Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K(+) channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (</=1 mM), which block only a few known K(+) channels including Kv3.1-Kv3.2, profoundly impaired action potential repolarization and high-frequency firing. Analysis of the spike trains evoked by steady depolarization revealed that, although TEA had little effect on the initial firing rate, it strongly reduced firing frequency later in the trains. These effects appeared to be specific to Kv3.1 and Kv3.2 channels, because blockade of dendrotoxin-sensitive Kv1 channels and BK Ca(2+)-activated K(+) channels, which also have high TEA sensitivity, produced opposite or no effects. Voltage-clamp experiments confirmed the presence of a Kv3.1-Kv3.2-like current in fast-spiking neurons, but not in other interneurons. Analysis of spike shape changes during the spike trains suggested that <em>Na</em>(+) channel inactivation plays a significant role in the firing-rate slowdown produced by TEA, a conclusion that was supported by computer simulations. These findings indicate that the unique properties of Kv3.1-Kv3.2 channels enable sustained high-frequency firing by facilitating the recovery of <em>Na</em>(+) channel inactivation and by minimizing the duration of the afterhyperpolarization in neocortical interneurons.

The ability of wheat to maintain a low sodium concentration ([Na(+)]) in leaves correlates with improved growth under saline conditions. This trait, termed Na(+) exclusion, contributes to the greater salt tolerance of bread wheat relative to durum wheat. To improve the salt tolerance of durum wheat, we explored natural diversity in shoot Na(+) exclusion within ancestral wheat germplasm. Previously, we showed that crossing of Nax2, a gene locus in the wheat relative Triticum monococcum into a commercial durum wheat (Triticum turgidum ssp. durum var. Tamaroi) reduced its leaf [Na(+)] (ref. 5). Here we show that a gene in the Nax2 locus, TmHKT1;5-A, encodes a Na(+)-selective transporter located on the plasma membrane of root cells surrounding xylem vessels, which is therefore ideally localized to withdraw Na(+) from the xylem and reduce transport of Na(+) to leaves. Field trials on saline soils demonstrate that the presence of TmHKT1;5-A significantly reduces leaf [Na(+)] and increases durum wheat grain yield by 25% compared to near-isogenic lines without the Nax2 locus.

Astrocytes exhibit excitability based on variations of their intracellular Ca2+ concentrations, which leads to glutamate release, that in turn can signal to adjacent neurons. This glutamate-mediated astrocyte-neuron signaling occurs at physiological intracellular Ca2+ levels in astrocytes and includes modulation of synaptic transmission. The mechanism underlying Ca2+-dependent glutamate release from astrocytes is most likely exocytosis, because astrocytes express the protein components of the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors complex, including synaptobrevin 2, syntaxin, and synaptosome-associated protein of 23 kDa. Although these proteins mediate Ca2+-dependent glutamate release from astrocytes, it is not well understood whether astrocytes express functional vesicular glutamate transporters (VGLUTs) that are critical for vesicle refilling. Here, we find in cultured and freshly isolated astrocytes the presence of brain-specific Na+-dependent inorganic phosphate cotransporter and differentiation-associated Na+-dependent inorganic phosphate cotransporter that have recently been identified as VGLUTs 1 and 2. Indirect immunocytochemistry showed a punctate pattern of VGLUT immunoreactivity throughout the entire cell body and processes, whereas pharmacological inhibition of VGLUTs abolished mechanically and agonist-evoked Ca2+-dependent glutamate release from astrocytes. Taken together, these data indicate that VGLUTs play a functional role in exocytotic glutamate release from astrocytes.

