bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of rat neuronal kainate receptors by cis‐unsaturated fatty acids
Authors: Wilding Timothy J. , Chai Yoon H. , Huettner James E.
Published: September 2004

Whole‐cell recordings from cultured rat hippocampal neurons, from freshly dissociated dorsal root ganglion (DRG) neurons and from human embryonic kidney (HEK) 293 cells expressing the glutamate receptor GluR6 subunit were used to study the modulation of kainate receptor channels by long chain fatty acids.

In all three cell types, application of cis‐unsaturated fatty acids caused a dose‐dependent reduction in whole‐cell currents evoked by kainate. Docosahexaenoic acid (DHA), arachidonic acid (AA), linolenic acid and linoleic acid all produced substantial inhibition at a concentration of 50 μm, whereas inhibition by linolenelaidic acid and linolelaidic acid was significantly weaker. Fully saturated fatty acids were essentially inactive.

With continuous exposure to active fatty acids, the peak current elicited by kainate declined over a time course of several minutes to reach a steady‐state level less than 50 % of the initial amplitude. Recovery was slow in control solution, but was speeded up by exposure to bovine serum albumin (0.5 mg ml−1), a protein that binds fatty acids with submicromolar affinity. The inhibition in neurons was half‐maximal with 5–15 μm AA or DHA, but potency was at least 10‐fold greater at GluR6 in HEK 293 cells.

Inhibition by AA or DHA was unaffected by extracellular nordihydroguaiaretic acid (10 μm), indomethacin (10 μm), 17‐octadecynoic acid (30 μm) or 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine dihydrochloride (H‐7; 10 μm). Furthermore, inclusion of H‐7 (100 μm), BAPTA (10 mm), AA (50 μm), antioxidants, or the protein kinase C inhibitor PKC19‐36 (20 μm) in the internal solution had little effect on whole‐cell currents and did not prevent inhibition of currents by extracellular application of AA or DHA.

We conclude that the inhibition produced by cis‐unsaturated fatty acids does not require conversion to oxidized metabolites or activation of PKC. Instead, active compounds may interact directly with an extracellular, or intramembraneous, site on kainate receptors.
Modulation of human erg K+ channel gating by activation of a G protein‐coupled receptor and protein kinase C
Authors: Barros Francisco , Gómez‐Varela David , Viloria Cristina G. , Palomero Teresa , Giráldez Teresa , de la Peña Pilar
Published: September 2004

Modulation of the human ether‐à‐go‐go‐related gene (HERG) K+ channel was studied in two‐electrode voltage‐clamped Xenopus oocytes co‐expressing the channel protein and the thyrotropin‐releasing hormone (TRH) receptor.

Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization‐induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH.

The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+.

The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with β‐phorbol 12‐myristate, 13‐acetate (PMA). The TRH‐induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA‐dependent regulation of the channels.

It is concluded that a PKC‐dependent pathway links G protein‐coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C‐activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.
Protein kinase C phosphorylation disengages human and mouse‐1a P‐glycoproteins from influencing the rate of activation of swelling‐activated chloride currents
Authors: Bond Tamara D. , Valverde Miguel A. , Higgins Christopher F.
Published: September 2004

Whole‐cell, swelling‐activated Cl− currents, ICl(swell), were characterized in Chinese hamster ovary (CHO) cells and found to exhibit time‐dependent inactivation at depolarizing potentials, tamoxifen and dideoxyforskolin sensitivity, and an anion permeability sequence: SCN− > I− > Br− > Cl− > F− > gluconate−.

CHO cells permanently transfected with either the human MDR1 or mouse mdr1a cDNAs demonstrated an increased rate of activation of ICl(swell) compared with parental cells or those permanently transfected with the mouse mdr1b cDNA. However, no differences in the magnitude of the currents were observed at steady state.

Pretreatment with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) did not affect ICl(swell) in MDR1 or mdr1a permanently transfected CHO cells. In contrast, pretreatment with TPA reduced ICl(swell) in MDR1(G185V)‐expressing transfected NIH3T3 fibroblasts. Subsequently, the CHO cell lines were shown to contain significantly reduced levels of protein kinase C (PKC), suggesting that PKC concentrations might be limiting in these cell lines, at least under whole‐cell patch clamp conditions.

Addition of purified PKC to the pipette solution, followed by a pretreatment with TPA, reduced the rate of ICl(swell) activation in human Pgp‐ and mouse Pgp1a‐expressing CHO cells to the levels observed in parental and mouse Pgp1b‐expressing cells. This confirms that PKC is limiting in these cells under whole‐cell, patch clamp conditions. Furthermore, these results suggest that PKC‐mediated phosphorylation of human Pgp and mouse Pgp1a disengages the influence which these Pgps have on ICl(swell).

