During wakefulness, neocortical neurons are subjected to an intense synaptic bombardment. To assess the consequences of this background activity for the integrative properties of pyramidal neurons, we constrained biophysical models with in vivo intracellular data obtained in anesthetized cats during periods of intense network activity similar to that observed in the waking state. In pyramidal cells of the parietal cortex (area 5-7), synaptic activity was responsible for an approximately fivefold decrease in input resistance (Rin), a more depolarized membrane potential (Vm), and a marked increase in the amplitude of Vm fluctuations, as determined by comparing the same cells before and after microperfusion of tetrodotoxin (TTX). The model was constrained by measurements of Rin, by the average value and standard deviation of the Vm measured from epochs of intense synaptic activity recorded with KAc or KCl-filled pipettes as well as the values measured in the same cells after TTX. To reproduce all experimental results, the simulated synaptic activity had to be of relatively high frequency (1-5 Hz) at excitatory and inhibitory synapses. In addition, synaptic inputs had to be significantly correlated (correlation coefficient approximately 0.1) to reproduce the amplitude of Vm fluctuations recorded experimentally. The presence of voltage-dependent K+ currents, estimated from current-voltage relations after TTX, affected these parameters by <10%. The model predicts that the conductance due to synaptic activity is 7-30 times larger than the somatic leak conductance to be consistent with the approximately fivefold change in Rin. The impact of this massive increase in conductance on dendritic attenuation was investigated for passive neurons and neurons with voltage-dependent Na+/K+ currents in soma and dendrites. In passive neurons, correlated synaptic bombardment had a major influence on dendritic attenuation. The electrotonic attenuation of simulated synaptic inputs was enhanced greatly in the presence of synaptic bombardment, with distal synapses having minimal effects at the soma. Similarly, in the presence of dendritic voltage-dependent currents, the convergence of hundreds of synaptic inputs was required to evoke action potentials reliably. In this case, however, dendritic voltage-dependent currents minimized the variability due to input location, with distal apical synapses being as effective as synapses on basal dendrites. In conclusion, this combination of intracellular and computational data suggests that, during low-amplitude fast electroencephalographic activity, neocortical neurons are bombarded continuously by correlated synaptic inputs at high frequency, which significantly affect their integrative properties. A series of predictions are suggested to test this model.

Transformation of mechanical energy into ionic currents is essential for touch, hearing and nociception. Although DEG/ENaC proteins are believed to form sensory mechanotransduction channels, the evidence for this role remains indirect. By recording from C. elegans touch receptor neurons in vivo, we found that external force evokes rapidly activating mechanoreceptor currents (MRCs) carried mostly by Na(+) and blocked by amiloride-characteristics consistent with direct mechanical gating of a DEG/ENaC channel. Like mammalian Pacinian corpuscles, these neurons depolarized with both positive and negative changes in external force but not with sustained force. Null mutations in the DEG/ENaC gene mec-4 and in the accessory ion channel subunit genes mec-2 and mec-6 eliminated MRCs. In contrast, the genetic elimination of touch neuron-specific microtubules reduced, but did not abolish, MRCs. Our findings link the application of external force to the activation of a molecularly defined metazoan sensory transduction channel.

Voltage-gated sodium (Na(v)) channels are essential for the rapid depolarization of nerve and muscle, and are important drug targets. Determination of the structures of Na(v) channels will shed light on ion channel mechanisms and facilitate potential clinical applications. A family of bacterial Na(v) channels, exemplified by the Na(+)-selective channel of bacteria (NaChBac), provides a useful model system for structure-function analysis. Here we report the crystal structure of Na(v)Rh, a NaChBac orthologue from the marine alphaproteobacterium HIMB114 (Rickettsiales sp. HIMB114; denoted Rh), at 3.05 Å resolution. The channel comprises an asymmetric tetramer. The carbonyl oxygen atoms of Thr 178 and Leu 179 constitute an inner site within the selectivity filter where a hydrated Ca(2+) resides in the crystal structure. The outer mouth of the Na(+) selectivity filter, defined by Ser 181 and Glu 183, is closed, as is the activation gate at the intracellular side of the pore. The voltage sensors adopt a depolarized conformation in which all the gating charges are exposed to the extracellular environment. We propose that Na(v)Rh is in an 'inactivated' conformation. Comparison of Na(v)Rh with Na(v)Ab reveals considerable conformational rearrangements that may underlie the electromechanical coupling mechanism of voltage-gated channels.

