersson, S.W. Hwang, P. McIntyre, T. Jegla, S. Bevan, A. Patapoutian, ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures, Cell 112 (2003) 819-829; S. Jordt, D. Julius, Molecular basis for species-specific sensitivity to "hot" chilli peppers, Cell 108 (2002) 421-430] the integration of these stimuli elicit pain [M. Tominaga, M.J. Caterina, A.B. Malmberg, T.A. Rosen, H. Gilbert, K. Skinner, B.E. Raumann, A.I. Basbaum, D. Julius, The cloned capsaicin receptor integrates multiple pain-producing stimuli, Neuron 21 (1998) 531-543; M. Bandell, A. Dubin, M. Petrus, A. Orth, J. Mathur, S. Hwang, A. Patapoutian, High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol, Nat. Neurosci. 9 (2006) 466-468; S. Zurborg, B. Yurgionas, JA. Jira, O. Caspani, P.A. Heppenstall, Direct activation of the ion channel TRPA1 by Ca(2+), Nat. Neurosci. 10 (2007) 277-279]. These stimuli include voltage, pH, agonist binding, and temperature. Understanding how each of these distinct physiological signals regulate channel opening will be informative about the mechanical linkages that can act either independently or in concert to influence channel activation. In this paper we show that thermoTRP channel-forming proteins are modular in the sense that certain structure or structures (modules) confer temperature-dependent regulation, whereas others confer voltage-dependent regulation. We also discuss the thermodynamic basis of heat and cold activation in an effort to elucidate what confer to these channels the capability to be gated by temperature directly.     

Molecular mechanisms of canalization: Hsp90 and beyond

The Hsp90 chaperone machine facilitates the maturation of a diverse set of ‘client’ proteins. Many of these Hsp90 clients are essential nodes in signal transduction pathways and regulatory circuits, accounting for the important role Hsp90 plays in organismal development and responses to the environment. Recent findings suggest a broader impact of the chaperone on phenotype: fully functional Hsp90 canalizes wild-type phenotypes by suppressing underlying genetic and epigenetic variation. This variation can be expressed upon challenging the Hsp90 machinery by environmental stress, genetic or pharmaceutical targeting of Hsp90. The existence of Hsp90-buffered genetic and epigenetic variation together with plausible release mechanisms has wide-ranging implication for phenotype and possibly evolutionary processes. Here, we discuss the role of Hsp90 in canalization and organismal plasticity, and highlight important questions for future experimental inquiry.     