These studies also demonstrate a functional distinction between the two mouse homologues, Pgp1a and Pgp1b. Although both can function as drug efflux pumps, only Pgp1a can act like human Pgp to influence ICl(swell).
Alcohols potentiate the function of 5‐HT3 receptor‐channels on NCB‐20 neuroblastoma cells by favouring and stabilizing the open channel state
Authors: Zhou Qing , Verdoorn Todd A. , Lovinger David M.
Published: September 2004

5‐HT3 receptor‐mediated ion current was recorded from NCB‐20 neuroblastoma cells using the whole‐cell patch‐clamp technique. Rapid drug superfusion was used to study the mechanism of alcohol potentiation of 5‐HT3 receptor function and to analyse effects of alcohols on receptor‐channel kinetics in detail.

Trichloroethanol (TCEt) increased in a dose‐dependent way the initial slope, 20‐80 % rise time and measured desensitization rate of the current induced by low concentrations (1‐2 μm) of 5‐HT. Ethanol (EtOH) and butanol (ButOH) had similar effects on the 5‐HT3 receptor‐induced current.

TCEt and ButOH decreased the measured desensitization rate of current induced by 10 μm 5‐HT, a maximally effective concentration of agonist. These alcohols also increased the relative amplitude of steady state to peak current induced by 2 or 10 μm 5‐HT, indicating a possible decrease in the intrinsic rate of desensitization.

TCEt also decreased the deactivation rate of the current activated by 2 μm 5‐HT after a short pulse of agonist application.

Current sweeps generated by 1 μm 5‐HT in the presence or absence of 10 mm TCEt or 100 mm EtOH were well fitted using a modified standard kinetic model derived from the nicotinic acetylcholine receptor. This analysis indicated that potentiation by alcohols could be accounted for by increases in the association rate constant coupled with decreases in the dissociation and desensitization rate constants.

This study suggests that alcohols potentiate 5‐HT3 receptor‐mediated current by both increasing the rate of channel activation and stabilizing the open state by decreasing the rates of channel deactivation and desensitization.
Fibre type‐specific gene expression activated by chronic electrical stimulation of adult mouse skeletal muscle fibres in culture
Authors: Liu Yewei , Schneider Martin F.
Published: September 2004

Fast‐twitch skeletal muscle fibres were enzymatically dissociated from adult mouse flexor digitorum brevis (FDB) muscles and maintained in culture without or with chronic low frequency stimulation (one 5 s train of 5 Hz pulses per minute) for up to 6 days. Single fibre reverse transcriptase‐polymerase chain reaction was conducted to coamplify β‐myosin heavy chain (β‐MHC) and α‐skeletal actin mRNA from the same fibre.

Chronic low frequency electrical stimulation of FDB fibres in culture increased the level of mRNA for β‐MHC. In unstimulated fibres there was a slight decline in the β‐MHC mRNA level. As an internal control there was no increase in the level of mRNA for α‐actin in the identical individual stimulated or unstimulated fibres.

Neither the percentage of fibres exhibiting β‐MHC protein nor the Ca2+ transients recorded from individual fibres subjected to the same pattern of stimulation showed any difference between stimulated and unstimulated fibres over the period in culture.

This system provides a convenient in vitro model system for studying activity‐dependent control of fibre type‐specific gene expression in adult skeletal muscle fibres in culture.
Membrane‐limited expression and regulation of Na+‐H+ exchanger isoforms by P2 receptors in the rat submandibular gland duct
Authors: Lee Min Goo , Schultheis Patrick J. , Yan Ming , Shull Gary E. , Bookstein Crescence , Chang Eugene , Tse Ming , Donowitz Mark , Park Keerang , Muallem Shmuel
Published: September 2004

Cell‐specific reverse transcriptase‐polymerase chain reaction (RT‐PCR), immunolocalization and microspectrofluorometry were used to identify and localize the Na+‐H+ exchanger (NHE) isoforms expressed in the submandibular gland (SMG) acinar and duct cells and their regulation by basolateral and luminal P2 receptors in the duct.

The molecular and immunofluorescence analysis showed that SMG acinar and duct cells expressed NHE1 in the basolateral membrane (BLM). Duct cells also expressed NHE2 and NHE3 in the luminal membrane (LM).

Expression of NHE3 was unequivocally established by the absence of staining in SMG from NHE3 knockout mice. NHE3 was expressed in the LM and in subluminal regions of the duct.

Measurement of the inhibition of NHE activity by the amiloride analogue HOE 694 (HOE) suggested expression of NHE1‐like activity in the BLM and NHE2‐like activity in the LM of the SMG duct. Several acute and chronic treatments tested failed to activate NHE activity with low affinity for HOE as expected for NHE3. Hence, the physiological function and role of NHE3 in the SMG duct is not clear at present.