Nine voltage-gated sodium channels are expressed in complex patterns in mammalian nerve and muscle. Three channels, Na(v)1.7, Na(v)1.8, and Na(v)1.9, are expressed selectively in peripheral damage-sensing neurons. Because there are no selective blockers of these channels, we used gene ablation in mice to examine the function of Na(v)1.7 (PN1) in pain pathways. A global Na(v)1.7-null mutant was found to die shortly after birth. We therefore used the Cre-loxP system to generate nociceptor-specific knockouts. Na(v)1.8 is only expressed in peripheral, mainly nociceptive, sensory neurons. We knocked Cre recombinase into the Na(v)1.8 locus to generate heterozygous mice expressing Cre recombinase in Na(v)1.8-positive sensory neurons. Crossing these animals with mice where Na(v)1.7 exons 14 and 15 were flanked by loxP sites produced nociceptor-specific knockout mice that were viable and apparently normal. These animals showed increased mechanical and thermal pain thresholds. Remarkably, all inflammatory pain responses evoked by a range of stimuli, such as formalin, carrageenan, complete Freund's adjuvant, or nerve growth factor, were reduced or abolished. A congenital pain syndrome in humans recently has been mapped to the Na(v)1.7 gene, SCN9A. Dominant Na(v)1.7 mutations lead to edema, redness, warmth, and bilateral pain in human erythermalgia patients, confirming an important role for Na(v)1.7 in inflammatory pain. Nociceptor-specific gene ablation should prove useful in understanding the role of other broadly expressed genes in pain pathways.

1. Intracellular recordings were obtained from neurons of the guinea pig sensorimotor cortical slice maintained in vitro. Under control recording conditions input resistances, time constants, and spiking characteristics of slice neurons were well within the ranges reported by other investigators for neocortical neurons in situ. However, resting potentials (mean of -75 mV) and spike amplitudes (mean of 93.5 mV) were 10-25 mV greater than has been observed in intact preparations. 2. Current-voltage relationships obtained under current clamp revealed a spectrum of membrane-rectifying properties at potentials that were subthreshold for spike generation. Ionic and pharmacologic analyses suggest that subthreshold membrane behavior is dominated by voltage-sensitive, very slowly inactivating conductances to K+ and Na+. 3. Action potentials were predominantly Na+ dependent under normal conditions but when outward K+ currents were reduced pharmacologically, it was possible, in most cells, to evoke a non-Na+-dependent, tetrodotoxin-(TTX) insensitive spike, which was followed by a prominent depolarizing after-potential. Both of these events were blocked by the Ca2+ current antagonists, Co2+ and Mn2+. 4. A small population of neurons generated intrinsic, all-or-none burst potentials when depolarized with current pulses or by synaptic activation. These cells were located at a narrow range of depths comprising layer IV and the more superficial parts of layer V. 5. Spontaneous excitatory synaptic potentials appeared in all neurons. Spontaneous inhibitory events were visible in only about 10% of the cells, and in those cases apparently reversed polarity at a level slightly positive to resting potential. Stimulation of the surface of the slice at low intensities evoked robust and usually concurrent excitatory and inhibitory synaptic potentials. Unitary inhibitory postsynaptic potentials (IPSPs) reversed at levels positive to rest. Stronger stimulation produced a labile, long-duration, hyperpolarizing IPSP with a reversal potential 15-20 mV negative to the resting level. 6. Neocortical neurons in vitro retain the basic membrane and synaptic properties ascribed to them in situ. However, the array of passive and active membrane behavior observed in the slice suggests that cortical neurons may be differentiated by specific functional properties as well as by their extensive morphological diversity.

Influenza virus neuraminidase (NA) plays an essential role in release and spread of progeny virions, following the intracellular viral replication cycle. To test whether NA could also facilitate virus entry into cell, we infected cultures of human airway epithelium with human and avian influenza viruses in the presence of the NA inhibitor oseltamivir carboxylate. Twenty- to 500-fold less cells became infected in drug-treated versus nontreated cultures (P < 0.0001) 7 h after virus application, indicating that the drug suppressed the initiation of infection. These data demonstrate that viral NA plays a role early in infection, and they provide further rationale for the prophylactic use of NA inhibitors.

