bluuebunny/crossref_metadata_2025 huggingface dataset:
Husserl's theory of wholes and parts and the methodology of nursing research
Authors: Schultz Gary S. , Cobb‐Stevens Richard
Published: September 2004

Abstract Whenever the name Edmund Husserl appears in the context of nursing research, what correctly comes to mind is the phenomenological approach to qualitative methodology. Husserl is not only considered the founder of phenomenology, but his broad concept development also contributed to the demise of positivism and inspired fruitful approaches to the social sciences. In this spirit of inspiration, it must be expressed that Husserl's theory of wholes and parts, and particularly his differentiation of parts into ‘pieces’ and ‘moments’, is very helpful in guiding the selection of research methods across the board in nursing science. The purpose of this paper is to highlight a frame of reference for nursing researchers to use in examining the essential nature of that which is being studied. This frame of reference is the Husserlian philosophy of ‘pieces’ and ‘moments’ in relation to the whole. ‘Pieces’ are independent parts of the whole that are subject to isolability in study, whereas ‘moments’ are nonindependent parts, which cannot be detached, presented, or studied apart from the whole. The intent is to propose this frame of reference as a philosophical base from which nursing researchers may better select among paradigms and methodological approaches in regard to the essential nature (‘pieces’ or ‘moments’) of that which they are researching.
Interdependence and dynamic linkages between the emerging stock markets of South Asia
Authors: Narayan Paresh , Smyth Russell , Nandha Mohan
Published: September 2004

The present article examines the dynamic linkages between the stock markets of Bangladesh, India, Pakistan and Sri Lanka using a temporal Granger causality approach by binding the relationship among the stock price indices within a multivariate cointegration framework. We also examine the impulse response functions. Our main finding is that in the long run, stock prices in Bangladesh, India and Sri Lanka Granger‐cause stock prices in Pakistan. In the short run there is unidirectional Granger causality running from stock prices in Pakistan to India, stock prices in Sri Lanka to India and from stock prices in Pakistan to Sri Lanka. Bangladesh is the most exogenous of the four markets, reflecting its small size and modest market capitalization.
A Computational Algebraic Approach to English Grammar
Authors: Lambek J.
Published: September 2004

Abstract.  Whereas type‐logical grammars treat syntactic derivations as logical proofs, usually represented by two‐dimensional diagrams, I here wish to defend the view that people process linguistic information by one‐dimensional calculations and will explore an algebraic approach based on the notion of a ‘‘pregroup,’’ a partially ordered monoid in which each element has both a left and a right ‘‘adjoint.’’ As a first approximation, say to English, one assigns to each word one or more ‘‘syntactic types,’’ elements of the free pregroup generated by a partially ordered set of ‘‘basic types,’’ in the expectation that the grammaticality of a string of words can be checked by a calculation on the corresponding types. This theoretical framework provides a simple foundation for a kind of feature checking that may be of general interest. According to G. A. Miller, there is a limit to the temporary storage capacity of our short‐term memory, which cannot hold more than seven (plus or minus two) ‘‘chunks’’ of information at any time. I explore here the possibility of identifying these chunks with ‘‘simple types,’’ which are obtained from basic types by forming iterated adjoints. In a more speculative vein, I attempt to find out how so‐called constraints on transformations can be framed in the present algebraic context.
Verbal Syntax and Morphology in Typically Developing Dutch Children and Children With Sli: How Developmental Data Can Play an Important Role In Morphological Theory
Authors: Wexler Kenneth , Schaeffer Jeannette , Bol Gerard
Published: September 2004

Abstract.  This paper is a study of normal and impaired (SLI) linguistic development, especially in verbal morphology and syntax. The paper studies both linguistic development and how developmental phenomena provide evidence for adult linguistic theory. We use extensive developmental data to test models of normal and impaired development that have been applied to other languages—in particular, the Agr/Tense Omission Model (ATOM). We develop detailed models of Dutch morphology that—together with ATOM—predict that Dutch‐speaking children will produce more root infinitives than English‐speaking children. Furthermore, the model predicts particular errors of tense and agreement will occur in Dutch but not English‐speaking children. These predictions are confirmed. We also investigate how developmental data can help us to solve a problem concerning the interpretation of features in adult morphology.
Inequality and the Right to Learn: Access to Qualified Teachers in California's Public Schools
Authors: Darling-Hammond Linda
Published: September 2004

As new standards for students are taking effect, large disparities continue to exist in the educational opportunities available to rich and poor students in most states. These disparities are especially pronounced in California, where thousands of students attend school in dilapidated buildings, without textbooks, materials, or qualified teachers. This article focuses on inequalities in children's access to qualified teachers, documenting the disproportionate assignment of untrained and uncredentialed teachers to students in high-minority, low-income schools and the effects that large concentrations of such teachers have on students’ opportunities to learn. Given the importance of teacher expertise to student achievement, and the existence of new standards to which students are held accountable, these inequalities threaten students’ basic rights to an education. The article outlines the legal rationale for insisting on access to qualified teachers for all students, analyzes the reasons for the current shortfalls in California, and proposes a set of remedies based on research and policy outcomes elsewhere.
Intracellular Mg2+ interacts with structural determinants of the narrow constriction contributed by the NR1‐subunit in the NMDA receptor channel
Authors: Wollmuth Lonnie P. , Kuner Thomas , Sakmann Bert
Published: September 2004