Current-clamp recordings were made from the deep cerebellar nuclei (DCN) of 12- to 15-day-old rats to understand the factors that mediate intrinsic spontaneous firing patterns. All of the cells recorded were spontaneously active with spiking patterns ranging continuously from regular spiking to spontaneous bursting with the former predominating. A robust rebound depolarization (RD) leading to a Na(+) spike burst was elicited after the offset of hyperpolarizing current injection. The voltage and time dependence of the RD was consistent with mediation by low-threshold voltage-gated Ca(2+) channels. In addition, induction of a RD also may be affected by activation of a hyperpolarization-activated cation current, I(h). A RD could be evoked efficiently after brief high-frequency bursts of inhibitory postsynaptic potentials (IPSPs) induced by stimulation of Purkinje cell axons. IPSP-driven RDs were typically much larger and longer than those elicited by direct hyperpolarizing pulses of approximately matched amplitude and duration. Intracellular perfusion of the Ca(2+) buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) dramatically enhanced the RD and its associated spiking, sometimes leading to a plateau potential that lasted several hundred milliseconds. The effects of BAPTA could be mimicked partly by application of apamin, a blocker of small conductance Ca(2+)-gated K(+) channels, but not by paxilline, which blocks large conductance Ca(2+)-gated K(+) channels. Application of both BAPTA and apamin, but not paxilline, caused cells that were regularly spiking to burst spontaneously. Taken together, our data suggest that there is a strong relationship between the ability of DCN cells to elicit a RD and their tendency burst spontaneously. The RD can be triggered by the opening of T-type Ca(2+) channels with an additional contribution of hyperpolarization-activated current I(h). RD duration is regulated by small-conductance Ca(2+)-gated K(+) channels. The RD also is modulated tonically by inhibitory inputs. All of these factors are in turn subject to alteration by extrinsic modulatory neurotransmitters and are, at least in part, responsible for determining the firing modes of DCN neurons.

1. When K(+) is removed from both sides of the somal membrane of Limnea neurones, time-dependent, voltage-dependent outward currents are observed at positive potentials. These currents can be carried by Tris(+) and tetraethylammonium (TEA(+)), as well as Cs(+), but the Cs currents are several times larger. The Cs currents are not affected by external or internal TEA, but are strongly reduced by 4-aminopyridine (4-AP) and all Ca blockers tried.2. The presence of these non-specific outward currents and their sensitivity to all treatments that eliminate the Ca currents prevent the complete isolation of Ca currents. The non-specific outward currents are most prominent at large positive potentials and as slow tail currents on stepping back to the holding potential.3. Ca currents are ;washed out' in well perfused cells. Typically the Ca current has decayed to less than one tenth of its original size after (1/2) h of perfusion. This wash-out is specific for the Ca current; Na and K currents persist for several hours.4. Once the Ca current has completely decayed, it is possible to study one type of non-specific current without overlapping inward currents. This current activates between 0 and +30 mV and appears to reverse near 0 mV.5. In spite of the probable presence of slowly activating outward currents, the net inward currents measured show little apparent inactivation. In all the cells studied the inward current evoked at +20 mV has never decayed by more than 50% during a 60 ms pulse. So the true inactivation of these Ca currents must be quite slow, with time constants of the order of 100 ms and larger.6. The activation of the Ca current agrees with m(2) kinetics. The rate of activation is the same for Ba currents as for Ca currents.7. When the membrane potential is stepped back to the holding level (-50 mV), the Ca current turns off quite rapidly with a time constant of about 100 mus (25 degrees C). The time constant for turning off the Ca current is not related to the time constant for turning on the Ca current at the same voltage as expected for m(2) kinetics in the Hodgkin and Huxley model. At -30 mV the tau(m) for turn-on is eight times larger than the tau(m) for turn-off.

There is growing evidence that oxidative stress contributes to hypertension. Oxidative stress can precede the development of hypertension. In almost all models of hypertension, there is oxidative stress that, if corrected, lowers BP, whereas creation of oxidative stress in normal animals can cause hypertension. There is overexpression of the p22(phox) and Nox-1 components of NADPH oxidase and reduced expression of extracellular superoxide dismutase (EC-SOD) in the kidneys of ANG II-infused rodents, whereas there is overexpression of p47(phox) and gp91(phox) and reduced expression of intracellular SOD with salt loading. Several mechanisms have been identified that can make oxidative stress self-sustaining. Reactive oxygen species (ROS) can enhance afferent arteriolar tone and reactivity both indirectly via potentiation of tubuloglomerular feedback and directly by microvascular mechanisms that diminish endothelium-derived relaxation factor/nitric oxide responses, generate a cyclooxygenase-2-dependent endothelial-derived contracting factor that activates thromboxane-prostanoid receptors, and enhance vascular smooth muscle cells reactivity. ROS can diminish the efficiency with which the kidney uses O(2) for Na(+) transport and thereby diminish the P(O(2)) within the kidney cortex. This may place a break on further ROS generation yet could further enhance vasculopathy and hypertension. There is a tight relationship between oxidative stress in the kidney and the development and maintenance of hypertension.