Peripheral pain pathways are activated by a range of stimuli. We used diphtheria toxin to kill all mouse postmitotic sensory neurons expressing the sodium channel Nav1.8. Mice showed normal motor activity and low-threshold mechanical and acute noxious heat responses but did not respond to noxious mechanical pressure or cold. They also showed a loss of enhanced pain responses and spontaneous pain behavior upon treatment with inflammatory insults. In contrast, nerve injury led to heightened pain sensitivity to thermal and mechanical stimuli indistinguishable from that seen with normal littermates. Pain behavior correlates well with central input from sensory neurons measured electrophysiologically in vivo. These data demonstrate that Na(v)1.8-expressing neurons are essential for mechanical, cold, and inflammatory pain but not for neuropathic pain or heat sensing.

Isolated cells from rat lacrimal glands were studied with patch-clamp techniques. Whole-cell and cell-attached recordings were obtained while the cells were stimulated by application of carbamylcholine or of the Ca ionophore, A23187. The results were compared with recordings of Ca-dependent channels obtained in isolated patches. Whole-cell recordings revealed two types of carbamylcholine-induced current. At low levels of stimulation, a specific class of Ca-dependent K channels was selectively activated ('BK channels'). With more intense stimulation an inward current, Ii, was obtained at the cell resting potential. Ii rose rather abruptly after a long delay. In several cells, Ii currents presented spontaneous oscillations. Both K and Ii current responses to carbamylcholine were due to activation of muscarinic receptors. Both responses were elicited by a rise of the intracellular Ca concentration. The immediate source of Ca was intracellular. Replacement of intracellular K with either Na or Cs blocked BK channels entirely, thus allowing the study of Ii currents free from K currents. Ii responses to carbamylcholine were, however, less frequently obtained in Na- or Cs-dialysed cells than in K-dialysed cells. In symmetrical NaCl solutions, Ii inverted at 0 mV. When replacing part of the intracellular or extracellular Cl with glutamate the reversal potential, Ei, was found to vary in the same direction as the equilibrium potential for Cl ions, ECl. In some experiments, Ei was close to ECl but in others Ei deviated strongly from ECl. These experiments suggested that Ii was mainly due to a Cl-selective conductance, and that another conductance type was contributing to Ii in variable proportions. It was found that, in K-free solutions, Ii had a reversal potential very close to ECl. Noise analysis showed that the Cl channels involved in Ii current had a unit conductance of about 1-2 pS in symmetrical NaCl solutions. At -60 mV, the mean channel open time derived from noise power spectra was about 200 ms. The activation of the Ca-dependent Cl channels was increased by depolarization. Voltage jumps elicited slow exponential relaxations. At -60 mV, the time constants of the relaxations were in the range 100-250 ms. Cell-attached recordings suggested that internal Ca activated three types of channel, depending on the Ca concentration: BK channels, 2-4 pS channels and 25 pS channels. Inside-out and outside-out patch conditions allowed a rough estimate to be made of the Ca concentration needed to activate each class of channel.(ABSTRACT TRUNCATED AT 400 WORDS)

Early afterdepolarizations (EADs) are a type of triggered activity found in heart muscle. We used voltage-clamped sheep cardiac Purkinje fibers to examine the mechanism underlying EADs induced near action potential plateau voltages with the Ca2+ current agonist Bay K 8644 and the effect of several interventions known to suppress or enhance these EADs. Bay K 8644 produced an inward shift of the steady-state current-voltage relation near plateau voltages. Tetrodotoxin, lidocaine, verapamil, nitrendipine, and raising [K]o abolish EADs and shift the steady-state current-voltage relations outwardly. Using a two-pulse voltage-clamp protocol, an inward current transient was present at voltages where EADs were induced. The voltage-dependence of availability of the inward current transient and of EAD induction were similar. The time-dependence of recovery from inactivation of the inward current transient and of EAD amplitude were nearly identical. Without recovery of the inward current transient, EADs could not be elicited. The inward current transient was enhanced with Bay K 8644 and blocked by nitrendipine, but was not abolished by tetrodotoxin or replacement of [Na]o with an impermeant cation. These results support a hypothesis that the induction of EADs near action potential plateau voltages requires 1) a conditioning phase controlled by the sum of membrane currents present near the action potential plateau and characterized by lengthening and flattening of the plateau within a voltage range where, 2) recovery from inactivation and reactivation of L-type Ca2+ channels to carry the depolarizing charge can occur. Our results suggest an essential role for the L-type Ca2+ "window" current and provide a framework for understanding the role of several membrane currents in the induction and block of EADs.