N‐methyl‐D‐aspartate (NMDA) receptor channels are blocked by intracellular Mg2+ in a voltage‐dependent manner. Amino acid residues positioned at or near the narrow constriction that interact with intracellular Mg2+ were identified in recombinant NR1‐NR2A channels expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells.

In the absence of extracellular Ca2+, the block of wild‐type channels by intracellular Mg2+ measured using macroscopic currents showed a voltage dependence (δ) of around 0.38 and a voltage‐independent affinity for the channel of 4 mM. These parameters were independent of the Mg2+ concentration (0.05‐10 mM), and were indistinguishable from those found for the reduction of single channel amplitudes under the same ionic conditions. Under biionic conditions with high intracellular Mg2+ and K+ extracellularly, Mg2+ was weakly permeant. Mg2+ efflux, however, attenuated the block only at positive potentials (> +80 mV).

Substitutions of the N‐site asparagine in the NR1‐subunit altered intracellular Mg2+ block over physiological membrane potentials (+10 to +50 mV). Substitution of glycine, glutamine or serine attenuated the extent of block whereas the negatively charged aspartate enhanced it, consistent with the side chain of the native asparagine at this position contributing to a blocking site for intracellular Mg2+.

Substitutions of the N‐site or N + 1 site asparagine in the NR2A‐subunit, which form a blocking site for extracellular Mg2+, also altered the block by intracellular Mg2+. However, for the NR2A‐subunit N‐site asparagine, the block was reduced but only at non‐physiological high potentials (> +70 mV).

The NR2A‐subunit N + 1 site asparagine, which together with the NR1‐subunit N‐site asparagine forms the narrow constriction of the channel, also contributed to a blocking site for intracellular Mg2+. However, it did so to a lesser extent than the NR1‐subunit N‐site and in a manner different from its contribution to a blocking site for extracellular Mg2+.

It is concluded that intracellular Mg2+ interacts with residues that form the narrow constriction in the NMDA receptor channel with the N‐site asparagine of the NR1‐subunit representing the dominant blocking site. Thus, intracellular Mg2+ interacts with different asparagine residues at the narrow constriction than extracellular Mg2+, although the two blocking sites are positioned very close to each other.
Non‐pore lining amino acid side chains influence anion selectivity of the human CFTR Cl− channel expressed in mammalian cell lines
Authors: Linsdell Paul , Zheng Shu‐Xian , Hanrahan John W.
Published: September 2004

The effects of individually mutating two adjacent threonine residues in the sixth membrane‐spanning region (TM6) of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel on permeation properties were examined using patch clamp recording from mammalian cell lines stably expressing human CFTR.

A number of mutations of T338 significantly affected the permeation properties of the channel. Increases and decreases in single channel conductance were observed for different mutants. Anion selectivity was strongly affected, with no two channel variants sharing the same selectivity sequence. Several mutations led to strong inward rectification of the macroscopic current‐voltage relationship. The effects of these mutations on permeation properties were correlated with the size of the amino acid side chain substituted, rather than its chemical nature.

Most mutations of T339 resulted in a lack of functional channel expression and apparent misprocessing of the protein. One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations.

These results suggest an important role for T338 in controlling the permeation properties of the CFTR Cl− channel. It is suggested that mutation of this residue alters the interaction between permeating anions and the channel pore via an indirect effect on the orientation of the TM6 helix.
Internal Mg2+ block of recombinant NMDA channels mutated within the selectivity filter and expressed in Xenopus oocytes
Authors: Kupper Jürgen , Ascher Philippe , Neyton Jacques
Published: September 2004

The NMDA receptor channel is blocked by both external and internal Mg2+ ions, which are assumed to bind inside the channel on each side of a central barrier. We have analysed the internal Mg2+ block in recombinant NR1‐NR2A NMDA receptors expressed in Xenopus oocytes. We have determined the effects of mutations of two asparagines that line the selectivity filter of the channel, one located within the NR1 subunit (N598) and the other within the NR2A subunit (N596).

The whole‐cell current‐voltage relation of wild‐type NMDA channels shows inward rectification that reflects the voltage‐dependent block produced by the internal Mg2+ of the oocyte. This inward rectification is slightly reduced in the NR2 mutant (N596S) but is abolished in the NR1 mutants (N598Q and N598S). This suggests that the NR1 asparagine plays a larger role than the NR2 asparagine in controlling the internal Mg2+ block.

