OBJECTIVE

Type D personality-a joint tendency toward negative affectivity (NA) and social inhibition (SI)-is related to poor cardiac prognosis, but there is no standard for assessing Type D. This study reports on the Type D Scale-14 (DS14) as a standard measure of NA, SI, and Type D.

METHODS

The study included 3813 participants (2508 from the general population, 573 cardiac patients, 732 hypertension patients). They all filled out the DS14, containing 7-item NA and SI subscales; 275 subjects also completed the NEO-FFI, and 121 patients filled out the DS14 twice.

RESULTS

Factor analysis of the DS14 yielded 2 dominant traits; all of the NA and SI items loaded between 0.62 to 0.82 on their corresponding factor (N = 3678). The NA scale covered dysphoria, worry, and irritability; the SI scale covered discomfort in social interactions, reticence, and lack of social poise. The NA and SI scales were internally consistent (alpha = 0.88/0.86; N = 3678), stable over a 3-month period (test-retest r = 0.72/0.82) and not dependent on mood and health status (N = 121). NA correlated positively with neuroticism (r = 0.68); SI correlated negatively with extraversion (r = -0.59/-0.65). Scale-level factor analysis confirmed the construct validity of the DS14 against the NEO-FFI. Using a cutoff of 10 (NA>> or =10 and SI>> or =10), 1027 subjects (28%) were classified as Type D, 21% in the general population versus 28% in coronary heart disease and 53% in hypertension (p < or = .001). Age, sex, and Type D (odds ratio, 3.98; 95% confidence interval, 3.2-4.6; p <.0001) were independently associated with cardiovascular morbidity.

CONCLUSIONS

The DS14 is a brief, psychometrically sound measure of negative affectivity and social inhibition that could readily be incorporated in epidemiologic and clinical research.

Iodide (I-) is an essential constituent of the thyroid hormones T3 and T4, and is accumulated by the thyroid. The transport of iodide, the first step in thyroid hormogenesis, is catalysed by the Na+/I- symporter, an intrinsic membrane protein that is crucial for the evaluation, diagnosis and treatment of thyroid disorders. Although several other important thyroid proteins involved in hormogenesis have been characterized, the Na+/I- symporter has not. Here we report the isolation of a complementary DNA clone that encodes this symporter, as a result of functional screening of a cDNA library from a rat thyroid-derived cell line (FRTL-5) in Xenopus laevis oocytes. Oocyte microinjection of an RNA transcript made in vitro from this cDNA clone elicited a more than 700-fold increase in perchlorate-sensitive Na+/I- symport activity over background. To our knowledge, this is the first iodide-transporting molecule to have its cDNA cloned, providing a missing link in the thyroid hormone biosynthetic pathway.

An attempt is made to elucidate the cellular mechanisms which may account for the well-documented correlation between sodium metabolism and peripheral vascular resistance. As a starting point, the evidence that the Na electrochemical gradient across the vascular smooth muscle cell plasma membrane (sarcolemma) plays an important role in cell calcium regulation is reviewed. Because there is significant resting tension ("tone") in most resistance vessels, the ionized Ca2+ level ([Ca2+]1) in the smooth muscle fibers in these vessels must be maintained above the contraction threshold. Consequently, the Ca transport system in the sarcolemma, presumably an Na-Ca exchange mechanism, must be set so as to hold [Ca2+]1 at this suprathreshold level. Any change in the Na gradient will then be reflected as a change in [Ca2+]1 and, therefore, in steady vessel wall tension and peripheral resistance. The correlation between Na metabolism and hypertension could then be accounted for if a circulating agent, perhaps the "natriuretic hormone," affects the Na gradient (across the sarcolemma) and, therefore, [Ca2+]1 and tension.

The mechanisms underlying many important properties of the human atrial action potential (AP) are poorly understood. Using specific formulations of the K+, Na+, and Ca2+ currents based on data recorded from human atrial myocytes, along with representations of pump, exchange, and background currents, we developed a mathematical model of the AP. The model AP resembles APs recorded from human atrial samples and responds to rate changes, L-type Ca2+ current blockade, Na+/Ca2+ exchanger inhibition, and variations in transient outward current amplitude in a fashion similar to experimental recordings. Rate-dependent adaptation of AP duration, an important determinant of susceptibility to atrial fibrillation, was attributable to incomplete L-type Ca2+ current recovery from inactivation and incomplete delayed rectifier current deactivation at rapid rates. Experimental observations of variable AP morphology could be accounted for by changes in transient outward current density, as suggested experimentally. We conclude that this mathematical model of the human atrial AP reproduces a variety of observed AP behaviors and provides insights into the mechanisms of clinically important AP properties.