The aim of this review is to provide a systematic overview on constitutively active G-protein-coupled receptors (GPCRs), a rapidly evolving area in signal transduction research. We will discuss mechanisms, pharmacological tools and methodological approaches to analyze constitutive activity. The two-state model defines constitutive activity as the ability of a GPCR to undergo agonist-independent isomerization from an inactive (R) state to an active (R*) state. While the two-state model explains basic concepts of constitutive GPCR activity and inverse agonism, there is increasing evidence for multiple active GPCR conformations with distinct biological activities. As a result of constitutive GPCR activity, basal G-protein activity increases. Until now, constitutive activity has been observed for more than 60 wild-type GPCRs from the families 1-3 and from different species including humans and commonly used laboratory animal species. Additionally, several naturally occurring and disease-causing GPCR mutants with increased constitutive activity relative to wild-type GPCRs have been identified. Alternative splicing, RNA editing, polymorphisms within a given species, species variants and coupling to specific G-proteins all modulate the constitutive activity of GPCRs, providing multiple regulatory switches to fine-tune basal cellular activities. The most important pharmacological tools to analyze constitutive activity are inverse agonists and Na(+) that stabilize the R state, and pertussis toxin that uncouples GPCRs from G(i)/G(o)-proteins. Constitutive activity is observed at low and high GPCR expression levels, in native systems and in recombinant systems, and has been reported for GPCRs coupled to G(s)-, G(i)- and G(q)-proteins. Constitutive activity of neurotransmitter GPCRs may provide a tonic support for basal neuronal activity. For the majority of GPCRs known to be constitutively active, inverse agonists have already been identified. Inverse agonists may be useful in the treatment of neuropsychiatric and cardiovascular diseases and of diseases caused by constitutively active GPCR mutants.

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, has been implicated in the neurotoxicity resulting from hypoxia-ischemia, and its inhibition has therapeutic potential for ischemic stroke. However, COX-2 inhibitors increase the risk of cardiovascular complications. We therefore sought to identify the downstream effectors of COX-2 neurotoxicity, and found that prostaglandin E(2) EP1 receptors are essential for the neurotoxicity mediated by COX-2-derived prostaglandin E(2). EP1 receptors disrupt Ca(2+) homeostasis by impairing Na(+)-Ca(2+) exchange, a key mechanism by which neurons cope with excess Ca(2+) accumulation after an excitotoxic insult. Thus, EP1 receptors contribute to neurotoxicity by augmenting the Ca(2+) dysregulation underlying excitotoxic neuronal death. Pharmacological inhibition or gene inactivation of EP1 receptors ameliorates brain injury induced by excitotoxicity, oxygen glucose deprivation and middle cerebral artery (MCA) occlusion. An EP1 receptor inhibitor reduces brain injury when administered 6 hours after MCA occlusion, suggesting that EP1 receptor inhibition may be a viable therapeutic option in ischemic stroke.