Single‐channel current‐voltage relations confirm that the internal Mg2+ block is reduced in both the NR1 and NR2 mutants. However, the reduction is small and is similar for the two families of mutants.

The discrepancy between whole‐cell and single‐channel data is partly due to differential effects of internal Mg2+ on the open probabilities of the two conductance states present in NR1 mutant channels.

The results suggest that mutations of NR1 and NR2 asparagines lower the central barrier to Mg2+. An additional contribution of the NR2 asparagine to the external Mg2+ binding site (and possibly to the external barrier that controls access to this site) may account for the marked relief of external Mg2+ block produced by the NR2 mutation.
Transfer of rapid inactivation and sensitivity to the class III antiarrhythmic drug E‐4031 from HERG to M‐eag channels
Authors: Herzberg Ian M. , Trudeau Matthew C. , Robertson Gail A.
Published: September 2004

The gating behaviour and pharmacological sensitivity of HERG are remarkably different from the corresponding properties of M‐eag, a structurally similar member of the Eag family of potassium channels. In contrast to HERG, M‐eag exhibits no apparent inactivation and little rectification, and is insensitive to the class III antiarrhythmic drug E‐4031.

We generated chimeric channels of HERG and M‐eag sequences and made point mutations to identify the region necessary for rapid inactivation in HERG. This region includes the P region and half of the S6 putative transmembrane domain, including sites not previously associated with inactivation and rectification in HERG.

Transfer of a small segment of the HERG polypeptide to M‐eag, consisting largely of the P region and part of the S6 transmembrane domain, is sufficient to confer rapid inactivation and E‐4031 sensitivity to M‐eag. This region differs from the corresponding region in M‐eag by only fifteen residues.

Previous hypotheses that rapid inactivation of HERG channels occurs by a C‐type inactivation mechanism are supported by the parallel effects on rates of HERG inactivation and Shaker C‐type inactivation by a series of mutations at two equivalent sites in the polypeptide sequences.

In addition to sites homologous to those previously described for C‐type inactivation in Shaker, inactivation in HERG involves a residue in the upstream P region not previously associated with C‐type inactivation. Although this site is equivalent to one implicated in P‐type inactivation in Kv2.1 channels, our data are most consistent with a single, C‐type inactivation mechanism.
Voltage‐controlled Ca2+ release in normal and ryanodine receptor type 3 (RyR3)‐deficient mouse myotubes
Authors: Dietze B. , Bertocchini F. , Barone V. , Struk A. , Sorrentino V. , Melzer W.
Published: September 2004

Primary cultured myotubes were derived from satellite cells of the diaphragm obtained from both normal mice (RyR3+/+) and mice with a targeted mutation eliminating expression of the type 3 isoform of the ryanodine receptor (RyR3−/−). Using the whole‐cell patch clamp technique, L‐type Ca2+ currents were measured during step depolarizations. Simultaneously, intracellular Ca2+ transients were recorded with the fluorescent indicator dye fura‐2.

After correction for non‐instantaneous binding of Ca2+ to the indicator dye and taking into account the dynamics of Ca2+ binding to intracellular constituents, an estimate of the time course of the Ca2+ release rate from the sarcoplasmic reticulum (SR) was obtained.

The calculated SR Ca2+ release flux exhibited a marked peak within less than 12 ms after the onset of the voltage‐clamp depolarization and fell rapidly thereafter to a five times lower, almost steady level. It declined rapidly after termination of the depolarization.

Signals in normal and RyR3‐deficient myotubes showed no significant difference in the activation of Ca2+ conductance and in amplitude, time course and voltage dependence of the Ca2+ efflux from the SR.

In conclusion, the characteristics of voltage‐controlled Ca2+ release reported here are similar to those of mature mammalian muscle fibres. In contrast to differences observed in the contractile properties of RyR3‐deficient muscle fibres, a contribution of RyR3 to excitation‐contraction coupling could not be detected in myotubes.
Regulation of kinetic and pharmacological properties of synaptic NMDA receptors depends on presynaptic exocytosis in rat hippocampal neurones
Authors: Lindlbauer Raimund , Mohrmann Ralf , Hatt Hanns , Gottmann Kurt
Published: September 2004

Using whole‐cell patch‐clamp recordings of NMDA EPSCs from co‐cultured rat hippocampal (CA region) neurones, developmental changes in the kinetic and pharmacological properties of synaptic NMDA receptors were investigated. During in vitro differentiation a fast decaying component increasingly contributed to NMDA EPSCs.

Extracellular Mg2+ (1 mM) strongly blocked NMDA EPSCs at all stages in culture. Using the NR2B subunit‐specific NMDA receptor antagonist ifenprodil (3 μM), we observed a developmental decrease in ifenprodil sensitivity of NMDA EPSCs. This suggests developmental changes in the expression of NMDA receptor subtypes.

