The pore of TRP channels: trivial or neglected?

Ultimate proof that a protein contributes to the pore of an ion channel is demonstrating that pore properties can be altered by mutations to the putative pore-forming region. To date this has only been achieved for a few TRP proteins, and only within the TRPV subfamily. The location and structure of the pore region and selectivity filter of most TRP proteins, including all members of the TRPM and TRPC subfamilies, is currently unknown. In this review we give a short overview of the limited current knowledge about TRP channel pores and argue that further study is needed, not only for a better understanding of the mechanisms underlying cation permeation, but also to establish that all members of the TRP superfamily indeed function as bona fide ion channels.     

How do calcium channels transport calcium ions?

Calcium channel activity is crucial for many fundamental physiological processes ranging from the heart beat to synaptic transmission. The channel-forming protein, of about 2000 amino acids, comprises four domains internally homologous to each other. Voltage-dependent Ca2+ channels are the most selective ion channels known. Under physiological conditions, they prefer Ca2+ over Na+ by a ratio of about 1000:1. To explain at the same time the exquisite ion selectivity and the large Ca2+ ion turnover rate of Ca2+ channels (approximately 3 x 10(6) ions/s), two kind models have been proposed. In one, the conduction pathway possesses two high-affinity binding sites. When two Ca2+ ions are bound to each site, the mutual repulsion between them speeds the exit rate for the ions, causing greater ion permeation through the pore. The second model hypothesizes the existence of a single site having a charged structure able to attract multiple, interacting ions, simultaneously. Recent studies that combine mutagenesis and electrophysiology show that the high-affinity binding site is formed by a ring of glutamate residues located in the pore forming region of the Ca2+ channel. As proposed in the second class of models, the results suggest that four glutamate residues, one glutamate donated by each repeat, combine to form a single high-affinity site. In this review the different conduction models for Ca2+ channels are discussed and confronted with structural data.     

Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels

Calcium entry through presynaptic voltage-gated calcium channels is essential for neurotransmitter release. The two major types of presynaptic calcium channels contain a synaptic protein interaction site that physically interacts with synaptic vesicle release proteins. This is thought to tighten the coupling between the sources of calcium entry and the neurotransmitter release machinery. Conversely, the binding of synaptic proteins to presynaptic calcium channels regulates calcium channel activity. Hence, presynaptic calcium channels act not only as the masters of the synaptic release process, but also as key targets for feedback inhibition.     

The regulation of sulphurated amino acid junctions: fact or fiction in the field of inflammation?

Summary.  The diet of industrialised countries is usually rich in amino acids, which are in part used as a source of calories. However, metabolic alterations are observed in diseased patients and a preferential retention of Sulphurated Amino Acids (SAA) occurs during the inflammatory response. Moreover, it has been demonstrated in a model of an acute sepsis phase of rats that the metabolism of Cysteine is modified. The liver converts Cysteine at a different ratio of Sulphate to Taurine (Tau) i.e. the sulphate production decreases while the Tau conversion increases. The Glutathione (GSH) concentration is greater in the liver, kidneys and other organs and the Cysteine incorporation into proteins is higher in the spleen, lungs and plasma (Acute Phase Proteins) while the Albumin level decreases. The pro-inflammatory cytokines such as Interleukin-1, Interleukin-6 and TNF-α are the main initiators that alter protein and amino acid metabolism. Another important phenomenon is the impairment of Methionine conversion to Cysteine during stress. For example, premature infants or AIDS patients are capable of synthesizing Cysteine from Methionine at a much lower rate. Thus, the metabolic flow through the trans-sulphuration path may be inadequate to meet the Cysteine demand under critical conditions. In this complex picture, an SAA supply may contribute to an immune system regulation. Received November 30, 2001 Accepted January 15, 2002 Published online August 30, 2002 Author's address: Francesco Santangelo, Zambon Group, via Lillo del Duca 10, I-20091 Bresso, Milan, Italy, Email: francesco.santangelo@zambongroup.com     

Company-sponsored egg freezing: an offer you can't refuse?

The aim of this article is to argue that one of the central arguments against company-sponsored non-medical egg freezing, namely that this practice is contrary to the reproductive autonomy of women, can be difficult to sustain under certain conditions. More specifically, we argue that company-sponsored egg freezing is not necessarily in conflict with the most common requirements for autonomous choice. That is, there is no reason to assume that employees cannot be adequately informed beforehand about what is scientifically known about the practice, and/or that they lack the required capacity to understand and process this information. Although they may feel a certain pressure to comply with the wishes of their employer, this concern can plausibly be alleviated through privacy regulations. In any event, such pressure is arguably not stronger than or relevantly different from other types of pressure on the labour market that most people readily accept as being ethically acceptable. Finally, we argue that company-sponsored non-medical egg freezing may mitigate certain types of oppressive socialization, although it may well perpetuate others, and should in any case arguably be dealt with through guidelines and counselling, which would ensure that women make autonomous choices when companies offer egg freezing.     

PhosphoSerine/threonine binding domains: you can't pSERious?