Moving beyond molecular mechanisms

A major goal in cell biology is to bridge the gap in our understanding of how molecular mechanisms contribute to cell and organismal physiology. Approaches well established in the physical sciences could be instrumental in achieving this goal. A better integration of the physical sciences with cell biology will therefore be an important step in our quest to decipher how cells work together to construct a living organism.Over the past 60 years, the field of cell biology has been firmly rooted in understanding the molecular basis of complex cellular processes including genome replication, migration, metabolism, and adhesion. This progress has been enabled by advances in molecular biology, biochemistry, physical chemistry, single-molecule physics, and microscopy. Bringing together these disciplines has been successful in identifying the molecular composition of macromolecular machines, characterizing the structure and physical properties of single proteins within cells, reconstituting complex macromolecular machinery in vitro, and imaging the dynamics and function of these machines in vivo.Despite this amazing progress, a major challenge facing cell biology is understanding how the chemical and physical properties of molecular machinery come together to guide cell processes. How do varied physical and chemical signals in the environment determine whether a cell survives, proliferates, or migrates? What circuitry allows for a complex body plan to be constructed out of a single-celled embryo? The signals in the environment are noisy, with fluctuations in both time and space. Moreover, as anyone who has tried to characterize cells is aware, cell phenotypes are variable both across individual cells and within a single cell over time. In the presence of all this noise, cells execute some processes exceedingly reliably (e.g., DNA segregation in cell division). Others, such as the determination of protrusive activity in a migrating cell, appear to be more variable. How does this complex network of stochastic chemical and mechanical machinery enable robust and complex decision making at the cell scale?The answers to these questions require knowledge of cell structure at the scale between single molecules and whole cells (Fig. 1). This intermediate, or mesoscopic, length scale has different names depending on who you ask. You can think of it as a “system” or interconnected network of biochemical interactions that provide a logic circuit as to how cells process a signal to decide on an output. It can be a subcellular machine consisting of a collection of macromolecules designed to work together for a desired mechanical output, such as cargo transport, DNA segregation, or cell movement. There is a significant gap in our understanding at this scale. To make an analogy between a cell and a car: most of us have a good understanding of the car’s component materials (e.g., rubber, metal), and in some cases we understand the individual machines that make up parts of the whole (e.g., the engine, transmission). However, we do not have a good understanding of the essential control parameters of the machines or how these are wired together to form productive, more complex machinery (e.g., creating the forward, backward, and turning motions). Understanding the control parameters that regulate macromolecular assemblies, and how these are wired together to enable complex cell outputs, represents an exciting frontier in cell biology.Open in a separate windowFigure 1.The scales of cell biology. Shown are images illustrating the range of scales in cell biology. At the smallest (∼10⁻⁹ m) is that of molecules represented by the structure of G-actin (left; reproduced from Paavilainen et al. 2008. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200803100) and the largest (10⁻⁵ to 10⁻⁴ meters) is that of cell physiology, represented by a migrating fibroblast with a labeled actin cytoskeleton (right; image courtesy of Patrick Oakes). In between these length scales reside: macromolecular assemblies (10⁻⁸ to 10⁻⁷ m) of individual proteins, represented by a schematic of an Arp2/3-mediated F-actin branch (second from the left); and organelles (10⁻⁷ to 10⁻⁵ m), such as lamellipodia (third from the left), which are formed by the integration of macromolecular assemblies into a mechanochemical machine depicted as a pathway diagram. At the next level are organelle systems (10⁻⁴ to 10⁻⁵ m) that integrate organelles together for a specific aspect of cell physiology, represented by a fluorescent image of actin overlaid with vectors of actin flow at the leading edge that result from the coordination of numerous regulatory organelles across the cell (second from the right; reproduced from Thievessen et al. 2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201303129). Understanding the processes at this intermediate scale will greatly aid in our knowledge of how molecules construct living cells.Many areas of the physical sciences have been devoted to studying how collections of objects work together to construct a material or machine. In this construction, new properties emerge that could not be predicted or understood by studies of objects in isolation. For instance, electrical engineers need to know how circuit elements are connected in order to predict the circuit response. Or, in condensed matter physics, interactions between atoms and/or molecules result in properties such as elasticity or viscosity. In these areas of science, it is well appreciated that knowledge of individual components (in isolation) cannot predict the output of the entire system. By analogy, this would imply that understanding the molecular components of a cell, which has been the gold standard of cell biology, is insufficient. As cell biology starts to address questions wherein cells are thought of as “systems,” “materials,” or “machines,” there are numerous challenges that can be informed by approaches that have proven successful in the studies of materials and machines in the physical world.

Developing a common community

Cell biology is an inherently multidisciplinary science, requiring approaches from genetics, chemistry, physics, applied mathematics, and engineering. While biochemical and genetic approaches have been successfully integrated into the field, other disciplines require more effort. Physical scientists that join the field of cell biology retain the training and language from their physical discipline, which has been specialized for specific purposes. Applied mathematicians, condensed matter physicists, and mechanical engineers all have unique perspectives on how to model complex biological phenomena (Fig. 2). This has led to the development of parallel theoretical and experimental approaches for modeling cell biological phenomena that are difficult to directly compare or rigorously test. A challenge for the future is to develop a community of researchers that will integrate these diverse physical approaches to identify strengths, resolve differences, and determine the best approaches for modeling cell behaviors.Open in a separate windowFigure 2.The integration of physical sciences with cell biology. A flow chart showing examples of how various disciplines from the physical sciences (bottom) have optimized a variety of theoretical/modeling tools (left) as well as experimental techniques (right) that have been applied to cell biological problems. However, these experimental and theoretical tools have been optimized for their home disciplines. A current challenge is to systematically have them benchmarked against each other and identify their weaknesses and strengths before using them to provide a new framework optimized for mesoscale cell biology.