To transiently block presynaptic exocytosis, we incubated presynaptic explants with tetanus toxin (TeTx) prior to cultivation. In TeTx‐pretreated cultures the occurrence of fast decaying components of NMDA EPSCs and the developmental decrease in ifenprodil sensitivity was inhibited. Our results indicate a regulatory role of presynaptic exocytosis in the expression of NMDA receptor subtypes.
The effect of rapid eye movement (REM) sleep on upper airway mechanics in normal human subjects
Authors: Rowley James A. , Zahn Brian R. , Babcock Mark A. , Badr M. Safwan
Published: September 2004

It has been proposed that the upper airway is more compliant during rapid eye movement (REM) sleep than during non‐rapid eye movement (NREM) sleep. The purpose of this study was to test this hypothesis in a group of subjects without sleep‐disordered breathing.

On the first night, the effect of sleep stage on the relationship of retropalatal cross‐sectional area (CSA; visualized with a fibre‐optic scope) to pharyngeal pressure (PPH) measured at the soft palate during eupnoeic breathing was studied. Breaths during REM sleep were divided into phasic (associated with eye movements) and tonic (not associated with eye movements). There was a significant decrease in pharyngeal CSA during NREM sleep compared with wakefulness. There was no further decrease observed during either tonic or phasic REM sleep. Pharyngeal compliance, defined as the slope of the regression CSA versus PPH, was significantly increased during NREM sleep compared with wakefulness and REM sleep, with the compliance during both tonic and phasic REM sleep being similar to that observed in wakefulness.

On the second night, the effect of sleep stage on pressure‐flow relationships of the upper airway was investigated. There was a trend towards the upper airway resistance being highest in NREM sleep compared with wakefulness and REM sleep.

We conclude that the upper airway is stiffer and less compliant during REM sleep than during NREM sleep. We postulate that this difference is secondary to differences in upper airway vascular perfusion between REM and NREM sleep.
Fatty acids stimulate cholecystokinin secretion via an acyl chain length‐specific, Ca2+‐dependent mechanism in the enteroendocrine cell line STC‐1
Authors: McLaughlin J. T. , Lomax R. B. , Hall L. , Dockray G. J. , Thompson D. G. , Warhurst G.
Published: September 2004

The present study has investigated whether fatty acids directly influence peptide release from enteroendocrine cells using STC‐1, a mouse intestinal endocrine tumour cell line, previously shown to release cholecystokinin (CCK) in response to other physiological stimuli.

Fatty acids elicited a chain length‐ and dose‐dependent stimulation of CCK secretion. Dodecanoic acid (C12) was most effective, producing up to a 5‐fold increase in CCK secretion. Fatty acids with less than ten carbon atoms did not increase secretion. The chain length dependence of these effects mimics closely fatty acid‐induced CCK secretion previously observed in humans in vivo.

Esterification of C12 abolished CCK secretion, indicating a critical role for a free carboxyl group in eliciting secretion. In contrast, modification of the methyl terminus had no effect on C12‐induced secretion. The non‐metabolizable C12 analogue 2‐bromododecanoic acid was equally effective.

C12 elicited a marked increase in intracellular calcium levels (200–300 nm) in STC‐1 cells which was abolished by the L‐type Ca2+ channel antagonist nicardipine. In contrast, C8 produced a smaller and more transient Ca2+ response. C12‐induced CCK secretion was also blocked by nicardipine.

These data suggest that fatty acids can interact directly with enteroendocrine cells to stimulate CCK secretion via increases in intracellular calcium mediated primarily by L‐type Ca2+ channels.
The dependence of Ag+ block of a potassium channel, murine Kir2.1, on a cysteine residue in the selectivity filter
Authors: Dart C. , Leyland M. L. , Barrett‐Jolley R. , Shelton P. A. , Spencer P. J. , Conley E. C. , Sutcliffe M. J. , Stanfield P. R.
Published: September 2004

Externally applied Ag+ (100‐200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1.

Mutation to serine of a cysteine residue at position 149 in the pore‐forming H5 region of Kir2.1 abolished Ag+ blockage.

To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current.

We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149.

Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild‐type channels; the rate was 4 times slower in channels formed from dimers.
Facilitation of rabbit α1B calcium channels: involvement of endogenous Gβγ subunits
Authors: Stephens Gary J. , Brice Nicola L. , Berrow Nicholas S. , Dolphin Annette C.
Published: September 2004

The α1B (N‐type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gβγ subunits. Using transient expression in COS‐7 cells of α1B together with the accessory subunits α2‐δ and β2a, we have examined the role of endogenous Gβγ subunits in the tonic modulation of α1B, and compared this with modulation by exogenously expressed Gβγ subunits.

Prepulse facilitation of control α1B/α2‐δ/β2a currents was always observed. This suggests the existence of tonic modulation of α1B subunits. To determine whether endogenous Gβγ is involved in the facilitation observed in control conditions, the βARK1 Gβγ‐binding domain (amino acids 495‐689) was overexpressed, in order to bind free Gβγ subunits. The extent of control prepulse‐induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPβS also reduced the control facilitation.