Glucose is the major energy source for mammalian cells as well as an important substrate for protein and lipid synthesis. Mammalian cells take up glucose from extracellular fluid into the cell through two families of structurallyrelated glucose transporters. The facilitative glucose transporter family (solute carriers SLC2A, protein symbol GLUT) mediates a bidirectional and energy-independent process of glucose transport in most tissues and cells, while the NaM(+)/glucose cotransporter family (solute carriers SLC5A, protein symbol SGLT) mediates an active, Na(+)-linked transport process against an electrochemical gradient. The GLUT family consists of thirteen members (GLUT1-12 and HMIT). Phylogenetically, the members of the GLUT family are split into three classes based on protein similarities. Up to now, at least six members of the SGLT family have been cloned (SGLT1-6). In this review, we report both the genomic structure and function of each transporter as well as intra-species comparative genomic analysis of some of these transporters. The affinity for glucose and transport kinetics of each transporter differs and ranges from 0.2 to 17mM. The ability of each protein to transport alternative substrates also differs and includes substrates such as fructose and galactose. In addition, the tissue distribution pattern varies between species. There are different regulation mechanisms of these transporters. Characterization of transcriptional control of some of the gene promoters has been investigated and alternative promoter usage to generate different protein isoforms has been demonstrated. We also introduce some pathophysiological roles of these transporters in human.

Three-dimensional cardiac mapping in rabbits with nonischemic cardiomyopathy has shown that ventricular arrhythmias initiate by a nonreentrant mechanism that may be due to triggered activity from delayed afterdepolarizations. Delayed afterdepolarizations are thought to be due to spontaneous release of Ca(2+) from the sarcoplasmic reticulum (SR) and consequent activation of an inward Na(+)/Ca(2+) exchange (NaCaX) current. The goal of this study was to determine whether there is enhanced NaCaX gene expression and functional activity that may contribute to nonreentrant activation. Heart failure (HF) was induced in rabbits by combined aortic insufficiency and aortic constriction. HF rabbits had left ventricular enlargement (left ventricular end-diastolic dimension increased from 1.43+/-0.03 to 1.97+/-0.05 cm) and severely depressed function (fractional shortening reduced from 37% to 26%, P<0.02). Heart-to-body weight was increased by 79% in HF. Western blots showed a 93% increase in NaCaX protein in HF (P<0.04). NaCaX mRNA (7-kb transcript) was increased by 104% relative to the 18S rRNA in HF. A 14-kb NaCaX transcript was also seen in the HF rabbits, raising total NaCaX mRNA to 2.7-fold compared with controls. The amplitude of caffeine-induced contractures, used to assess SR Ca(2+) load, was not significantly different in HF. Relaxation and [Ca(2+)](i) decline during caffeine-induced contractures is attributable to Ca(2+) transport by NaCaX and was 61% and 45% faster in HF (P<0.05), respectively. NaCaX current measured under controlled voltage clamp conditions was also 2-fold higher in HF cells. SR Ca(2+)-ATPase mRNA and protein levels and Ca(2+) current density were not significantly altered in HF. Twitch amplitudes from HF myocytes were 26% smaller compared with control (P<0.02), but twitch relaxation and [Ca(2+)](i) decline (due largely to SR Ca(2+)-ATPase) were not altered. Thus myocytes and myocardium from HF rabbits exhibit enhanced NaCaX expression and function. The enhanced NaCaX activity may contribute to depressed contractions, increased transient inward current (for a given SR Ca(2+) release), delayed afterdepolarizations, and nonreentrant initiation of ventricular tachycardia in this arrhythmogenic model of HF.