Activation of P2 receptors resulted in activation of an NHE‐independent, luminal H+ transport pathway that markedly and rapidly acidified the cells. This pathway could be blocked by luminal but not basolateral Ba2+.

Stimulation of P2U receptors expressed in the BLM activated largely NHE1‐like activity, and stimulation of P2Z receptors expressed in the LM activated largely NHE2‐like activity.

The interrelation between basolateral and luminal NHE activities and their respective regulation by P2U and P2Z receptors can be used to co‐ordinate membrane transport events in the LM and BLM during active Na+ reabsorption by the SMG duct.
The Na+‐phosphate cotransport system (NaPi‐II) with a cleaved protein backbone: implications on function and membrane insertion
Authors: Kohl Beate , Wagner Carsten A. , Huelseweh Birgit , Busch Andreas E. , Werner Andreas
Published: September 2004

Renal handling of inorganic phosphate (Pi) involves a Na+‐Pi cotransport system which is well conserved between vertebrates. The members of this protein family, denoted NaPi‐II, share a topology with, it is thought, eight transmembrane domains. The transporter is proposed to be proteolytically cleaved within a large hydrophilic loop in vivo.

The consequences of an interrupted backbone were tested by constructing cDNA clones encoding different N‐ (1‐3 and 1‐5) and C‐terminal (4‐8 and 6‐8) complementary fragments of NaPi‐II from winter flounder. When the cognate fragments were used in combination (1‐3 plus 4‐8; 1‐5 plus 6‐8) they comprised the full complement of the putative transporter domains.

None of the four individual fragments or the 1‐5 plus 6‐8 combination when expressed in Xenopus oocytes increased Pi flux. Coexpression of fragments 1‐3 plus 4‐8 stimulated transport activity identical to that for expressed wild‐type NaPi‐II with regard to pH dependency and Km for Na+ and Pi binding; however, the maximal transport rate (vmax) was lower.

Immunohistochemistry on cryosections confined the functionally active 1‐3 plus 4‐8 combination to the oocyte membrane. This was not the case for the 1‐5 plus 6‐8 combination or any of the individual fragments, all of which failed to induce fluorescence.

A second immunohistochemical approach using intact oocytes allowed determination of the extracellular regions of the protein. Epitopes within the loop between transmembrane domains 3 and 4 enhanced fluorescence. Neither N‐ nor C‐terminal tags induced fluorescence.
Thiophosphorylation of myosin light chain increases rigor stiffness of rabbit smooth muscle
Authors: Khromov A. S. , Somlyo A. V. , Somlyo A. P.
Published: September 2004

The effect of thiophosphorylation of the regulatory myosin light chain (MLC20) on rigor stiffness was determined in permeabilized rabbit bladder smooth muscle.

Rigor stiffness of α‐toxin‐permeabilized smooth muscle was significantly increased by thiophosphorylation of MLC20. This increase may have been due to partial shortening (melting) in the proximal rod region and/or stiffening of the regulatory domain of the myosin head.

We suggest that phosphorylation of MLC20, by increasing the stiffness of the S1 lever arm and/or S2 hinge regions of the myosin molecule, favours separation of the two phosphorylated heads and consequent deinhibition of motor domain activity.
Basic fibroblast growth factor induction of neuronal ion channel expression in ascidian ectodermal blastomeres
Authors: Inazawa Takeshi , Okamura Yasushi , Takahashi Kunitaro
Published: September 2004

Cleavage‐arrested anterior animal (a4‐2) blastomeres isolated from eight‐cell embryos of Halocynthia aurantium differentiated into neuronal type cells expressing neuron‐specific ion channels when they were treated with basic fibroblast growth factor (bFGF). This induction process was very similar to that when a4‐2 blastomeres were cultured in contact with anterior vegetal (A4‐1) blastomeres from the same embryos or when treated with subtilisin, a serine protease.

Other growth factors, transforming growth factor (TGF) β1, activin A, epidermal growth factor (EGF) and nerve growth factor (NGF), had no effect on the default epidermal differentiation of cleavage‐arrested a4‐2 blastomeres.

Messenger RNA of the ascidian neuronal Na+ channel, TuNa I, was detected using RT‐PCR in a4‐2‐derived partial embryos of Halocynthia aurantium as well as in the cleavage‐arrested a4‐2 blastomeres treated with bFGF, confirming the neural inducer activity of bFGF during ascidian embryogenesis.

bFGF was effective at concentrations as low as 1 ng ml−1 in inducing neuronal ion channels in cleavage‐arrested a4‐2 blastomeres. EC50 for neuronal differentiation was estimated to be around 8 ng ml−1, and the maximum effect of 90 % neuronalization was obtained with above 100 ng ml−1.

For induction of neuronal differentiation, bFGF was required to be continuously present 8 to 14 h after fertilization. A similar time window was required for cell‐contact induction, but it was considerably shorter for subtilisin induction.