Co‐expression of the Gβ1γ2 subunit, together with the α1B/α2‐δ/β2a calcium channel combination, resulted in a marked degree of depolarizing prepulse‐reversible inhibition of the whole‐cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid‐point of the voltage dependence of activation. Activation of endogenous Gβγ subunits by dialysis with GTPγS produced a smaller degree of prepulse‐reversible inhibition.

The rate of reinhibition of α1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gβ1γ2 than for the decay of facilitation in control cells. Furthermore, βARK1 (495‐689) co‐expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gβγ in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined.

These findings indicate that, in this system, the voltage‐dependent facilitation of α1B that is observed under control conditions occurs as a result of endogenous free Gβγ binding to α1B.
Modulation of ATP‐gated non‐selective cation channel (P2X1 receptor) activation and desensitization by the actin cytoskeleton
Authors: Parker Karen E.
Published: September 2004

ATP‐gated non‐selective cation channels from the rat vas deferens (P2X1 receptors) were stably expressed in HEK 293 cells, assayed by patch clamp on the first day after passage of the culture, and found to have whole‐cell current kinetics markedly faster in both activation and desensitization than those found in the native vas deferens tissue, in agreement with previous reports.

By the second day after passage of the culture, however, the whole‐cell current kinetics of the expressed receptors shifted, slowing in both activation and desensitization. The kinetic change correlated with a change in phenotype of the host cells from round to flat, and the slower kinetics were similar to native P2X1 currents recorded from dissociated rat vas deferens smooth muscle cells. Two point mutations in a pore‐like domain near or within the second transmembrane domain of the P2X1 receptor appeared to confer on the receptor the inability to effect this change in kinetics over time.

Treatment of cells on day 3 after passage with cytochalasins B or D caused a reversion to the rapid kinetics phenotype, implicating the actin cytoskeleton in the development of the native kinetics. P2X1 receptors may therefore require interaction with an intact actin cytoskeleton for native kinetics, and the mutants may be defective either in interaction with the actin skeleton or in coupling the interaction to gating.
The selectivity filter of a potassium channel, murine Kir2.1, investigated using scanning cysteine mutagenesis
Authors: Dart C. , Leyland M. L. , Spencer P. J. , Stanfield P. R. , Sutcliffe M. J.
Published: September 2004

We have produced a structural model of the pore‐forming H5 (or P) region of the strong inward rectifier K+ channel, Kir2.1, based initially on an existing molecular model of the pore region of the voltage‐gated K+ channel, Kv1.3.

Cysteine‐scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residues in H5 whose side chains line the ion conduction pathway.

Mutations made in eight positions within the highly conserved H5 region resulted in apparently non‐functional channels. Constructing covalently linked dimers, which carry a cysteine substitution in only one of the linked subunits, rescued six of these mutants; a covalently linked tetramer, carrying a cysteine substitution on only one of the linked subunits, rescued a further mutant.

Our results using the dimers and tetramers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100‐200 nM) and therefore that their side chains line the channel pore.

We conclude that the topology of the Kir pore is similar, but not identical, to that of Kv channels. Additionally, the molecular model suggests that selectivity may be conferred both by aromatic residues (Tyr145 and Phe147) via cation‐π interactions and by backbone carbonyl groups (Thr142 and Gly144).
Properties of human glycine receptors containing the hyperekplexia mutation α1(K276E), expressed in Xenopus oocytes
Authors: Lewis T. M. , Sivilotti L. G. , Colquhoun D. , Gardiner R. M. , Schoepfer R. , Rees M.
Published: September 2004

Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single‐channel properties of receptors that contain a mutation, α1(K276E), associated with an atypical form of hyperekplexia.

Equilibrium concentration‐response curves showed that recombinant human α1(K276E)β receptors had a 29‐fold lower glycine sensitivity than wild‐type α1β receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged.

Both wild‐type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively).

Channel openings were shorter for the α1(K276E)β mutant than for the wild‐type α1β, with mean overall apparent open times of 0.82 and 6.85 ms, respectively.

The main effect of the α1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose‐response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single‐channel bursts, and the lack of an effect of the mutation on the strychnine‐binding site.
Multiple sites of action of neomycin, Mg2+ and spermine on the NMDA receptors of rat hippocampal CA1 pyramidal neurones
Authors: Lu Wei‐Yang , Xiong Zhi‐Gang , Orser Beverley A. , MacDonald John F.
Published: September 2004

The effects of neomycin on NMDA‐evoked currents in isolated CA1 hippocampal pyramidal neurones were investigated and single channel activity was examined in outside‐out patches taken from cultured hippocampal neurones. The effects of neomycin on two combinations of NMDA receptor subunits (NR1a‐NR2A and NR1a‐NR2B) expressed in human embryonic kidney (HEK293) cells were also studied.

Neomycin (0.01–1 mm) caused a potentiation of NMDA‐activated currents in all neurones examined. No evidence of a voltage‐dependent depression was observed in whole‐cell recordings.

