bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.