We discuss whether activation of receptor tyrosine kinase is a common pathway for neural induction by bFGF, subtilisin, and cell‐contact with A4‐1 blastomeres.
Metabotropic glutamate group II receptors activate a G protein‐coupled inwardly rectifying K+ current in neurones of the rat cerebellum
Authors: Knoflach Frédéric , Kemp John A.
Published: September 2004

The effects of the group II metabotropic glutamate receptor (mGluR) agonists DCG‐IV and LY354740 were examined in neurones freshly dissociated from the rat cerebellum and olfactory bulb, using the whole‐cell configuration of the patch‐clamp technique.

Under experimental conditions in which K+ currents would be inward, rapid application of DCG‐IV and LY354740 to interneurones expressing the group II mGluRs induced an inward current in a subpopulation of interneurones of the cerebellum, the unipolar brush cells.

The currents induced by DCG‐IV and LY354740 had the major characteristics of a G protein‐coupled inwardly rectifying K+ channel (GIRK) current; namely, rapid activation and deactivation upon agonist application and removal, G protein dependence, strong inward rectification, Cs+ and Ba2+ sensitivity, and K+ selectivity.

In Golgi cells of the cerebellum and interneurones of the accessory olfactory bulb, which also express group II mGluRs, LY354740 did not induce GIRK activation but inhibited voltage‐gated Ca2+ channel currents.

These results demonstrate that, in unipolar brush cells, native group II mGluRs can functionally couple to activation of GIRKs. Thus, the absence of coupling in the majority of CNS neurones examined to date may be due to restricted cellular co‐localization or co‐expression of the appropriate proteins.
Two mechanisms for inward rectification of current flow through the purinoceptor P2X2 class of ATP‐gated channels
Authors: Zhou Zhen , Hume Richard I.
Published: September 2004

The ATP receptor subunit P2X2 was expressed in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. ATP‐activated currents were studied with two‐electrode voltage clamp recordings from oocytes, whole‐cell recordings from HEK 293 cells, and outside‐out patch clamp recordings from both cell types. The steady‐state current‐voltage (I‐V) relation showed profound inward rectification in all recording configurations.

Recordings from outside‐out patches demonstrated that inward rectification does not require intracellular Mg2+ or polyamines, and that inward rectification was present when the same solution was used on both sides of the patch.

Voltage jump experiments were performed to evaluate the voltage dependence of channel gating. After fast voltage jumps, instantaneous current jumps were followed by substantial relaxations to the steady state. The time course of the current relaxations could be fitted by single exponential functions. The instantaneous I‐V relation was less inwardly rectifying than the steady‐state I‐V relation; however, it was not linear.

Single channel recordings indicated that the single channel conductance became smaller when the membrane potential became more positive. This decrease could quantitatively account for inward rectification of the instantaneous I‐V relation.

We conclude that inward rectification of P2X2 is due to two mechanisms: voltage‐dependent gating and voltage dependence of the single channel conductance.
Characterization of a Ca2+‐activated K+ current in insulin‐secreting murine βTC‐3 cells
Authors: Kozak J. Ashot , Misler Stanley , Logothetis Diomedes E.
Published: September 2004

The whole‐cell perforated‐patch recording mode was used to record a Ca2+‐dependent K+ current (IK(Ca)) in mouse βTC‐3 insulin‐secreting cells.

Depolarizing voltage steps (to potentials where Ca2+ currents are activated) evoked a slowly activating, outward current, which exhibited a slow deactivation (in seconds) upon subsequent hyperpolarization.

This current was shown to increase with progressively longer depolarizing voltage steps. It could be reversibly abolished by the removal of Ca2+ from the external medium or by application of Ca2+ channel blockers, such as Cd2+ and nifedipine. It was concluded that the depolarization‐evoked current was activated by Ca2+.

Variations in external K+ concentration led to shifts in the reversal potential of the Ca2+‐dependent current as predicted by the Nernst equation for a K+‐selective current.

The Ca2+‐activated K+ current was insensitive to external TEA (10 mM), a concentration sufficient to block the large‐conductance Ca2+‐dependent (maxi‐KCa) channel in β‐cells. It was also insensitive to apamin, tubocurarine and scyllatoxin (leiurotoxin I), specific blockers of small‐conductance KCa channels.

The current was blocked by quinine, a non‐specific KCa channel blocker and, surprisingly, by charybdotoxin (ChTX; 100 nM) but not iberiotoxin, a charybdotoxin analogue, which blocks the maxi‐KCa channel. It was sensitive to block by clotrimazole and could be potently and reversibly potentiated by micromolar concentrations of niflumic acid. Thus, the current exhibited unique pharmacological characteristics, not conforming to the known KCa channel classes.

The ChTX‐sensitive KCa channel was permeable to Tl+, K+, Rb+ and NH4+ but not Cs+ ions.