The fundamental biological importance of protein phosphorylation is underlined by the existence of more than 500 protein kinase genes within the human genome. In many cases, phosphorylation on serine, threonine, and tyrosine residues creates binding surfaces for a variety of phospho-amino acid binding proteins/modules. Here, we review the insights into serine/threonine phosphorylation-dependent signal transduction processes provided by structures of several of these proteins and their complexes.     

Fast inactivation of voltage-dependent calcium channels. A hinged-lid mechanism?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.     

Toxic effects of antimalarial drugs in Paramecium : role of calcium channels

The antimalarial drugs, quinacrine, quinine and mefloquine, as well as the structurally-similar compound, W-7, inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi. These drugs are also toxic to paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. To test this hypothesis, the toxicity of the antimalarials and W-7 was compared in paramecia with and without calcium channels. Since calcium channels are located on the cilia, calcium channels were removed from the paramecia by deciliating the cells. Deciliated cells were found to be less susceptible to the lethal effects of the antimalarials and W-7 than their ciliated counterparts. Moreover, Pawns, mutants of P. tetraurelia that possess cilia but lack functional calcium channels, were also less susceptible to the antimalarials than wild-type cells. Thus, calcium channels may be one site of toxic action of the antimalarial drugs in paramecia and perhaps in other protists. Accepted: 27 December 1996     

The long tale of the calcium activated Cl− channels in olfactory transduction

Ca²⁺-activated Cl^? currents have been implicated in many cellular processes in different cells, but for many years, their molecular identity remained unknown. Particularly intriguing are Ca²⁺-activated Cl^? currents in olfactory transduction, first described in the early 90s. Well characterized electrophysiologically, they carry most of the odorant-induced receptor current in the cilia of olfactory sensory neurons (OSNs). After many attempts to determine their molecular identity, TMEM16B was found to be abundantly expressed in the cilia of OSNs in 2009 and having biophysical properties like those of the native olfactory channel. A TMEM16B knockout mouse confirmed that TMEM16B was indeed the olfactory Cl^? channel but also suggested a limited role in olfactory physiology and behavior.

The question then arises of what the precise role of TMEM16b in olfaction is. Here we review the long story of this channel and its possible roles.     

The evolution of voltage-gated sodium channels: were algal toxins involved?

Many algal toxins target voltage-gated sodium (Na⁺) channels where they act to block or otherwise perturb the function of this critical component of an animal’s neuromuscular system. However, many eukaryotes, particularly those that are routinely exposed to algal toxins, have toxin-insensitive or toxin-resistant Na⁺ channels. Here we provide a brief overview of the structure, function and evolution of voltage-gated Na⁺ channels, and the structural basis for toxin-insensitivity, as a prelude to a discussion of whether the early evolution of Na⁺ channels may have been influenced by the presence of algal toxins in the environment.     

Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM

Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF(3)(-)-CheY, a phosphorylated CheY (CheY approximately P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY approximately P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF(3)(-)-CheY-FliM(16) complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg(2+)-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF(3)(-)-activated CheY. This structure makes clear the active role that the beta4-alpha4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.     

The cell: locus or object of inquiry?

Research in many fields of biology has been extremely successful in decomposing biological mechanisms to discover their parts and operations. It often remains a significant challenge for scientists to recompose these mechanisms to understand how they function as wholes and interact with the environments around them. This is true of the eukaryotic cell. Although initially identified in nineteenth-century cell theory as the fundamental unit of organisms, researchers soon learned how to decompose it into its organelles and chemical constituents and have been highly successful in understanding how these carry out many operations important to life. The emphasis on decomposition is particularly evident in modern cell biology, which for the most part has viewed the cell as merely a locus of the mechanisms responsible for vital phenomena. The cell, however, is also an integrated system and for some explanatory purposes it is essential to recompose it and understand it as an organized whole. I illustrate both the virtues of decomposition (treating the cell as a locus) and recomposition (treating the cell as an object) with recent work on circadian rhythms. Circadian researchers have both identified critical intracellular operations that maintain endogenous oscillations and have also addressed the integration of cells into multicellular systems in which cells constitute units.     

The evolution of regeneration: adaptive or inherent?

If regeneration were adaptive, it would have arisen autonomously by natural selection from non-regenerative antecedents. Unless each episode coincidentally reinvented the same method of regeneration independently, one would expect the various lineages to differ basically from each other, which they do not. On the other hand, if regeneration were inherent to metazoan life, a derivative of embryogenesis, its various expressions should be as much like each other as they resemble the development of embryonic appendage buds, which they do. It follows that the uneven distribution of regeneration must have been due to its extinction here and there, not as a negative adaptation by natural selection but as a pleiotropic epiphenomenon linked to more useful adaptations with which it was incompatible. In vertebrate evolution, these adaptations have included the transition from aquatic to terrestrial habitats and the modification of poikilothermic to homeothermic metabolism. The former advance rendered the regeneration of weight-bearing limbs impractical; the latter favored rapid wound healing and scar formation which effectively precluded blastema formation. If the latent capacity for regeneration persists in non-regenerative appendages, as would seem to be the case, then the restoration of its overt expression should be possible if the mechanisms of its inhibition could be discovered and eventually rendered ineffectual.