Vesicular glutamate transporter 1 (VGluT1) is one of the best markers for glutamatergic neurons, because it accumulates transmitter glutamate into synaptic vesicles. Differentiation-associated Na(+)-dependent inorganic phosphate cotransporter (DNPI) shows 82% amino acid identity to VGluT1, and is another candidate for vesicular glutamate transporters. Here, we report the immunocytochemical localization of DNPI and compare it with that of VGluT1 in the adult rat brain. Both DNPI and VGluT1 immunoreactivities were found mostly in neuropil, presumably in axon terminals, throughout the brain. In the telencephalic regions, intense DNPI immunoreactivity was observed in the glomeruli of the olfactory bulb, layer IV of the neocortex, granular layer of the dentate gyrus, presubiculum, and postsubiculum. In contrast, VGluT1 immunoreactivity was intense in the olfactory tubercle, layers I-III of the neocortex, piriform cortex, entorhinal cortex, hippocampus, dentate gyrus, and subiculum. In the thalamic nuclei, DNPI-immunoreactive terminal-like profiles were much larger than VGluT1-immunoreactive ones, suggesting that DNPI immunoreactivity was subcortical in origin. DNPI immunoreactivity was much more intense than VGluT1 immunoreactivity in many brainstem and spinal cord regions, except the pontine nuclei, interpeduncular nucleus, cochlear nuclei, and external cuneate nucleus. In the molecular layer of the cerebellar cortex, climbing-like fibers showed intense DNPI immunoreactivity, whereas neuropil contained dense VGluT1-immnoreactive deposits. Both DNPI and VGluT1 immunoreactivities were observed as mossy fiber terminal-like profiles in the cerebellar granular layer. DNPI and VGluT1 immunoreactivities appeared associated with synaptic vesicles in the axon terminals forming asymmetric synapses in several regions examined electron microscopically. The present results indicate that DNPI and VGluT1 are used by different neural components in most, if not all, brain regions, suggesting the complementary functions of DNPI and VGluT1.

OBJECTIVE

Small nerve fiber neuropathy (SFN) often occurs without apparent cause, but no systematic genetic studies have been performed in patients with idiopathic SFN (I-SFN). We sought to identify a genetic basis for I-SFN by screening patients with biopsy-confirmed idiopathic SFN for mutations in the SCN9A gene, encoding voltage-gated sodium channel Na(V)1.7, which is preferentially expressed in small diameter peripheral axons.

METHODS

Patients referred with possible I-SFN, who met the criteria of ≥2 SFN-related symptoms, normal strength, tendon reflexes, vibration sense, and nerve conduction studies, and reduced intraepidermal nerve fiber density (IENFD) plus abnormal quantitative sensory testing (QST) and no underlying etiology for SFN, were assessed clinically and by screening of SCN9A for mutations and functional analyses.

RESULTS

Twenty-eight patients who met stringent criteria for I-SFN including abnormal IENFD and QST underwent SCN9A gene analyses. Of these 28 patients with biopsy-confirmed I-SFN, 8 were found to carry novel mutations in SCN9A. Functional analysis revealed multiple gain of function changes in the mutant channels; each of the mutations rendered dorsal root ganglion neurons hyperexcitable.

CONCLUSIONS

We show for the first time that gain of function mutations in sodium channel Na(V)1.7, which render dorsal root ganglion neurons hyperexcitable, are present in a substantial proportion (28.6%; 8 of 28) of patients meeting strict criteria for I-SFN. These results point to a broader role of Na(V)1.7 mutations in neurological disease than previously considered from studies on rare genetic syndromes, and suggest an etiological basis for I-SFN, whereby expression of gain of function mutant sodium channels in small diameter peripheral axons may cause these fibers to degenerate.