The ChTX‐sensitive IK(Ca) could be activated by the muscarinic agonists in the presence or absence of external Ca2+, presumably by releasing Ca2+ from internal stores.

Acutely isolated porcine islet cells also exhibited a slow IK(Ca) resembling that described in βTC‐3 cells in kinetic properties, insensitivity to TEA (5 mM) and sensitivity to quinidine, an analogue of quinine. The porcine IK(Ca), however, was not sensitive to block by 100‐200 nM ChTX. It is likely, that species differences account for pharmacological differences between the mouse and porcine slow IK(Ca).
Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel
Authors: Mathews Ceri J. , Tabcharani Joseph A. , Chang Xiu‐Bao , Jensen Timothy J. , Riordan John R. , Hanrahan John W.
Published: September 2004

The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild‐type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA).

The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP‐PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate.

The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co‐operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline.

Wild‐type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non‐hydrolysable analogue AMP‐PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels.

We conclude that phosphorylation at ‘weak’ PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP‐PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.
Stealth ryanodine‐sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells
Authors: Paltauf‐Doburzynska Jolanta , Posch Karla , Paltauf Guenther , Graier Wolfgang F.
Published: September 2004

The involvement of ryanodine‐sensitive Ca2+ release (RsCR) in bradykinin (Bk)‐induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (NOS) activation was assessed in freshly isolated bovine coronary artery endothelial cells.

Using deconvolution microscopy fura‐2 was found throughout the whole cytosol, while the cell membrane impermeable dye FFP‐18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]pn) and subplasmalemmal Ca2+ concentration ([Ca2+]sp) were monitored using fura‐2 and FFP‐18.

Inhibition of Na+−Ca2+ exchange by lowering extracellular Na+ concentration augmented the Bk‐induced [Ca2+]pn signal in Ca2+‐free solution. This effect was abolished when RsCR was prevented with 25 μmmu;mol l−1 ryanodine, while inhibition of RsCR had no effect on Bk‐induced increase in [Ca2+]pn without inhibition of Na+−Ca2+ exchange.

Initiating RsCR by 200 nmol l−1 ryanodine increased [Ca2+]sp, while [Ca2+]pn remained constant. However, when Na+−Ca2+ exchange was prevented, ryanodine was also able to elevate [Ca2+]pn.

Blockage of RsCR diminished Ca2+ extrusion in response to stimulation with Bk in normal Na+‐containing solution.

Inhibition of RsCR blunted Bk‐activated CCE, while inhibition of Na+−Ca2+ exchange during stimulation enhanced CCE.

Although direct activation of RsCR failed to activate NOS, inhibition of RsCR diminished the effect of ATP and Bk on NOS, while the effect of thapsigargin remained unchanged.

These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and NOS. Thus, the function of the subplasmalemmal Ca2+ control unit must be extended as a regulator for CCE and NOS.
Local calcium release in mammalian skeletal muscle
Authors: Shirokova Natalia , García Jesús , Ríos Eduardo
Published: September 2004

Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres mechanically dissected from fast twitch muscle of rats or frogs, voltage clamped in a two Vaseline‐gap chamber.

Interventions that elicited Ca2+ sparks in frog skeletal muscle (low voltage depolarizations, application of caffeine) generated in rat fibres images consistent with substantial release from triadic regions, but devoid of resolvable discrete events. Ca2+ sparks were never observed in adult rat fibres. In contrast, sparks of standard morphology were abundant in myotubes from embryonic mice.

Depolarization‐induced gradients of fluorescence between triadic and surrounding regions (which are proportional to Ca2+ release flux) peaked at about 20 ms and then decayed to a steady level. Gradients were greater in frog fibres than in rat fibres. The ratio of peak over steady gradient (R) was steeply voltage dependent in frogs, reaching a maximum of 4.8 at −50 mV (n= 7). In rats, R had an essentially voltage‐independent value of 2.3 (n= 5).

Ca2+‐induced Ca2+ release, resulting in concerted opening of several release channels, is thought to underlie Ca2+ sparks and the peak phase of release in frog skeletal muscle. A diffuse ‘small event’ release, similar to that observed in these rats, is also present in frogs and believed to be directly activated by voltage. The present results suggest that in these rat fibres there is little contribution by CICR to Ca2+ release triggered by depolarization, and a lack of concerted channel opening.
Characterization of the putative chloride channel xClC‐5 expressed in Xenopus laevis oocytes and comparison with endogenous chloride currents
Authors: Schmieder S. , Lindenthal S. , Banderali U. , Ehrenfeld J.
Published: September 2004

We recently cloned a putative chloride channel (xClC‐5) from the renal cell line A6, which induced the appearance of a Cl− conductance not found in control oocytes after homologous expression in Xenopus oocytes. With the aim of increasing the Xenopus oocyte xClC‐5 expression, we constructed a new plasmid in which the native 5′ and 3′ non‐coding regions of xClC‐5 were replaced by the non‐coding regions of the Xenopusβ‐globin sequence and in which a Kozak consensus site was introduced before the initiator ATG.