Headache attacks and autonomic dysfunctions characterize migraine, a very common, disabling disorder with a prevalence of 12% in the general population of Western countries. About 20% of individuals affected with migraine experience aura, a visual or sensory-motor neurological dysfunction that usually precedes or accompanies the headache. Although the mode of transmission is controversial, population-based and twin studies have implicated genetic factors, especially in migraine with aura. Familial hemiplegic migraine is a hereditary form of migraine characterized by aura and some hemiparesis. Here we show that mutations in the gene ATP1A2 that encodes the alpha2 subunit of the Na+/K+ pump are associated with familial hemiplegic migraine type 2 (FHM2) linked to chromosome 1q23 (OMIM 602481). Functional data indicate that the putative pathogenetic mechanism is triggered by a loss of function of a single allele of ATP1A2. This is the first report associating mutations of Na+K+ pump subunits to genetic diseases.

1. Membrane parameters of an isolated neural cell body have been determined by voltage clamp analysis. Data are expressed as membrane ion-specific conductances, leak conductance, and capacitance.2. Three ionic currents are present: Inward, I(I); and two operationally distinct outward currents, I(K) and I(A). Both outward currents are apparently carried by potassium ions.3. Hodgkin-Huxley-like equations were solved for the discharge of two sequential action potentials in response to a constant stimulus current. The digital computer solutions are compared with action potential data recorded from the investigated cell.4. The computed and experimentally measured relationships between firing frequency and stimulus current intensity are compared and are linear over the same portion of the total frequency range.5. Cell behaviour in the latter part of the interspike interval is dominated by the conductance g(A) while g(Na) and g(K) largely determine the character of the action potential and the initial portion of the interspike interval.6. Prehyperpolarization of the membrane activates g(A) and the membrane response to depolarizing current differs markedly from the response elicited when no prehyperpolarization is imposed.

The high metabolic rate of tumours often leads to acidosis and hypoxia in poorly perfused regions. Tumour cells have thus evolved the ability to function in a more acidic environment than normal cells. Key pH regulators in tumour cells include: isoforms 2, 9 and 12 of carbonic anhydrase, isoforms of anion exchangers, Na+/HCO3- co-transporters, Na+/H+ exchangers, monocarboxylate transporters and the vacuolar ATPase. Both small molecules and antibodies targeting these pH regulators are currently at various stages of clinical development. These antitumour mechanisms are not exploited by the classical cancer drugs and therefore represent a new anticancer drug discovery strategy.

The epithelial Na(+) channel (ENaC) plays an essential role in the regulation of whole body Na(+) balance and blood pressure. The cell surface expression of this channel, a complex of three subunits (alpha, beta and gamma ENaC), has been shown to be regulated by hormones such as aldosterone and vasopressin and by intracellular signaling, including ubiquitylation and/or phosphorylation. However, the molecular mechanisms involving phosphorylation in the regulation of ENaC are unclear. Here we show by expression studies in Xenopus laevis oocytes that the aldosterone-induced Sgk1 kinase interacts with the ubiquitin protein ligase Nedd4-2 in a PY motif-dependent manner and phosphorylates Nedd4-2 on Ser444 and, to a lesser extent, Ser338. Such phosphorylation reduces the interaction between Nedd4-2 and ENaC, leading to elevated ENaC cell surface expression. These data show that phosphorylation of an enzyme involved in the ubiquitylation cascade (Nedd4-2) controls cell surface density of ENaC and propose a paradigm for the control of ion channels. Moreover, they suggest a novel and complete signaling cascade for aldosterone-dependent regulation of ENaC.