In outside‐out patch recordings relatively low concentrations (30 and 100 μm) of neomycin caused a voltage‐dependent reduction in single channel current amplitude as well as a large increase in the frequency of channel opening.

In saturating concentrations of glycine, neomycin enhanced NMDA‐activated currents and this glycine‐independent enhancement was confirmed using recombinant NR1a‐NR2B receptors. Neomycin substantially increased the potency of glycine for the receptor by reducing the rate of dissociation of glycine from the receptor. Neomycin demonstrated a glycine‐dependent enhancement of currents mediated by the NR1a‐NR2A combination of subunits but a paradoxical depression was observed in saturating concentrations of glycine.

Neomycin increased the rate of deactivation of glutamate‐activated currents consistent with neomycin causing a reduction in the affinity of the receptor for agonist.

These results indicate that neomycin has multiple and complex effects on NMDA receptors.
Antisense to MDR1 mRNA reduces P‐glycoprotein expression, swelling‐activated Cl− current and volume regulation in bovine ciliary epithelial cells
Authors: Wang Liwei , Chen Lixin , Walker Veronica , Jacob Tim J. C.
Published: September 2004

Native ciliary epithelial cells from the ciliary epithelium of the eye exhibit anti‐P‐glycoprotein (P‐gp) immunofluorescence.

We have used an antisense ‘knock‐down’ approach to investigate the relationship between P‐gp and the volume‐activated chloride current (ICl,swell) and its role in volume regulation.

An antisense oligonucleotide to the human multidrug resistance (MDR1) gene, taken up by the cells in a dose‐dependent manner, reduced P‐gp immunofluorescence, inhibited ICl,swell and significantly increased the latency of activation of ICl,swell.

Increasing the hypotonic stress did not result in an increased activation of ICl,swell. MDR1 antisense ‘knock‐down’ also reduced the ability of the cells to volume regulate following a hypotonic challenge.

These cells are known to express at least two volume‐activated chloride channels, and the data suggest that P‐gp is involved in the activation pathway of a subset of channels that contribute to whole‐cell ICl,swell and participate in volume regulation.
GABAA and GABAC receptors in adult porcine cones: evidence from a photoreceptor‐glia co‐culture model
Authors: Picaud Serge , Pattnaik Bikash , Hicks David , Forster Valérie , Fontaine Valérie , Sahel José , Dreyfus Henri
Published: September 2004

Edge contrast enhancement is an integrated visual function based on the complex centre‐surround organization of the cone photoreceptor light response. While centre responses result from direct light activation, surround responses are thought to result from lateral inhibition mediated by horizontal cells. This feedback signal has been attributed to GABAA receptors which have been found in lower vertebrate cones.

In order to study the GABA response of adult mammalian photoreceptors, we designed a culture system consisting of isolated photoreceptors seeded on a layer of retinal glial cells. Mature rods and cones required the presence of Müller glial cells to survive and develop neurites; they degenerated in the absence of glial cells.

Cone photoreceptors generated large GABA responses whereas rod photoreceptors did not respond to GABA applications.

Cone GABA responses consisted of two distinct components, one suppressed by the GABAA receptor blockers bicuculline and SR95531, and the second by the GABAC receptor antagonists TPMPA and imidazole‐4‐acetic acid (I4AA). Pentobarbital greatly increased the GABAA receptor component whereas it did not affect, or even reduced, the GABAC receptor component. During long GABA applications, GABAA receptor currents desensitized by 78 %, contrasting with the sustained GABAC response.

Expression of GABAC receptors in cone photoreceptors was confirmed by anti‐ρ‐subunit immunolabelling of porcine retinal sections.

These results indicate that both GABAA and GABAC receptors may participate in the feedback synapse from horizontal cells to cone photoreceptors in the mammalian retina.
Voltage‐dependent inactivation of the human K+ channel KvLQT1 is eliminated by association with minimal K+ channel (minK) subunits
Authors: Tristani‐Firouzi Martin , Sanguinetti Michael C.
Published: September 2004

The time course and voltage dependence of inactivation of KvLQT1 channels expressed in Xenopus oocytes were studied using two‐microelectrode voltage‐clamp techniques.

Tail current analysis was used to characterize the kinetics of channel inactivation and deactivation. The time constant for recovery from channel inactivation was voltage dependent and varied from 30 ± 2 ms at −90 mV to 36 ± 1 ms at −30 mV. The time constant for deactivation varied from 186 ± 21 to 986 ± 43 ms over the same voltage range.

Inactivation of KvLQT1 channels was incomplete, reducing fully activated current by 35 % at +40 mV. Inactivation of KvLQT1 channels was half‐maximal at −18 ± 2 mV.