We then compared the induced currents Inative (induced by injection of cRNA presenting the native non‐coding regions of xClC‐5) and Iβ‐globin (induced by injection of cRNA presenting the non‐coding regions of the Xenopusβ‐globin sequence) investigating anion selectivity and anion blocker sensitivity. Several differences were found: (1) expression yield and oocyte surviving rate were largely increased by injecting (β) xClC‐5 cRNA, (2) the Iβ‐globin outward rectification score was 2.6 times that of Inative, (3) the anion conductivity sequence was nitrate > bromide > chloride > iodide >> gluconate for Iβ‐globin and iodide > bromide > nitrate > chloride >> gluconate for Inative, (4) 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB), anthracene‐9‐carboxylic acid (9‐AC), DIDS, lanthanum ions, cAMP and ionomycin‐induced [Ca2+]i increase inhibited Inative but had no effect on Iβ‐globin, and (5) Inative showed considerable similarity to the previously reported endogenous current appearing after ClC‐6 or pICln cRNA injection.

Comparison of Inative with the endogenous chloride current ICl,swell which develops under hyposmotic conditions demonstrated several similarities in their electrophysiological and pharmacological characteristics but were nevertheless distinguishable.

In vitro translation assays demonstrated that protein synthesis was much greater using the (β) xClC‐5 construct than that of xClC‐5. Furthermore, immunoreactivity of membrane preparations of Xenopus oocytes was only observed with the (β) xClC‐5 construct, its intensity being positively correlated with Iβ‐globin levels.

In addition, the current induced in (β) xClC‐5 cRNA‐injected oocytes presented a very marked pH dependence (inhibition by acid external media) with a pKa value (negative log of the acid dissociation constant) of 5.67.

In conclusion, Iβ‐globin may be due to the presence of xClC‐5 in the oocyte plasma membrane playing a role as an anion channel whereas Inative may represent an endogenous current induced by xClC‐5 cRNA injection. The use of antibodies will facilitate the tissue and subcellular localization of xClC‐5 and the identification of its physiological role.
Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in mammalian cells
Authors: Lansdell K. A. , Delaney S. J. , Lunn D. P. , Thomson S. A. , Sheppard D. N. , Wainwright B. J.
Published: September 2004

To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full‐length cDNA encoding wild‐type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells.

Like human CFTR, murine CFTR formed Cl− channels that were regulated by cAMP‐dependent phosphorylation and intracellular ATP. However, murine CFTR Cl− channels had a reduced single‐channel conductance and decreased open probability (Po) compared with those of human CFTR.

Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant.

For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long‐lived closed state.

5′‐Adenylylimidodiphosphate (AMP‐PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl− channels open, although AMP‐PNP increased the Po of murine CFTR.

The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.
Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes
Authors: Zhou Zhuan , Matlib Mohammed A. , Bers Donald M.
Published: September 2004

Ventricular myocytes isolated from ferret or cat were loaded with the acetoxymethyl ester form of indo‐1 (indo‐1 AM) such that ∼75 % of cellular indo‐1 was mitochondrial. The intramitochondrial indo‐1 concentration was 0.5‐2 mm.

Myocytes were also voltage clamped (membrane capacitance, Cm= 100 pF) and a typical wash‐out time constant of cytosolic indo‐1 by a patch pipette was found to be ∼300 s. Depolarizations to +110 mV produced graded and progressive cellular Ca2+ load via Na+‐Ca2+ exchange.

During these relatively slow Ca2+ transients, cell contraction (ΔL) paralleled fluorescence ratio signals (R) such that ΔL could be used as a bioassay of cytosolic [Ca2+] ([Ca2+]c), where [Ca2+]CL is the inferred signal which is delayed by ∼200 ms from true [Ca2+]c.

In myocytes without Mn2+ quench, the kinetics of the total cellular indo‐1 signal, ΔR (including cytosolic and mitochondrial components), match ΔL during stimulations at low basal [Ca2+]i. However, after progressive Ca2+ loading, ΔR kinetics deviate from ΔL dramatically. The deviation can be completely blocked by a potent mitochondrial Ca2+ uniport blocker, Ru360.

When cytosolic indo‐1 is quenched by Mn2+, initial moderate stimulation triggers contractions (ΔL), but no change in indo‐1 signal, indicating both the absence of cytosolic Ca2+‐sensitive indo‐1 and unchanged mitochondrial [Ca2+] (Δ[Ca2+]m). Subsequent stronger stimulation evoked larger ΔL and also ΔR. The threshold [Ca2+]c for mitochondrial Ca2+ uptake was 300‐500 nM, similar to that without Mn2+ quench.