Precision in language

One of the simplest solutions to implement is to develop a consistent and precise language to describe measurements or ideas. In my field, which centers on how mechanical forces are sensed and generated by cells, terms like “mechanosensing” or even “stiffness sensing” are used without precision, resulting in confusion of what is known versus just “thought to be true.” Precision of language is essential for standardizing experimental protocols and measurements and in being able to clearly communicate conclusions and ideas.

Construction and validation of physical methods

One historical role of physical scientists in biology has been the introduction of new experimental and analytical tools. Some of these tools, such as microscopy and scattering techniques, have been developed extensively. However, in other cases, the nature of measurements require small apparatuses that can be difficult to replicate or operate (magnetic tweezers are a notorious example), making it difficult for other laboratories to build upon this knowledge. Similar issues arise in analysis and methods. It is extremely important for these methods to be used and validated by different laboratories to confirm results independently and by many individuals so that the language used to describe physical concepts and results can be made more precise. Being able to directly compare two different measurement techniques so that the same parameters can be used is essential for resolving discrepancies.Even though the goal is to understand cell physiology, model testing will require physical characterization that may not immediately inform a biological process. To use an analogous example: the work in basic materials science of magnetism that needed to be performed before we could construct and build computer hard drives. It is my hope that the cell biology community will remain interested in these advances in characterization of biological materials and systems, as they are crucial to uncovering synergies that are not currently apparent.

Feedback between modeling and experiments

In the physical sciences, research has evolved so that individuals typically focus on either theory or experimentation. Of course, each of these can be further subdivided into analytical theory versus computer modeling, as well as sample preparation versus characterization. This specialization has emerged as both the questions and fields themselves become more mature. It also has led to a vigorous feedback between theoretical prediction, experimental measurement, and new materials development. To be useful, models need to be falsifiable. There is increasing evidence that many of the models used in biology are over-parameterized and, consequently, difficult (or impossible) to falsify. That is, when parameters are assigned with molecular-level details, the number of parameters quickly becomes large. In these scenarios, changes in the parameter value have little effect on the model predictions and make it difficult to verify the accuracy of the model (for more details, see http://www.lassp.cornell.edu/sethna/Sloppy/). Identifying order parameters that encompass the physical quantities or metrics (e.g., elastic modulus, organelle transport) that make up many of the molecular details is essential for developing models with fewer control parameters. Such order parameters will provide crucial insight into understanding regulation of the individual macromolecular machinery.The word mechanism in cell biology typically refers to a molecular mechanism that is explored rigorously by genetic and biochemical testing. Understanding the physical mechanism requires both identification of the parameters controlling a system and then elucidation of the regulation of parameter values. Thus, seldom does a single molecular mechanism tie directly into a physical parameter. Moreover, understanding how molecular interactions give rise to a single physical parameter is not straightforward, and may require years of work. It is quite natural to apply models and approaches that we have used to engineer machines, such as the flow of decision making in electrical circuits or mechanic designs. However, cells are working under different sets of constraints, and a future challenge of understanding cellular machines is that completely different design principles may be used.Establishing a culture that encourages dynamic feedback between theory, experimentation, and physiology is crucial to advancing the integration of physical sciences with cell biology. A potentially very exciting possibility is that understanding the physical mechanisms controlling biological machines will enable a completely new set of design principles that provide insight into how living cells are able to respond and adapt to highly variable environments. This will enable understanding of how these states change during disease progression and the capability of engineering biological cells to maintain a healthy phenotype.     

Boron transport mechanisms: collaboration of channels and transporters

Boron (B) is an essential element for plants, but is also toxic when present in excess. B deficiency and toxicity are both major agricultural problems worldwide, and elucidating the molecular mechanisms of B transport should allow us to develop technology to alleviate B deficiency and toxicity problems. Recent milestones include the identification of a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, from Arabidopsis thaliana. Both proteins were shown to be required for plant growth under B limitation. In addition, BOR1 homologs are required for B homeostasis in mammalian cells and B-toxicity tolerance in yeast and plants. Here, we discuss how transgenic approaches show promise for generating crops that are tolerant of B deficiency and toxicity.     