Voltage-gated sodium channels (NaCh) are colocalized with isoforms of the membrane-skeletal protein ankyrinG at axon initial segments, nodes of Ranvier, and postsynaptic folds of the mammalian neuromuscular junction. The role of ankyrinG in directing NaCh localization to axon initial segments was evaluated by region-specific knockout of ankyrinG in the mouse cerebellum. Mutant mice exhibited a progressive ataxia beginning around postnatal day P16 and subsequent loss of Purkinje neurons. In mutant mouse cerebella, NaCh were absent from axon initial segments of granule cell neurons, and Purkinje cells showed deficiencies in their ability to initiate action potentials and support rapid, repetitive firing. Neurofascin, a member of the L1CAM family of ankyrin-binding cell adhesion molecules, also exhibited impaired localization to initial segments of Purkinje cell neurons. These results demonstrate that ankyrinG is essential for clustering NaCh and neurofascin at axon initial segments and is required for physiological levels of sodium channel activity.

Basal extracellular glutamate sampled in vivo is present in micromolar concentrations in the extracellular space outside the synaptic cleft, and neither the origin nor the function of this glutamate is known. This report reveals that blockade of glutamate release from the cystine-glutamate antiporter produced a significant decrease (60%) in extrasynaptic glutamate levels in the rat striatum, whereas blockade of voltage-dependent Na+ and Ca2+ channels produced relatively minimal changes (0-30%). This indicates that the primary origin of in vivo extrasynaptic glutamate in the striatum arises from nonvesicular glutamate release by the cystine-glutamate antiporter. By measuring [35S]cystine uptake, it was shown that similar to vesicular release, the activity of the cystine-glutamate antiporter is negatively regulated by group II metabotropic glutamate receptors (mGluR2/3) via a cAMP-dependent protein kinase mechanism. Extracellular glutamate derived from the antiporter was shown to regulate extracellular levels of glutamate and dopamine. Infusion of the mGluR2/3 antagonist (RS)-1-amino-5-phosphonoindan-1-carboxylic acid (APICA) increased extracellular glutamate levels, and previous blockade of the antiporter prevented the APICA-induced rise in extracellular glutamate. This suggests that glutamate released from the antiporter is a source of endogenous tone on mGluR2/3. Blockade of the antiporter also produced an increase in extracellular dopamine that was reversed by infusing the mGluR2/3 agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxlylate, indicating that antiporter-derived glutamate can modulate dopamine transmission via mGluR2/3 heteroreceptors. These results suggest that nonvesicular release from the cystine-glutamate antiporter is the primary source of in vivo extracellular glutamate and that this glutamate can modulate both glutamate and dopamine transmission.

Efficient and rapid propagation of action potentials in myelinated axons depends on the molecular specialization of the nodes of Ranvier. The nodal region is organized into several distinct domains, each of which contains a unique set of ion channels, cell-adhesion molecules and cytoplasmic adaptor proteins. Voltage-gated Na+ channels - which are concentrated at the nodes - are separated from K+ channels - which are clustered at the juxtaparanodal region - by a specialized axoglial contact that is formed between the axon and the myelinating cell at the paranodes. This local differentiation of myelinated axons is tightly regulated by oligodendrocytes and myelinating Schwann cells, and is achieved through complex mechanisms that are used by another specialized cell-cell contact - the synapse.

OBJECTIVE

What is the optimal management of women with endometriosis based on the best available evidence in the literature?

CONCLUSIONS

Using the structured methodology of the Manual for ESHRE Guideline Development, 83 recommendations were formulated that answered the 22 key questions on optimal management of women with endometriosis.

BACKGROUND

The European Society of Human Reproduction and Embryology (ESHRE) guideline for the diagnosis and treatment of endometriosis (2005) has been a reference point for best clinical care in endometriosis for years, but this guideline was in need of updating.

METHODS

This guideline was produced by a group of experts in the field using the methodology of the Manual for ESHRE Guideline Development, including a thorough systematic search of the literature, quality assessment of the included papers up to January 2012 and consensus within the guideline group on all recommendations. To ensure input from women with endometriosis, a patient representative was part of the guideline development group. In addition, patient and additional clinical input was collected during the scoping and review phase of the guideline.

METHODS

NA.

RESULTS

The guideline provides 83 recommendations on diagnosis of endometriosis and on the treatment of endometriosis-associated pain and infertility, on the management of women in whom the disease is found incidentally (without pain or infertility), on prevention of recurrence of disease and/or painful symptoms, on treatment of menopausal symptoms in patients with a history of endometriosis and on the possible association of endometriosis and malignancy.