At high Ca2+ loads where Δ[Ca2+]m is detected, the time course of [Ca2+]m was different from that of [Ca2+]c. Peak [Ca2+]m after stimulation has an ∼1 s latency with respect to [Ca2+]c, and [Ca2+]m decline is extremely slow.

Upon a Ca2+ influx which increased [Ca2+]c by 0.4 μm and [Ca2+]m by 0.2 μm, total mitochondrial Ca2+ uptake was ∼13 μmol (l mitochondria)−1.

With Mn2+ quench of cytosolic indo‐1, there was no mitochondrial uptake of Mn2+ until the point at which mitochondrial Ca2+ uptake became apparent. However, after mitochondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ even during relaxation, when [Ca2+]c is low.

It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca2+ during individual contractions, unless resting [Ca2+]c exceeds 300‐500 nM. At high cell Ca2+ loads and [Ca2+]c, mitochondrial Ca2+ transients occur during the twitch, but with much slower kinetics than those of [Ca2+]c.
Respiration‐modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem‐spinal cord of the neonatal rat
Authors: Oyamada Y. , Ballantyne D. , Mückenhoff K. , Scheid P.
Published: September 2004

The activity of locus coeruleus (LC) neurones (n= 126) was examined in whole‐cell (conventional and amphotericin B‐perforated patch) recordings, and the relationship of this activity to the respiratory discharge recorded on the C4 or C5 phrenic nerve roots was determined at different CO2 concentrations (2 and 8 %; bath pH 7.8 and 7.2) in the in vitro brainstem‐spinal cord preparation of the neonatal rat (1–5 days old).

In most neurones (n= 105) ongoing activity was modulated at respiratory frequency. Typically, this consisted of a phase of depolarization and increased discharge frequency synchronous with the phrenic burst, followed by a phase of hyperpolarization and inhibition of discharge (n= 94 of 105). The incidence of respiratory modulation decreased from 91 % on P1 to 57 % on P5.

Bath application of the non‐nmDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5 μmmu;m) or the nmDA receptor antagonist DL‐2‐amino‐5‐phosphonovaleric acid (APV; 100 μmmu;m) abolished both phases of respiratory modulation. The hyperpolarizing phase alone was abolished by the adrenoceptor antagonists idazoxan (5 μmmu;m) or phentolamine (0.8 μmmu;m). These results indicate that excitatory amino acid pathways are involved in the transmission of both the excitatory and inhibitory components and that the latter involves in addition an α2‐adrenoceptor‐mediated pathway.

Increasing the CO2 concentration from 2 to 8 % resulted in a shortening of expiratory duration and weakening or loss of respiratory‐phased inhibition; this was accompanied by depolarization, increased discharge frequency and, in those neurones where they were initially present (60 %), an increase in the frequency of subthreshold membrane potential oscillations. The depolarizing response was retained in the presence of tetrodotoxin (TTX, 0.2–1.0 μmmu;m).

These results indicate that in this neonatal preparation LC neurones form part of the synaptically connected brainstem respiratory network, and that the LC constitutes a site of CO2‐ or pH‐dependent chemoreception.
Developmental changes in the expression of low‐voltage‐activated Ca2+ channels in rat visual cortical neurones
Authors: Tarasenko A. N. , Isaev D. S. , Eremin A. V. , Kostyuk P. G.
Published: September 2004

The functional properties of low‐voltage‐activated (LVA) Ca2+ channels were studied in pyramidal neurones from different rat visual cortical layers in order to investigate changes in their properties during early postnatal development. Ca2+ currents were recorded in brain slices using the whole‐cell patch‐clamp technique in rats from three age groups: 2, 3 and 12 days old (postnatal day (P) 2, P3 and P12).

It was demonstrated that LVA Ca2+ currents are present in neurones from superficial (I‐II) and deep (V‐VI) visual cortex layers of P2 and P3 rats. No LVA Ca2+ currents were observed in neurones from the middle (III‐IV) layers of these rats. The LVA Ca2+ currents observed in P2 and P3 neurones from both superficial and deep layers could be completely blocked by nifedipine (100 μM) and were insensitive to Ni2+ (25 μM).

The density of LVA Ca2+ currents decreased rapidly during the early stages of postnatal development, while the density of high‐voltage‐activated (HVA) Ca2+ currents progressively increased up to the twelfth postnatal day. No LVA Ca2+ currents were found in P12 neurones from any of the layers. Only HVA Ca2+ currents with high sensitivity to F− applied through the patch pipette were observed.

The kinetics of LVA Ca2+ currents could be well approximated by the m2h Hodgkin‐Huxley equation with an inactivation time constant of 24 ± 6 ms. The steady‐state inactivation curve fitted by a Boltzmann function had the following parameters: membrane potential at half‐inactivation, ‐86.9 mV; steepness coefficient,3.4 mV.