Na(+) channel clustering at nodes of Ranvier in the developing rat optic nerve was analyzed to determine mechanisms of localization, including the possible requirement for glial contact in vivo. Immunofluorescence labeling for myelin-associated glycoprotein and for the protein Caspr, a component of axoglial junctions, indicated that oligodendrocytes were present, and paranodal structures formed, as early as postnatal day 7 (P7). However, the first Na(+) channel clusters were not seen until P9. Most of these were broad, and all were excluded from paranodal regions of axoglial contact. The number of detected Na(+) channel clusters increased rapidly from P12 to P22. During this same period, conduction velocity increased sharply, and Na(+) channel clusters became much more focal. To test further whether oligodendrocyte contact directly influences Na(+) channel distributions, nodes of Ranvier in the hypomyelinating mouse Shiverer were examined. This mutant has oligodendrocyte-ensheathed axons but lacks compact myelin and normal axoglial junctions. During development Na(+) channel clusters in Shiverer mice were reduced in numbers and were in aberrant locations. The subcellular location of Caspr was disrupted, and nerve conduction properties remained immature. These results indicate that in vivo, Na(+) channel clustering at nodes depends not only on the presence of oligodendrocytes but also on specific axoglial contact at paranodal junctions. In rats, ankyrin-3/G, a cytoskeletal protein implicated in Na(+) channel clustering, was detected before Na(+) channel immunoreactivity but extended into paranodes in non-nodal distributions. In Shiverer, ankyrin-3/G labeling was abnormal, suggesting that its localization also depends on axoglial contact.

Two vesicular glutamate transporters (VGluTs) have been identified at the molecular level very recently and revealed to possess similar pharmacological characteristics for glutamate uptake. Vesicular glutamate transporter 1 (VGluT1), which was originally named brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI), is mainly expressed in telencephalic regions, whereas vesicular glutamate transporter 2 (VGluT2), formerly referred to as differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI), is produced principally in diencephalic and lower brainstem regions. Since no other proteins show as high molecular similarity to VGluT1 or VGluT2 as the two transporters exhibit, it is likely that the mammalian central nervous system use only two gene products for vesicular glutamate uptake. Immunoelectron-microscopic analysis has revealed that the two VGluTs are located on synaptic vesicles in axon terminals making an asymmetric type of synapses, supporting that they serve as vesicular transporters in excitatory terminals. Furthermore, mRNA and immunoreactivity for VGluTs are distributed largely in a complementary fashion to distinct populations of excitatory neurons; for example, in the cerebral cortex, thalamocortical axon terminals use VGluT2, whereas excitatory axon terminals of corticocortical or intracortical fibers seem to apply VGluT1 for glutamate uptake. This complementary distribution might suggest that the two VGluTs have an as yet unknown difference in functions.

A class of K channels in cardiac muscle is reversibly blocked by intracellular adenosine 5'-triphosphate (ATP). The characteristics of this K channel were studied by recording single-channel currents in ventricular cells isolated enzymatically from guinea-pig heart. The reversal potential of single-channel currents agreed well with the K equilibrium potential. Blockers of other K channels, such as tetraethylammonium and 4-aminopyridine, decreased the mean open time of the channel. The chord conductance increased as the 0.24th power of the K concentration on the outer surface of the membrane, and showed a marked inward-going rectification on strong depolarizations. The degree of rectification was larger with increasing Na concentration on the inner side of the membrane. The kinetics of the channel were almost voltage independent, but depended on the concentration of intracellular ATP. The conductance of the channel was not affected by ATP. When channel kinetics were examined in the presence of ATP, the distribution of open times and closed times was fitted well with a sum of two exponential components. When ATP concentration was increased, the time constants obtained from the open-time histogram decreased and those from the closed-time histogram increased, resulting in a decrease of the open-state probability. The channel was blocked by ATP, adenosine 5'-diphosphate,5'-adenylylimidodiphosphate, guanosine 5'-triphosphate and uridine 5'-triphosphate, but not by adenosine 5'-monophosphate, creatine phosphate, creatine or adenosine. Plots of the open-state probability versus the ATP concentration revealed Michaelis-Menten saturation kinetics with strong co-operativity of multiple receptor sites (Hill coefficient 3-4, concentration of half-saturation 0.5 mM). It was concluded that this K channel has three or four receptor sites selective for triphosphate nucleotide on the inner surface of the membrane, and that the channel is blocked through the binding of agonists to the receptors.