The onset of KvLQT1 channel inactivation during a single depolarization to +20 mV was exponential (τ= 130 ± 10 ms), and developed after a delay of ≈75 ms. In contrast, when inactivation was reinduced following transient recovery of channels to the open state(s), the onset of inactivation was immediate and 10 times faster. These findings suggest multiple open states, and a sequential gating model for KvLQT1 channel activation and inactivation (C1⇌ Cn⇌ O1⇌ O2⇌ I).

Delayed rectifier K+ (IKs) channels formed by heteromultimeric coassembly of KvLQT1 and minimal K+ channel (minK) subunits did not inactivate. Thus, minK subunits eliminate, or greatly slow, the gating associated with channel inactivation when coassembled with KvLQT1.
Calcium‐dependent inactivation of high‐threshold calcium currents in human dentate gyrus granule cells
Authors: Nägerl U. Valentin , Mody Istvan
Published: September 2004

Dentate gyrus granule cells acutely dissociated from hippocampal slices obtained from chronic temporal lobe epilepsy (TLE) patients displayed a high‐voltage activated (HVA) Ca2+ conductance with a pronounced Ca2+‐dependent inactivation.

Inactivation time constants and peak HVA Ca2+ current (ICa) amplitudes did not differ between perforated patch and whole‐cell recordings without added exogenous Ca2+ buffers, indicating that the Ca2+‐dependent characteristics of ICa inactivation were well preserved in whole‐cell recordings.

Inactivation time constants correlated with whole‐cell ICa, and were increased when Ca2+ was replaced with Ba2+ in the external solution or 5 mm BAPTA was added to the pipette solution.

In recordings without added exogenous Ca2+ buffers, the time course of ICa inactivation was comparable between human TLE and kindled rat granule cells. Conversely, the time course of ICa in human TLE granule cells loaded with 5 mm intracellular BAPTA resembled that observed in buffer‐free recordings from control rat neurones.

The loss of a putative intraneuronal Ca2+ buffer, the Ca2+‐binding protein calbindin (CB), from human granule cells during TLE may result in the pronounced Ca2+‐dependent ICa inactivation. This process could serve a neuroprotective role by significantly decreasing Ca2+ entry during prolonged trains of action potentials known to occur during seizures.
Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures
Authors: Chen Yu‐Hwa , Wu Mei‐Lin , Fu Wen‐Mei
Published: September 2004

The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve‐muscle co‐cultures.

Spontaneous and evoked ACh release of motoneurons was monitored by using whole‐cell voltage‐clamped myocytes. Intracellular alkalinization with 15 mm NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release.

Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release.

Impulse‐evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification.

Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification.

We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.
Interaction of memantine and amantadine with agonist‐unbound NMDA‐receptor channels in acutely isolated rat hippocampal neurons
Authors: Sobolevsky Alexander I. , Koshelev Sergey G. , Khodorov Boris I.
Published: September 2004

Using whole‐cell patch‐clamp techniques, the mechanisms of NMDA channel blockade by amino‐adamantane derivatives (AADs) memantine (3,5‐dimethyl‐aminoadamantane, MEM) and amantadine (1‐aminoadamantane, AM) have been studied in rat hippocampal neurons acutely isolated by the vibrodissociation method. A rapid concentration‐jump technique was used to replace superfusing solutions.

The aspartate (Asp)‐induced channel opening greatly accelerated but was not a prerequisite for the recovery from the block by MEM: it was able to leave the channel without agonist assistance. The co‐agonist (glycine) as well as the competitive NMDA antagonist dl‐2‐amino‐7‐phosphonoheptanoic acid (APV), did not affect this recovery. Membrane depolarization accelerated it, strongly suggesting that this process proceeded via the hydrophilic pathway of the channel.

A comparison of the kinetics of the recovery from the block by AADs in the presence and absence of the agonist prompted a hypothesis that the blocker trapped in the channel increased the probability of its transition to the open state.

Both MEM and AM were able to block NMDA channels not only in the presence but also in the absence of Asp, although in the latter case the effective blocking concentrations were much higher and the rate of the block development was much smaller than in the former case. The extent of the block increased with the duration of the blocker application. Glycine enhanced this block, while APV attenuated it. The MEM‐induced blockade of agonist‐unbound channels was enhanced by membrane hyperpolarization and weakened by external Mg2+. These findings strongly suggested that the blocker reached its binding sites via the same hydrophilic pathway both in the presence and absence of the agonist.

A comparative analysis of the channel unblocking kinetics in the presence of Asp after their blockade with or without the agonist assistance led us to conclude that in the two cases AADs were bound to the same blocking sites in the channel.
The channel opening rate of adult‐ and fetal‐type mouse muscle nicotinic receptors activated by acetylcholine
Authors: Maconochie David J. , Steinbach Joe Henry
Published: September 2004

expressing either the fetal (Q‐F18) or the adult (Q‐A33) complement of nicotinic acetylcholine receptor subunits derived from mouse skeletal muscle. Pulses of ACh were applied to outside‐out patches of cell membrane by means of a fast perfusion system, at concentrations from 100 nM to 10 mM. We obtained current records with intracellular potentials of ‐60 and +40 mV. The goal of this study was to estimate the channel opening rate.