CONCLUSIONS

We identified several areas in care of women with endometriosis for which robust evidence is lacking. These areas were addressed by formulating good practice points (GPP), based on the expert opinion of the guideline group members.

CONCLUSIONS

Since 32 out of the 83 recommendations for the management of women with endometriosis could not be based on high level evidence and therefore were GPP, the guideline group formulated research recommendations to guide future research with the aim of increasing the body of evidence.

BACKGROUND

The guideline was developed and funded by ESHRE, covering expenses associated with the guideline meetings, with the literature searches and with the implementation of the guideline. The guideline group members did not receive payment. All guideline group members disclosed any relevant conflicts of interest (see Conflicts of interest).

BACKGROUND

NA.

In Arabidopsis thaliana, the Salt Overly Sensitive 2 (SOS2) gene is required for intracellular Na(+) and K(+) homeostasis. Mutations in SOS2 cause Na(+) and K(+) imbalance and render plants more sensitive toward growth inhibition by high Na(+) and low K(+) environments. We isolated the SOS2 gene through positional cloning. SOS2 is predicted to encode a serine/threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase. Sequence analyses of sos2 mutant alleles reveal that both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2 are functionally essential. The steady-state level of SOS2 transcript is up-regulated by salt stress in the root. Autophosphorylation assays show that SOS2 is an active protein kinase. In the recessive sos2-5 allele, a conserved glycine residue in the kinase catalytic domain is changed to glutamate. This mutation abolishes SOS2 autophosphorylation, indicating that SOS2 protein kinase activity is required for salt tolerance.

The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na(+), K(+), Ca(2+) and Mg(2+)) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa >>or=10(7)MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (M(r) 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

Glycosaminogycans (GAGs) are involved in numerous vital functions in the human body. Mapping the GAG concentration in vivo is desirable for the diagnosis and monitoring of a number of diseases such as osteoarthritis, which affects millions of individuals. GAG loss in cartilage is typically an initiating event in osteoarthritis. Another widespread pathology related to GAG is intervertebral disk degeneration. Currently existing techniques for GAG monitoring, such as delayed gadolinium-enhanced MRI contrast (dGEMRIC), T(1)(rho), and (23)Na MRI, have some practical limitations. We show that by exploiting the exchangeable protons of GAG one may directly measure the localized GAG concentration in vivo with high sensitivity and therefore obtain a powerful diagnostic MRI method.

Evolution of animal morphology, physiology and behaviour is shaped by the selective pressures to which they are subject. Some selective pressures act to increase the benefits accrued whilst others act to reduce the costs incurred, affecting the cost/benefit ratio. Selective pressures therefore produce a trade-off between costs and benefits that ultimately influences the fitness of the whole organism. The nervous system has a unique position as the interface between morphology, physiology and behaviour; the final output of the nervous system is the behaviour of the animal, which is a product of both its morphology and physiology. The nervous system is under selective pressure to generate adaptive behaviour, but at the same time is subject to costs related to the amount of energy that it consumes. Characterising this trade-off between costs and benefits is essential to understanding the evolution of nervous systems, including our own. Within the nervous system, sensory systems are the most amenable to analysing costs and benefits, not only because their function can be more readily defined than that of many central brain regions and their benefits quantified in terms of their performance, but also because recent studies of sensory systems have begun to directly assess their energetic costs. Our review focuses on the visual system in particular, although the principles we discuss are equally applicable throughout the nervous system. Examples are taken from a wide range of sensory modalities in both vertebrates and invertebrates. We aim to place the studies we review into an evolutionary framework. We combine experimentally determined measures of energy consumption from whole retinas of rabbits and flies with intracellular measurements of energy consumption from single fly photoreceptors and recently constructed energy budgets for neural processing in rats to assess the contributions of various components to neuronal energy consumption. Taken together, these studies emphasize the high costs of maintaining neurons at rest and whilst signalling. A substantial proportion of neuronal energy consumption is related to the movements of ions across the neuronal cell membrane through ion channels, though other processes such as vesicle loading and transmitter recycling also consume energy. Many of the energetic costs within neurons are linked to 3Na(+)/2K(+) ATPase activity, which consumes energy to pump Na(+) and K(+) ions across the cell membrane and is essential for the maintenance of the resting potential and its restoration following signalling. Furthermore, recent studies in fly photoreceptors show that energetic costs can be related, via basic biophysical relationships, to their function. These findings emphasize that neurons are subject to a law of diminishing returns that severely penalizes excess functional capacity with increased energetic costs. The high energetic costs associated with neural tissue favour energy efficient coding and wiring schemes, which have been found in numerous sensory systems. We discuss the role of these efficient schemes in reducing the costs of information processing. Assessing evidence from a wide range of vertebrate and invertebrate examples, we show that reducing energy expenditure can account for many of the morphological features of sensory systems and has played a key role in their evolution.

Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na(+)/H(+) exchange. Yet, the functional relationship between Na(+)/H(+) exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na(+)/H(+) exchange, elevating cytosolic pH and stimulating Na(+) influx. Remarkably, although inhibition of Na(+)/H(+) exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na(+) influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na(+)/H(+) exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na(+)/H(+) exchange.

The transmural potential difference, short-circuit current, and Na fluxes have been investigated in an in vitro preparation of isolated rabbit ileum. When the tissue is perfused with a physiological buffer, the serosal surface is electrically positive with respect to the mucosal surface and the initial potential difference in the presence of glucose averages 9 mv. Unidirectional and net Na fluxes have been determined under a variety of conditions, and in each instance, most if not all of the simultaneously measured short-circuit current could be attributed to the active transport of Na from mucosa to serosa. Active Na transport is dependent upon the presence of intact aerobic metabolic pathways and is inhibited by low concentrations of ouabain in the serosal medium. A method is described for determining whether a unidirectional ionic flux is the result of passive diffusion alone, in the presence of active transport of that ion in the opposite direction. Using this method we have demonstrated that the serosa-to-mucosa flux of Na may be attributed to passive diffusion with no evidence for the presence of carrier-mediated exchange diffusion or the influence of solvent-drag.

The permeability of K channels to various cations is studied in myelinated nerve. Ionic currents under voltage clamp are measured in Ringer solution containing tetrodotoxin and a high concentration of the test ion. Reversal potentials for current in K channels are determined and used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The ratios P(Tl):P(K):P(Rb):P(NHNH4) are 2.3:1.00:0.92:0.13. No other ions are found to be measurably permeant including Li(+), Na(+), Cs(+), methylamine, guanidine, hydrazine, or hydroxylamine. The ratio P(Na)/P(K) is less than 0.01. Potassium conductance is depressed at pH values below 5.0. Leakage conductance is higher in K, Rb, Cs, NH(4), and Tl Ringer than in Na Ringer, but the selectivity sequence probably is not the same as for K channels. The hypothesis is offered that the narrowest part of the K channel is a circle of oxygen atoms about 3 A in diameter with low electrostatic field strength.

Voltage-sensitive ion channels mediate action potentials in electrically excitable cells and play important roles in signal transduction in other cell types. In the past several years, their protein components have been identified, isolated, and restored to functional form in the purified state. Na+ and Ca2+ channels consist of a principal transmembrane subunit, which forms the ion-conducting pore and is expressed with a variable number of associated subunits in different cell types. The principal subunits of voltage-sensitive Na+, Ca2+, and K+ channels are homologous members of a gene family. Models relating the primary structures of these principal subunits to their functional properties have been proposed, and experimental results have begun to define a functional map of these proteins. Coordinated application of biochemical, biophysical, and molecular genetic methods should lead to a clear understanding of the molecular basis of electrical excitability.