Despite striking advances in functional brain imaging, the cellular and molecular mechanisms that underlie the signals detected by these techniques are still largely unknown. The basic physiological principle of functional imaging is represented by the tight coupling existing between neuronal activity and the associated local increase in both blood flow and energy metabolism. Positron emission tomography (PET) signals detect blood flow, oxygen consumption and glucose use associated with neuronal activity; the degree of blood oxygenation is currently thought to contribute to the signal detected with functional magnetic resonance imaging, while magnetic resonance spectroscopy (MRS) identifies the spatio-temporal pattern of the activity-dependent appearance of certain metabolic intermediates such as glucose or lactate. Recent studies, including those of neurotransmitter-regulated metabolic fluxes in purified preparations and analyses of the cellular localization of enzymes and transporters involved in energy metabolism, as well as in vivo microdialysis and MRS approaches have identified the neurotransmitter glutamate and astrocytes, a specific type of glial cell, as pivotal elements in the coupling of synaptic activity with energy metabolism. Astrocytes are ideally positioned to sense increases in synaptic activity and to couple them with energy metabolism. Indeed they possess specialized processes that cover the surface of intraparenchymal capillaries, suggesting that astrocytes may be a likely site of prevalent glucose uptake. Other astrocyte processes are wrapped around synaptic contacts which possess receptors and reuptake sites for neurotransmitters. Glutamate stimulates glucose uptake into astrocytes. This effect is mediated by specific glutamate transporters present on these cells. The activity of these transporters, which is tightly coupled to the synaptic release of glutamate and operates the clearance of glutamate from the extracellular space, is driven by the electrochemical gradient of Na+. This Na(+)-dependent uptake of glutamate into astrocytes triggers a cascade of molecular events involving the Na+/K(+)-ATPase leading to the glycolytic processing of glucose and the release of lactate by astrocytes. The stoichiometry of this process is such that for one glutamate molecule taken up with three Na+ ions, one glucose molecule enters an astrocyte, two ATP molecules are produced through aerobic glycolysis and two lactate molecules are released. Within the astrocyte, one ATP molecule fuels one 'turn of the pump' while the other provides the energy needed to convert glutamate to glutamine by glutamine synthase. Evidence has been accumulated from structural as well as functional studies indicating that, under aerobic conditions, lactate may be the preferred energy substrate of activated neurons. Indeed, in the presence of oxygen, lactate is converted to pyruvate, which can be processed through the tricarboxylic acid cycle and the associated oxidative phosphorylation, to yield 17 ATP molecules per lactate molecule. These data suggest that during activation the brain may transiently resort to aerobic glycolysis occurring in astrocytes, followed by the oxidation of lactate by neurons. The proposed model provides a direct mechanism to couple synaptic activity with glucose use and is consistent with the notion that the signals detected during physiological activation with 18F-deoxyglucose (DG)-PET may reflect predominantly uptake of the tracer into astrocytes. This conclusion does not question the validity of the 2-DG-based techniques, rather it provides a cellular and molecular basis for these functional brain imaging techniques.

We surveyed the genetic diversity among avian influenza virus (AIV) in wild birds, comprising 167 complete viral genomes from 14 bird species sampled in four locations across the United States. These isolates represented 29 type A influenza virus hemagglutinin (HA) and neuraminidase (NA) subtype combinations, with up to 26% of isolates showing evidence of mixed subtype infection. Through a phylogenetic analysis of the largest data set of AIV genomes compiled to date, we were able to document a remarkably high rate of genome reassortment, with no clear pattern of gene segment association and occasional inter-hemisphere gene segment migration and reassortment. From this, we propose that AIV in wild birds forms transient "genome constellations," continually reshuffled by reassortment, in contrast to the spread of a limited number of stable genome constellations that characterizes the evolution of mammalian-adapted influenza A viruses.

Intracellular concentrations of redox-active molecules can significantly increase in the heart as a result of activation of specific signal transduction pathways or the development of certain pathophysiological conditions. Changes in the intracellular redox environment can affect many cellular processes, including the gating properties of ion channels and the activity of ion transporters. Because cardiac contraction is highly dependent on intracellular Ca(2+) levels ([Ca(2+)](i)) and [Ca(2+)](i) regulation, redox modification of Ca(2+) channels and transporters has a profound effect on cardiac function. The sarcoplasmic reticulum (SR) Ca(2+) release channel, or ryanodine receptor (RyR), is one of the well-characterized redox-sensitive ion channels in the heart. The redox modulation of RyR activity is mediated by the redox modification of sulfhydryl groups of cysteine residues. Other key components of cardiac excitation-contraction (e-c) coupling such as the SR Ca(2+) ATPase and L-type Ca(2+) channel are subject to redox modulation. Redox-mediated alteration of the activity of ion channels and pumps is directly involved in cardiac pathologies such as ischemia-reperfusion injury. Significant bursts of reactive oxygen species (ROS) generation occur during reperfusion of the ischemic heart, and changes in the activity of the major components of [Ca(2+)](i) regulation, such as RyR, Na(+)-Ca(2+) exchange and Ca(2+) ATPases, are likely to play an important role in ischemia-related Ca(2+) overload. This article summarizes recent findings on redox regulation of cardiac Ca(2+) transport systems and discusses contributions of this redox regulation to normal and pathological cardiac function.

In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.

OBJECTIVE

To compare the efficacy of neoadjuvant (NA) docetaxel (DOC) with anthracycline-based therapy and determine the efficacy of NA DOC in patients with breast cancer initially failing to respond to anthracycline-based NA chemotherapy (CT).