Because little is known about the human trait of affiliation, we provide a novel neurobehavioral model of affiliative bonding. Discussion is organized around processes of reward and memory formation that occur during approach and consummatory phases of affiliation. Appetitive and consummatory reward processes are mediated independently by the activity of the ventral tegmental area (VTA) dopamine (DA)-nucleus accumbens shell (NAS) pathway and the central corticolimbic projections of the u-opiate system of the medial basal arcuate nucleus, respectively, although these two projection systems functionally interact across time. We next explicate the manner in which DA and glutamate interact in both the VTA and NAS to form incentive-encoded contextual memory ensembles that are predictive of reward derived from affiliative objects. Affiliative stimuli, in particular, are incorporated within contextual ensembles predictive of affiliative reward via: (a) the binding of affiliative stimuli in the rostral circuit of the medial extended amygdala and subsequent transmission to the NAS shell; (b) affiliative stimulus-induced opiate potentiation of DA processes in the VTA and NAS; and (c) permissive or facilitatory effects of gonadal steroids, oxytocin (in interaction with DA), and vasopressin on (i) sensory, perceptual, and attentional processing of affiliative stimuli and (ii) formation of social memories. Among these various processes, we propose that the capacity to experience affiliative reward via opiate functioning has a disproportionate weight in determining individual differences in affiliation. We delineate sources of these individual differences, and provide the first human data that support an association between opiate functioning and variation in trait affiliation.

1. Intracellular pH (pH(i)) of surface fibres of the mouse soleus muscle was measured in vitro by recessed-tip pH-sensitive micro-electrodes. pH(i) was displaced in an acid direction by removal of external (NH(4))(2)SO(4) after a short exposure, and the mechanism of recovery from this acidification was investigated.2. Removal of external K caused a very slow acidification (probably due to the decreasing Na gradient) but had no effect on the rate of pH(i) recovery following acidification. This indicates that K(+)-H(+) exchange is not involved in the pH(i) regulating system.3. Short applications of 10(-4)M ouabain had no obvious effect on pH(i) and did not alter the rate of pH(i) recovery following acidification. This suggests that there is no direct connexion between the regulation of pH(i) and the Na pump.4. Reduction of external Ca from 10 to 1 mM caused a transient fall in pH(i), but the rate of pH(i) recovery following acidification was unaffected. This suggests that Ca(2+)-H(+) exchange is not involved in the pH(i) regulating system.5. An 11% reduction in external Na caused a significant slowing of pH(i) recovery following acidification. 90% or complete removal of external Na almost stopped pH(i) recovery. This suggests that Na(+)-H(+) exchange is involved in pH(i) regulation.6. Amiloride (10(-4)M) reversibly reduced the rate of pH(i) recovery to much the same extent as removal of external Na. Its effect was not additive to that of removal of external Na.7. Internal Na ion concentration ([Na(+)](i)), measured using Na(+)-sensitive micro-electrodes, fell on application of (NH(4))(2)SO(4) and increased on its removal. The increase transiently raised [Na(+)](i) above the level recorded before (NH(4))(2)SO(4) application. This overshoot of [Na(+)](i) was almost completely inhibited by amiloride. This is consistent with the involvement of Na(+)-H(+) exchange in the pH(i) regulating system.8. Removal of external CO(2) or application of SITS (10(-4)M) caused some slowing of the rate of pH(i) recovery following acidification by removal of (NH(4))(2)SO(4). The effect of SITS was additive to that of Na-free Ringer or amiloride. These results suggest that Cl(-)-HCO(3) (-) exchange is also involved in the pH(i) regulating system and that it is a separate mechanism. Under the conditions used, Cl(-)-HCO(3) (-) exchange formed about 20% of the pH(i) regulating system.9. Decreasing the temperature from 37 to 28 degrees C not only caused an increase in pH(i), but also considerably slowed the rate of pH(i) recovery following acidification. We have calculated a Q(10) for Na(+)-H(+) exchange of 1.4 and for Cl(-)-HCO(3) (-) exchange, 6.9.10. We conclude that the pH(i) regulating system is comprised of two separate ionic exchange mechanisms. The major mechanism is Na(+)-H(+) exchange, which is probably driven by the transmembrane Na gradient. The other mechanism is Cl(-)-HCO(3) (-) exchange, which probably requires metabolic energy.