Mechanistic and therapeutic perspectives for cardiac arrhythmias:beyond ion channels

Cardiac arrhythmias are among the most common causes of death in the world. Foundational studies established the critical role of ion channel disorders in arrhythmias, yet defects in ion channels themselves, such as mutations, may not account for all arrhythmias. Despite the progress made in recent decades, the antiarrhythmic drugs currently available have limited effectiveness,and the majority of these drugs can have proarrhythmic effects. This review describes novel knowledge on cellular mechanisms that cause cardiac arrhythmias, focuses on the dysfunction of subcellular organelles and intracellular logistics, and discusses potential strategies and challenges for developing novel, safe and effective treatments for arrhythmias.     

Transient receptor potential ion channels and animal sensation: lessons from Drosophila functional research

Ion channels of the transient receptor potential (TRP) superfamily are non-selective cationic channels with six transmembrane domains. The TRP channel made its first debut as a light-gated Ca2+ channel in Drosophila. Recently, research on animal sensation in Drosophila disclosed other members of the TRP family that are required for touch sensation and hearing as well as the sensation of painful stimuli.     

Update on peripheral mechanisms of pain: beyond prostaglandins and cytokines

The peripheral nociceptor is an important target of pain therapy because many pathological conditions such as inflammation excite and sensitize peripheral nociceptors. Numerous ion channels and receptors for inflammatory mediators were identified in nociceptors that are involved in neuronal excitation and sensitization, and new targets, beyond prostaglandins and cytokines, emerged for pain therapy. This review addresses mechanisms of nociception and focuses on molecules that are currently favored as new targets in drug development or that are already targeted by new compounds at the stage of clinical trials - namely the transient receptor potential V1 receptor, nerve growth factor, and voltage-gated sodium channels - or both.     

Responses of human skin temperature and thermal sensation to step change of air temperature

1. 1. The purpose of this paper is to clarify the non-linearity of the human physiological and psychological responses to step change of air temperature by impulse response analysis using Discrete Fourier Transformation.

2. 2. Experiments were conducted to investigate the effect of thermal transients on human responses.

3. 3. Experimental conditions were as follows: lowering air temperature from 30 to 20°C and raising air temperature from 20 to 30°C.

4. 4. The responses of local skin temperature on lowering air temperature from 30 to 20°C are not necessarily opposite to the responses found on raising air temperature from 20 to 30°C.

5. 5. From impulse response analysis using Discrete Fourier Transformation, skin temperature responses to the opposite air temperature change do not necessarily coincide with each other whenever the same temperature stimulus is occurred.

Impaired pain sensation in mice lacking Aquaporin-1 water channels

Aquaporin-1 (AQP1), a membrane water channel, is expressed in choroid plexus where it contributes to cerebrospinal fluid production. Here, we show that AQP1 is also expressed in the dorsal horn of the spinal cord and the trigeminal nucleus caudalis, regions that process pain information. Within the dorsal root and trigeminal sensory ganglia, AQP1 is concentrated in small diameter cell bodies, most of which give rise to unmyelinated C-fibers. To study the role of AQP1 in pain signaling, we compared acute pain responses in wild-type mice and in mice lacking AQP1. AQP1^−/− mice had reduced responsiveness to thermal and capsaicin chemical stimuli, but not to mechanical stimuli or formalin. These results provide evidence for AQP1 expression in nociceptive neurons and suggest that AQP1 may play a role in pain signal transduction.     