METHODS

Patients with large or locally advanced breast cancer received four pulses of cyclophosphamide 1,000 mg/m(2), doxorubicin 50 mg/m(2), vincristine 1.5 mg/m(2), and prednisolone 40 mg (4 x CVAP) for 5 days. Clinical tumor response was assessed. Those who responded (complete response [CR] or partial response [PR]) were randomized to receive further 4 x CVAP or 4 x DOC (100 mg/m(2)). All nonresponders received 4 x DOC.

RESULTS

One hundred sixty-two patients were enrolled; 145 patients completed eight cycles of NA CT. One hundred two patients (66%) achieved a clinical response (PR or CR) after 4 x CVAP. After randomization, 50 patients received 4 x CVAP and 47 patients received 4 x DOC. In patients who received eight cycles of CT, the clinical CR (cCR) and clinical PR (cPR) (94% v 66%) and pathologic CR (pCR) (34% v 16%) response rates were higher (P =.001 and P =.04) in those who received further DOC. Intention-to-treat analysis demonstrated cCR and cPR (85% v 64%; P =.03) and pCR (31% v 15%; P =.06). Axillary lymph node examination revealed residual tumor in 33% of patients who received 8 x CVAP and 38% of patients who received further DOC. In patients who failed to respond to the initial CVAP, 4 x DOC resulted in a cCR and cPR rate of 55% and a pCR rate of 2%. Forty-four percent of these patients had residual tumor within axillary lymph nodes.

CONCLUSIONS

NA DOC resulted in substantial improvement in responses to DOC.

Renal salt loss in Bartter's syndrome is caused by impaired transepithelial transport in the loop of Henle. Sodium chloride is taken up apically by the combined activity of NKCC2 (Na+-K--2Cl- cotransporters) and ROMK potassium channels. Chloride ions exit from the cell through basolateral ClC-Kb chloride channels. Mutations in the three corresponding genes have been identified that correspond to Bartter's syndrome types 1-3. The gene encoding the integral membrane protein barttin is mutated in a form of Bartter's syndrome that is associated with congenital deafness and renal failure. Here we show that barttin acts as an essential beta-subunit for ClC-Ka and ClC-Kb chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either ClC-Kb or barttin compromise currents through heteromeric channels. Currents can be stimulated further by mutating a proline-tyrosine (PY) motif on barttin. This work describes the first known beta-subunit for CLC chloride channels and reveals that heteromers formed by ClC-K and barttin are crucial for renal salt reabsorption and potassium recycling in the inner ear.

Originally considered an enigmatic protein, the sigma-1 receptor has recently been identified as a unique ligand-regulated molecular chaperone in the endoplasmic reticulum of cells. This discovery causes us to look back at the many proposed roles of this receptor, even before its molecular function was identified, in many diseases such as methamphetamine or cocaine addiction, amnesia, pain, depression, Alzheimer's disease, stroke, retinal neuroprotection, HIV infection, and cancer. In this review, we examine the reports that have clearly shown an agonist-antagonist relationship regarding sigma-1 receptors in models of those diseases and also review the relatively known mechanisms of action of sigma-1 receptors in an attempt to spur the speculation of readers on how the sigma-1 receptor at the endoplasmic reticulum might relate to so many diseases. We found that the most prominent action of sigma-1 receptors in biological systems including cell lines, primary cultures, and animals is the regulation and modulation of voltage-regulated and ligand-gated ion channels, including Ca(2+)-, K(+)-, Na(+), Cl(-), and SK channels, and NMDA and IP3 receptors. We found that the final output of the action of sigma-1 receptor agonists is to inhibit all above-mentioned voltage-gated ion channels, while they potentiate ligand-gated channels. The inhibition or potentiation induced by agonists is blocked by sigma-1 receptor antagonists. Other mechanisms of action of sigma-1 receptors, and to some extent those of sigma-2 receptors, were also considered. We conclude that the sigma-1 and sigma-2 receptors represent potential fruitful targets for therapeutic developments in combating many human diseases.