Excitotoxicity contributes to neuronal degeneration in many acute CNS diseases, including ischemia, trauma, and epilepsy, and may also play a role in chronic diseases, such as amyotrophic lateral sclerosis (ALS). Key mediators of excitotoxic damage are Ca ions (Ca(2+)), which under physiological conditions govern a multitude of cellular processes, including cell growth, differentiation, and synaptic activity. Consequently, homeostatic mechanisms exist to maintain a low intracellular Ca(2+) ion concentration so that Ca(2+) signals remain spatially and temporally localized. This permits multiple independent Ca-mediated signaling pathways to occur in the same cell. In excitotoxicity, excessive synaptic release of glutamate can lead to the disregulation of Ca(2+) homeostasis. Glutamate activates postsynaptic receptors, including the ionotropic N-methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) proprionate (AMPA), and kainate receptors. Upon their activation, these open their associated ion channel to allow the influx of Ca(2+) and Na(+) ions. Although physiological elevations in intracellular Ca(2+) are salient to normal cell functioning, the excessive influx of Ca(2+) together with any Ca(2+) release from intracellular compartments can overwhelm Ca(2+)-regulatory mechanisms and lead to cell death. Although Ca(2+) disregulation is paramount to neurodegeneration, the exact mechanism by which Ca(2+) ions actually mediate excitotoxicity is less clear. One hypothesis outlined in this review suggests that Ca(2+)-dependent neurotoxicity occurs following the activation of distinct signaling cascades downstream from key points of Ca(2+) entry at synapses, and that triggers of these cascades are physically co-localized with specific glutamate receptors. Thus, we summarize the importance of Ca(2+) regulation in mammalian neurons and the excitotoxicity hypothesis, and focus on the molecular determinants of glutamate receptor-mediated excitotoxic mechanisms.

Sodium-potassium ATPase is an ATP-powered ion pump that establishes concentration gradients for Na(+) and K(+) ions across the plasma membrane in all animal cells by pumping Na(+) from the cytoplasm and K(+) from the extracellular medium. Such gradients are used in many essential processes, notably for generating action potentials. Na(+), K(+)-ATPase is a member of the P-type ATPases, which include sarcoplasmic reticulum Ca(2+)-ATPase and gastric H(+), K(+)-ATPase, among others, and is the target of cardiac glycosides. Here we describe a crystal structure of this important ion pump, from shark rectal glands, consisting of alpha- and beta-subunits and a regulatory FXYD protein, all of which are highly homologous to human ones. The ATPase was fixed in a state analogous to E2.2K(+).P(i), in which the ATPase has a high affinity for K(+) and still binds P(i), as in the first crystal structure of pig kidney enzyme at 3.5 A resolution. Clearly visualized now at 2.4 A resolution are coordination of K(+) and associated water molecules in the transmembrane binding sites and a phosphate analogue (MgF(4)(2-)) in the phosphorylation site. The crystal structure shows that the beta-subunit has a critical role in K(+) binding (although its involvement has previously been suggested) and explains, at least partially, why the homologous Ca(2+)-ATPase counter-transports H(+) rather than K(+), despite the coordinating residues being almost identical.

Glutamate transport across plasma membranes of neurons, glial cells and epithelial cells of the small intestine and kidney proceeds by high- and low-affinity transport systems. High-affinity (Km 2-50 microM) transport systems have been described that are dependent on Na+ but not Cl- ions and have a preference for L-glutamate and D- and L-aspartate. In neurons high-affinity glutamate transporters are essential for terminating the postsynaptic action of glutamate by rapidly removing released glutamate from the synaptic cleft. We have isolated a complementary DNA encoding an electrogenic Na(+)- but not Cl(-)-dependent high-affinity glutamate transporter (named EAAC1) from rabbit small intestine by expression in Xenopus oocytes. We find EAAC1 transcripts in specific neuronal structures in the central nervous system as well as in the small intestine, kidney, liver and heart. The function and pharmacology of the expressed protein are characteristic of the high-affinity glutamate transporter already identified in neuronal tissues. The abnormal glutamate transport that is associated with certain neurodegenerative diseases and which occurs during ischaemia and anoxia could be due to abnormalities in the function of this protein.

Calcium-activated nonselective (CAN) cation channels are expressed in various excitable and nonexcitable cells supporting important cellular responses such as neuronal bursting activity, fluid secretion, and cardiac rhythmicity. We have cloned and characterized a second form of TRPM4, TRPM4b, a member of the TRP channel family, as a molecular candidate of a CAN channel. TRPM4b encodes a cation channel of 25 pS unitary conductance that is directly activated by [Ca2+]i with an apparent K(D) of approximately 400 nM. It conducts monovalent cations such as Na+ and K+ without significant permeation of Ca2+. TRPM4b is activated following receptor-mediated Ca2+ mobilization, representing a regulatory mechanism that controls the magnitude of Ca2+ influx by modulating the membrane potential and, with it, the driving force for Ca2+ entry through other Ca2+-permeable pathways.