Ligand-gated ion channels: mechanisms underlying ion selectivity

Anion/cation selectivity is a critical property of ion channels and underpins their physiological function. Recently, there have been numerous mutagenesis studies, which have mapped sites within the ion channel-forming segments of ligand-gated ion channels that are determinants of the ion selectivity. Site-directed mutations to specific amino acids within or flanking the M2 transmembrane segments of the anion-selective glycine, GABA(A) and GABA(C) receptors and the cation-selective nicotinic acetylcholine and serotonin (type 3) receptors have revealed discrete, equivalent regions within the ion channel that form the principal selectivity filter, leading to plausible molecular mechanisms and mathematical models to describe how ions preferentially permeate these channels. In particular, the dominant factor determining anion/cation selectivity seems to be the sign and exposure of charged amino acids lining the selectivity filter region of the open channel. In addition, the minimum pore diameter, which can be influenced by the presence of a local proline residue, also makes a contribution to such ion selectivity in LGICs with smaller diameters increasing anion/cation selectivity and larger ones decreasing it.     

Trafficking mechanisms and regulation of TRPC channels

TRPC channels are Ca²⁺-permeable cation channels which are regulated downstream from receptor-coupled PIP₂ hydrolysis. These channels contribute to a wide variety of cellular functions. Loss or gain of channel function has been associated with dysfunction and aberrant physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their effects on the downstream cellular processes. Such interactions also compartmentalize the Ca²⁺ signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in trafficking of the seven members of the TRPC family (TRPC1–TRPC7).     

Effects of temperature and wind on facial temperature, heart rate, and sensation.

Skin temperature measurements of the face have shown that the cheek cools faster than the nose and the nose faster than the forehead. The cooling effect of wind is maximum at wind speeds between 4.5 and 6.7 m/s. Cold winds produce significant bradycardia, which is, however, much more pronounced during the expiratory phase of respiration. A significant correlation was noted between cooling of face and the reflex bradycardia observed. Similarly, a very significant correlation was noted between drop in skin temperature and subjective evaluation of cold discomfort. Consequently, the drop in skin temperature, reflex bradycardia, and subjective evaluation are parameters which are directly affected by cold wind and can be used as adequate indicators of the degree of discomfort. When comparing the present results with the windchill index, it was found that in the zone described as "dangerously cold" the index fits well with the physiological measurements. In the zone described as "bitterly cold," the index by comparison with actual skin temperature measurements and subjective evaluation underestimates the cooling effects of combined temperature and wind by approximately 10 degrees C.     

Thirsty plants and beyond: structural mechanisms of abscisic acid perception and signaling

Abscisic acid (ABA) is a plant hormone with important functions in stress protection and physiology. Recently, the PYR/PYL/RCAR family of intracellular ABA receptors was identified. These receptors directly link ABA perception to a canonical ABA signaling pathway, in which ABA-bound receptors bind and inhibit type 2C phosphatases. High resolution crystal structures of members of this family have been solved in all relevant states: as apo receptors, bound to ABA, and as receptor-ABA-phosphatase complexes. Together, these structures provide a detailed gate-latch-lock mechanism of ABA recognition, receptor-PP2C interaction, and inhibition of the PP2C phosphatase activity and provide a basis for the design of synthetic ABA agonists for stress protection of crop plants.     

Regional differences in temperature sensation and thermal comfort in humans

Sensations evoked by thermal stimulation (temperature-related sensations) can be divided into two categories, "temperature sensation" and "thermal comfort." Although several studies have investigated regional differences in temperature sensation, less is known about the sensitivity differences in thermal comfort for the various body regions. In the present study, we examined regional differences in temperature-related sensations with special attention to thermal comfort. Healthy male subjects sitting in an environment of mild heat or cold were locally cooled or warmed with water-perfused stimulators. Areas stimulated were the face, chest, abdomen, and thigh. Temperature sensation and thermal comfort of the stimulated areas were reported by the subjects, as was whole body thermal comfort. During mild heat exposure, facial cooling was most comfortable and facial warming was most uncomfortable. On the other hand, during mild cold exposure, neither warming nor cooling of the face had a major effect. The chest and abdomen had characteristics opposite to those of the face. Local warming of the chest and abdomen did produce a strong comfort sensation during whole body cold exposure. The thermal comfort seen in this study suggests that if given the chance, humans would preferentially cool the head in the heat, and they would maintain the warmth of the trunk areas in the cold. The qualitative differences seen in thermal comfort for the various areas cannot be explained solely by the density or properties of the peripheral thermal receptors and thus must reflect processing mechanisms in the central nervous system.