The oxidation of glucose represents a major source of metabolic energy for mammalian cells. However, because the plasma membrane is impermeable to polar molecules such as glucose, the cellular uptake of this important nutrient is accomplished by membrane-associated carrier proteins that bind and transfer it across the lipid bilayer. Two classes of glucose carriers have been described in mammalian cells: the Na(+)-glucose cotransporter and the facilitative glucose transporter. The Na(+)-glucose cotransporter transports glucose against its concentration gradient by coupling its uptake with the uptake of Na+ that is being transported down its concentration gradient. Facilitative glucose carriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport. cDNAs have been isolated from human tissues encoding a Na(+)-glucose-cotransporter protein and five functional facilitative glucose-transporter isoforms. The Na(+)-glucose cotransporter is expressed by absorptive epithelial cells of the small intestine and is involved in the dietary uptake of glucose. The same or a related protein may be responsible for the reabsorption of glucose by the kidney. Facilitative glucose carriers are expressed by most if not all cells. The facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions. The GLUT1 (erythrocyte) and GLUT3 (brain) facilitative glucose-transporter isoforms may be responsible for basal or constitutive glucose uptake. The GLUT2 (liver) isoform mediates the bidirectional transport of glucose by the hepatocyte and is responsible, at least in part, for the movement of glucose out of absorptive epithelial cells into the circulation in the small intestine and kidney. This isoform may also comprise part of the glucose-sensing mechanism of the insulin-producing beta-cell. The subcellular localization of the GLUT4 (muscle/fat) isoform changes in response to insulin, and this isoform is responsible for most of the insulin-stimulated uptake of glucose that occurs in muscle and adipose tissue. The GLUT5 (small intestine) facilitative glucose-transporter isoform is expressed at highest levels in the small intestine and may be involved in the transcellular transport of glucose by absorptive epithelial cells. The exon-intron organizations of the human GLUT1, GLUT2, and GLUT4 genes have been determined. In addition, the chromosomal locations of the genes encoding the Na(+)-dependent and facilitative glucose carriers have been determined. Restriction-fragment-length polymorphisms have also been identified at several of these loci.(ABSTRACT TRUNCATED AT 400 WORDS)

In mammals, active transport of organic solutes across plasma membranes was thought to be primarily driven by the Na+ gradient. Here we report the cloning and functional characterization of a H(+)-coupled transporter of oligopeptides and peptide-derived antibiotics from rabbit small intestine. This new protein, named PepT1, displays an unusually broad substrate specificity. PepT1-mediated uptake is electrogenic, independent of extracellular Na+, K+ and Cl-, and of membrane potential. PepT1 messenger RNA was found in intestine, kidney and liver and in small amounts in brain. In the intestine, the PepT1 pathway constitutes a major mechanism for absorption of the products of protein digestion. To our knowledge, the PepT1 primary structure is the first reported for a proton-coupled organic solute transporter in vertebrates and represents an interesting evolutionary link between prokaryotic H(+)-coupled and vertebrate Na(+)-coupled transporters of organic solutes.

Ionic fluxes in Na channels of myelinated axons show ionic competition, block, and deviations from simple flux independence. These phenomena are particularly evident when external Na+ ions are replaced by other permeant or impermeant ions. The observed currents require new flux equations not based on the concepts of free diffusion. A specific permeability model for the Na channel is developed from Eyring rate theory applied to a chain of saturable binding sites. There are four energy barriers in the pore and only one ion is allowed inside at a time. Deviations from independence arise from saturation. The model shows that ionic permeability ratios measured from zero-current potentials can differ from those measured from relative current amplitudes or conductances. The model can be fitted to experiments with various external sodium substitutes by varying only two parameters: For each ion the height of the major energy barrier (the selectivity filter) determines the biionic zero-current potential and the depth of the energy well (binding site) just external to that barrier then determines the current amplitudes. Voltage clamp measurements with myelinated nerve fibers are given showing numerous examples of deviations from independence in ionic fluxes. Strong blocks of ionic currents by guanidinium compounds and Tl+ ions are fitted by binding within the channel with apparent dissociation constants in the range 50-122 mM. A small block with high Na+ concentrations can be fitted by Na+ ion binding with a dissociation constant of 368 mM. The barrier model is given a molecular interpretation that includes stepwise dehydration of the permeating ion as it interacts with an ionized carboxylic acid.