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor appears to play a pivotal role in enabling glutamate to express its neurotoxic potential in a variety of neurological disorders. Our results show that the transition of glutamate from neurotransmitter to neurotoxin is facilitated when cellular energy is limited in cultured cerebellar neurons. Omission of glucose, exclusion of oxygen, or inclusion of inhibitors of oxidative phosphorylation or of the sodium/potassium pump, enables the excitatory amino acids glutamate or NMDA to express their neurotoxic potential. We interpret these results as demonstrating that glucose metabolism, ATP production, and functioning Na+,K+-ATPases are necessary to generate a resting potential sufficient to maintain the voltage-dependent Mg2+ block of the NMDA receptor channel; relief of the Mg2+ block enables the excitatory amino acids to act persistently at the NMDA receptor, resulting in the opening of ion channels and subsequent neuronal damage. These findings are discussed in the context of perturbations or abnormalities which lead to decreased availability or utilization of glucose and oxygen in the brain which may trigger endogenous excitatory amino acids to become neurotoxic by this mechanism.

The cytotoxic response of cells in culture is dependant on the degree of functionalization of the single-walled carbon nanotube (SWNT). After characterizing a set of water-dispersible SWNTs, we performed in vitro cytotoxicity screens on cultured human dermal fibroblasts (HDF). The SWNT samples used in this exposure include SWNT-phenyl-SO(3)H and SWNT-phenyl-SO(3)Na (six samples with carbon/-phenyl-SO(3)X ratios of 18, 41, and 80), SWNT-phenyl-(COOH)(2) (one sample with carbon/-phenyl-(COOH)(2) ratio of 23), and underivatized SWNT stabilized in 1% Pluronic F108. We have found that as the degree of sidewall functionalization increases, the SWNT sample becomes less cytotoxic. Further, sidewall functionalized SWNT samples are substantially less cytotoxic than surfactant stabilized SWNTs. Even though cell death did not exceed 50% for cells dosed with sidewall functionalized SWNTs, optical and atomic force microscopies show direct contact between cellular membranes and water-dispersible SWNTs; i.e. the SWNTs in aqueous suspension precipitate out and selectively deposit on the membrane.

We have shown that ouabain activates Src, resulting in subsequent tyrosine phosphorylation of multiple effectors. Here, we tested if the Na+/K+-ATPase and Src can form a functional signaling complex. In LLC-PK1 cells the Na+/K+-ATPase and Src colocalized in the plasma membrane. Fluorescence resonance energy transfer analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pulldown assay showed a direct, ouabain-regulated, and multifocal interaction between the 1 subunit of Na+/K+-ATPase and Src. Although the interaction between the Src kinase domain and the third cytosolic domain (CD3) of 1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na+/K+-ATPase or GST-CD3 inhibited Src activity. Addition of ouabain, but not vanadate, to the purified Na+/K+-ATPase/Src complex freed the kinase domain and restored the Src activity. Consistently, exposure of intact cells to ouabain apparently increased the distance between the Na+/K+-ATPase and Src. Concomitantly, it also stimulated tyrosine phosphorylation of the proteins that are associated with the Na+/K+-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a nonreceptor tyrosine kinase.

The sodium/glucose cotransporter family (SLCA5) has 220 or more members in animal and bacterial cells. There are 11 human genes expressed in tissues ranging from epithelia to the central nervous system. The functions of nine have been revealed by studies using heterologous expression systems: six are tightly coupled plasma membrane Na(+)/substrate cotransporters for solutes such as glucose, myo-inositol and iodide; one is a Na(+)/Cl(-)/choline cotransporter; one is an anion transporter; and another is a glucose-activated ion channel. The exon organization of eight genes is similar in that each comprises 14-15 exons. The choline transporter (CHT) is encoded in eight exons and the Na(+)-dependent myo-inositol transporter (SMIT) in one exon. Mutations in three genes produce genetic diseases (glucose-galactose malabsorption, renal glycosuria and hypothyroidism). Members of this family are multifunctional membrane proteins in that they also behave as uniporters, urea and water channels, and urea and water cotransporters. Consequently it is a challenge to determine the role(s) of these genes in human physiology and pathology.