By fitting sums of exponentials to averaged responses, we estimated the rate of development of the current on the application of acetylcholine. The rate constant of the predominant exponential component (the on‐rate) ranges over 3 orders of magnitude, from around 100 s−1 (fetal) at low concentrations of ACh to over 100 000 s−1 (fetal and adult) at the highest concentrations.

We establish that our measurement of the on‐rate is not limited by technical constraints, and can therefore be related to the rate constants of a kinetic scheme. Our observations are consistent with a model having a rate‐limiting channel opening step with a forwards rate constant (β) of 80 000 s−1 on average for adult receptors and 60 000 s−1 for fetal receptors, and a minimum opening to closing ratio (β/α) of around 33 (adult) or 50 (fetal). The channel opening rate, β, varies from around 30 000 s−1 to well over 100 000 s−1 for different patches. The large variation cannot all be ascribed to errors of measurement, but indicates patch to patch variation.
Evidence that mitochondria buffer physiological Ca2+ loads in lizard motor nerve terminals
Authors: David Gavriel , Barrett John N. , Barrett Ellen F.
Published: September 2004

Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG‐5N). Mitochondrial [Ca2+] was measured using rhod‐2, bath loaded as dihydrorhod‐2.

In response to 100‐250 stimuli at 25‐100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation.

When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+].

After addition of carbonyl cyanide m‐chlorophenyl hydrazone (CCCP, 1‐2 μm) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+].

These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.
N‐ and L‐type calcium channel involvement in depolarization‐induced suppression of inhibition in rat hippocampal CA1 cells
Authors: Lenz R. A. , Wagner J. J. , Alger B. E.
Published: September 2004

We investigated depolarization‐induced suppression of inhibition (DSI) under whole‐cell voltage clamp in CA1 pyramidal neurons of rat hippocampal slices. DSI, a transient reduction in monosynaptic evoked GABAAergic IPSCs lasting for ∼1 min, was induced by depolarizing the pyramidal cell to −10 or 0 mV for 1 or 2 s.

Raising extracellular Ca2+ concentration increased DSI, and varying the DSI‐inducing voltage step showed that the voltage dependence of DSI was like that of high‐voltage‐activated Ca2+ channels.

The P‐ and Q‐type Ca2+ channel blocker ω‐agatoxin TK (200 nm and 1 μm) and the R‐ and T‐type Ca2+ channel blocker Ni2+ (100 μm) reduced IPSCs without reducing DSI.

The specific N‐type Ca2+ channel antagonist ω‐conotoxin GVIA (250 nm) reduced IPSC amplitudes and almost completely abolished DSI.

Blocking L‐type Ca2+ channels with nifedipine (10 μm) had no effect on IPSCs or DSI induced by our standard protocol, but reduced DSI induced by the unclamped Na+‐ and Ca2+‐dependent spikes that occurred when 2(triethylamino)‐N‐(2,6‐dimethylphenyl)acetamide (QX‐314) was omitted from the recording pipette solution.

Although intracellular Ca2+ stores were not measured, DSI was not affected by cyclopiazonic acid (CPA, 20–40 μm), a blocker of Ca2+ uptake into intracellular stores.

We conclude that DSI is initiated by Ca2+ influx through N‐ and, under certain conditions, L‐type Ca2+ channels.
The erg inwardly rectifying K+ current and its modulation by thyrotrophin‐releasing hormone in giant clonal rat anterior pituitary cells
Authors: Bauer Christiane K.
Published: September 2004

The voltage‐dependent inwardly rectifying K+ current (IK,IR) of clonal rat anterior pituitary cells (GH3/B6) was investigated in solutions with physiological K+ gradient using giant polynuclear cells.

I
K,IR was isolated by the use of the selective erg (ether‐à‐go‐go‐related gene) channel blocker E‐4031. In external 5 mM K+ solution, IK,IR carried steady‐state outward current in the potential range between –60 and 0 mV, with a maximum current amplitude at –40 mV. Negative to the K+ equilibrium potential, EK, large transient inward currents occurred.

A selective pharmacological block of IK,IR induced a sustained depolarization of the membrane potential when Ca2+ action potentials were blocked, confirming the contribution of IK,IR to the resting membrane potential of GH3/B6 cells.

Thyrotrophin‐releasing hormone (TRH) reduced effectively the sustained outward and the transient inward IK,IR. The magnitude of a TRH‐induced depolarization of the membrane potential was consistent with an almost complete reduction of IK,IR.

The results demonstrate that the TRH‐induced reduction of IK,IR is able to mediate the resting potential depolarization, suggesting that the increase in the frequency of action potentials occurring during the second phase of the TRH response in GH cells should be sustained by IK,IR inhibition. Moreover, this is the first evidence of a ligand‐induced physiological modulation of an erg‐mediated current.