The mtDNA variation of 321 individuals from 17 Native American populations was examined by high-resolution restriction endonuclease analysis. All mtDNAs were amplified from a variety of sources by using PCR. The mtDNA of a subset of 38 of these individuals was also analyzed by D-loop sequencing. The resulting data were combined with previous mtDNA data from five other Native American tribes, as well as with data from a variety of Asian populations, and were used to deduce the phylogenetic relationships between mtDNAs and to estimate sequence divergences. This analysis revealed the presence of four haplotype groups (haplogroups A, B, C, and D) in the Amerind, but only one haplogroup (A) in the Na-Dene, and confirmed the independent origins of the Amerinds and the Na-Dene. Further, each haplogroup appeared to have been founded by a single mtDNA haplotype, a result which is consistent with a hypothesized founder effect. Most of the variation within haplogroups was tribal specific, that is, it occurred as tribal private polymorphisms. These observations suggest that the process of tribalization began early in the history of the Amerinds, with relatively little intertribal genetic exchange occurring subsequently. The sequencing of 341 nucleotides in the mtDNA D-loop revealed that the D-loop sequence variation correlated strongly with the four haplogroups defined by restriction analysis, and it indicated that the D-loop variation, like the haplotype variation, arose predominantly after the migration of the ancestral Amerinds across the Bering land bridge.

Sodium (Na+) at high millimolar concentrations in soils is toxic to most higher plants and severely reduces agricultural production worldwide. However, the molecular mechanisms for plant Na+ uptake remain unknown. Here, the wheat root high-affinity potassium (K+) uptake transporter HKT1 was shown to function as a high-affinity K(+)-Na+ cotransporter. High-affinity K+ uptake was activated by micromolar Na+ concentrations; moreover, high-affinity Na+ uptake was activated by K+ (half-activation constant, 2.8 microM K+). However, at physiologically detrimental concentrations of Na+, K+ accumulation mediated by HKT1 was blocked and low-affinity Na+ uptake occurred (Michaelis constant, approximately 16 mM Na+), which correlated to Na+ toxicity in plants. Point mutations in the sixth putative transmembrane domain of HKT1 that increase Na+ tolerance were isolated with the use of yeast as a screening system. Na+ uptake and Na+ inhibition of K+ accumulation indicate a possible role for HKT1 in physiological Na+ toxicity in plants.

It is clear from the literature reviewed that modifications in membrane lipid composition play a major role in the adaptation of diverse organisms to specific environments and physiological circumstances. Acyl chain and molecular species restructuring in phospholipids are the most ubiquitous adaptations to environmental insult, being implicated in membrane adjustments to temperature, pressure, water activity, pH and salinity. In contrast, other adaptations (e.g. modulation of anionic phospholipids (salinity adaptation), trehalose content (dehydration) and the PC/PE ratio (temperature acclimation] appear to be more context specific. Although the volume of correlative data relating membrane composition to environmental state is impressive, several questions must be explicitly addressed in future research if a mechanistic understanding of the role of lipids in fine tuning membrane function is to be achieved. These include: (1) Adaptation thresholds--How much environmental variation is required before an acclimatory response is initiated, and is the extent of membrane perturbation induced by such minimally effective stimuli similar for different stress vectors? Interspecific comparisons of the Na+/K(+)-ATPase of fish collected at different depths indicate that species must be separated in depth by a distance corresponding to a pressure difference of 20 MPa before pressure adaptation is evident. Assuming a dT/dP value of 0.23 (Table 1), a 20 MPa change in pressure corresponds to ca. a 5 degrees C change in temperature, which agrees well with the minimal temperature change required to elicit changes in the lipid composition of plasma membranes in kidney tissue of thermally-acclimating trout. A pressure of 20 MPa also corresponds approximately to the maximum depth from which deep sea animals survive being brought to the surface. Collectively, these observations suggest that the minimally effective stimuli for both temperature and pressure adaptation are similar. Comparable data are not available for other environmental variables. (2) Signal transduction--What signals are being sensed and how are they transduced into an adaptational response? In some cases, it is clear that the enzymes of lipid metabolism respond directly (either by a variation in catalytic rate or substrate preference) to variations in the physical environment in an apparently adaptive manner (e.g. refer Sections VI.A.1 and VI.B.2). It seems unlikely, however, that such direct effects can explain the totality of the adaptive capacity of organisms, especially given the evidence for the induction of desaturase synthesis in cold adaptation (refer to Section VI.A.2).(ABSTRACT TRUNCATED AT 400 WORDS)