Mouse embryo dorsal root ganglion neurones were grown in tissue culture and voltage-clamped with two micro-electrodes. Hyperpolarizing voltage commands from holding potentials of -50 to -60 mV evoked slow inward current relaxations which were followed by inward tail currents on repolarization to the holding potential. These relaxations are due to the presence of a time- and voltage-dependent conductance provisionally termed Gh. Gh activates over the membrane potential range -60 to -120 mV. The presence of Gh causes time-dependent rectification in the current-voltage relationship measured between -60 and -120 mV. Gh does not inactivate within this range and thus generates a steady inward current at hyperpolarized membrane potentials. The current carried by Gh increases when the extracellular K+ concentration is raised, and is greatly reduced in Na+-free solutions. Current-voltage plots show considerably less inward rectification in Na+-free solution; conversely inward rectification is markedly enhanced when the extracellular K+ concentration is raised. The reversal potential of Ih is close to -30 mV in media of physiological composition. Tail-current measurement suggests that Ih is a mixed Na+-K+ current. Low concentrations of Cs+ reversibly block Ih and produce outward rectification in the steady-state current-voltage relationship recorded between membrane potentials of -60 and -120 mV. Cs+ also reversibly abolishes the sag and depolarizing overshoot that distort hyperpolarizing electrotonic potentials recorded in current-clamp experiments. Impermeant anion substitutes reversibly block Ih; this block is different from that produced by Cs+ or Na+-free solutions: Cs+ produces outward rectification in the steady-state current-voltage relationship recorded over the Ih activation range; in Na+-free solutions inward rectification, of reduced amplitude, can still be recorded since Ih is a mixed Na+-K+ current; in anion-substituted solutions the current-voltage relationship becomes approximately linear. It is concluded that in dorsal root ganglion neurones anomalous rectification is generated by the time-and voltage-dependent current Ih. The possible function of Ih in sensory neurones is discussed.

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na(+) sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain [K(+)](int) in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K(+)/Na(+) than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K(+) deficiency on culture medium with low Ca(2+) (0.15 mM) and <200 microM K(+). Interestingly, the capacity of hkt1 mutations to suppress the Na(+) hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca(2+) (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na(+) entry and high affinity K(+) uptake. The hkt1 mutations have revealed the existence of another Na(+) influx system(s) whose activity is reduced by high [Ca(2+)](ext).

Conceptual models of carcinogenesis typically consist of an evolutionary sequence of heritable changes in genes controlling proliferation, apoptosis, and senescence. We propose that these steps are necessary but not sufficient to produce invasive breast cancer because intraductal tumour growth is also constrained by hypoxia and acidosis that develop as cells proliferate into the lumen and away from the underlying vessels. This requires evolution of glycolytic and acid-resistant phenotypes that, we hypothesise, is critical for emergence of invasive cancer. Mathematical models demonstrate severe hypoxia and acidosis in regions of intraductal tumours more than 100 microm from the basement membrane. Subsequent evolution of glycolytic and acid-resistant phenotypes leads to invasive proliferation. Multicellular spheroids recapitulating ductal carcinoma in situ (DCIS) microenvironmental conditions demonstrate upregulated glucose transporter 1 (GLUT1) as adaptation to hypoxia followed by growth into normoxic regions in qualitative agreement with model predictions. Clinical specimens of DCIS exhibit periluminal distribution of GLUT-1 and Na(+)/H(+) exchanger (NHE) indicating transcriptional activation by hypoxia and clusters of the same phenotype in the peripheral, presumably normoxic regions similar to the pattern predicted by the models and observed in spheroids. Upregulated GLUT-1 and NHE-1 were observed in microinvasive foci and adjacent intraductal cells. Adaptation to hypoxia and acidosis may represent key events in transition from in situ to invasive cancer.

Neurons in the suprachiasmatic nucleus (SCN) function as part of a central timing circuit that drives daily changes in our behaviour and underlying physiology. A hallmark feature of SCN neuronal populations is that they are mostly electrically silent during the night, start to fire action potentials near dawn and then continue to generate action potentials with a slow and steady pace all day long. Sets of currents are responsible for this daily rhythm, with the strongest evidence for persistent Na(+) currents, L-type Ca(2+) currents, hyperpolarization-activated currents (I(H)), large-conductance Ca(2+) activated K(+) (BK) currents and fast delayed rectifier (FDR) K(+) currents. These rhythms in electrical activity are crucial for the function of the circadian timing system, including the expression of clock genes, and decline with ageing and disease. This article reviews our current understanding of the ionic and molecular mechanisms that drive the rhythmic firing patterns in the SCN.