It is concluded that, in visual cortical neurones, LVA Ca2+ channels are expressed only in the neurones of deep and superficial layers over a short period during the earliest postnatal stages. These channels are nifedipine sensitive and similar in functional properties to those in the laterodorsal (LD) thalamic nucleus. However, the cortical neurones do not express another (‘slow’) type of LVA Ca2+ channel, which is permanently present in LD thalamic neurones after the second postnatal week, indicating that the developmental time course of cortical and thalamic cellsdifferent.
Encoding properties induced by a persistent voltage‐gated muscarinic sodium current in rabbit sympathetic neurones
Authors: Gola Maurice , Delmas Patrick , Chagneux Hélène
Published: September 2004

A time‐ and voltage‐dependent Na+‐selective current termed INa,M is activated by muscarinic agonists or splanchnic nerve stimulation in sympathetic neurones of rabbit coeliac and superior mesenteric ganglia. The firing patterns induced by INa,M were investigated in patch‐clamped neurones within intact ganglia, and compared with those generated by a neuronal model including INa,M.

I
Na,M was characterized by voltage‐dependent low‐threshold activation and high‐threshold inactivation functions. The overlapping functions produced a persistent U‐shaped current between −100 and −20 mV, which peaked at the cell resting potential. The activation and inactivation kinetics were fitted to single exponentials with time constants of ≈100 and 400 ms, respectively.

Activating INa,M with muscarinic agonists or nerve stimulation depolarized and fired the neurones. The depolarization was paralleled by an apparent increase in input membrane resistance. The model showed that this paradox resulted from the turning off of INa,M during resistance tests, which also accounted for the all‐or‐none slow hyperpolarizing responses to current pulses.

I
Na,M gave the neurones an N‐shaped I‐V relationship capable of producing complex firing patterns. Under given conditions, carbachol‐treated neurones could either fire regularly or remain silent at ≈‐80 mV, i.e. they displayed bistability. Transitions from one state to the other were triggered with short current pulses. The transitions resulted from the turning on and off of INa,M.

Firing reduced INa,M, an effect abolished by blocking Ca2+ channels or adding BAPTA (40 mM) to the pipette. The Ca2+‐related negative regulation of INa,M may have mediated endogenous bursting activity. Burst firing was generated by the model upon introducing Ca2+ regulation of INa,M.

The results demonstrate that INa,M gives prevertebral sympathetic neurones a wide repertoire of firing patterns: pacemaker‐like properties, bistability and burst firing capability. They suggest that the INa,M‐related encoding properties may provide sympathetic neurotransmission with new potentialities.
The open duration of fetal ACh receptor‐channel changes during mouse muscle development
Authors: Grassi Francesca , Epifano Olga , Mileo Anna Maria , Barabino Benedetta , Eusebi Fabrizio
Published: September 2004

We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the γ subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5.

We studied unitary ACh‐evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6).

At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24‐26 °C.

On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the γs subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period.
Cytoplasmic terminus domains of Kir6.x confer different nucleotide‐dependent gating on the ATP‐sensitive K+ channel
Authors: Takano Makoto , Xie Lai‐Hua , Otani Hideo , Horie Minoru
Published: September 2004

In order to investigate the structural basis for the nucleotide‐dependent gating of ATP‐sensitive K+ channels (KATP), Kir6.1 (uKATP‐1), Kir6.2 (BIR1) and chimeric channels were co‐expressed with a common subtype of sulphonylurea receptor, SUR1, in COS7 cells. Representing the amino terminal domain‐transmembrane domain‐carboxyl‐terminal domain of Kir6.1 as 1–1–1 and of Kir6.2 as 2‐2‐2, chimeric Kir6.x channels were constructed by swapping the amino and/or carboxyl terminal domains between Kir6.1 and Kir6.2 to give the chimeric x‐1‐x channels 1‐1‐2, 2‐1‐1 and 2‐1‐2, and the chimeric x‐2‐x channels 2‐2‐1, 1‐2‐2 and 1‐2‐1.

Inside‐out patch clamp experiments revealed that both wild‐type Kir6.1 and Kir6.2 formed inwardly rectifying K+ channels. Single‐channel conductances were 36.3 and 66.1 pS, respectively. Chimeric x‐1‐x channels, whose transmembrane domain was that of Kir6.1, showed similar ion‐pore properties to wild‐type Kir6.1. Likewise, chimeric x‐2‐x channels had similar ion‐pore properties to wild‐type Kir6.2.

Wild‐type Kir6.1 and Kir6.2 possessed distinct gating properties towards intracellular nucleotides. The activity of Kir6.1 was entirely dependent on Mg2+ and nucleotide diphosphates (NDPs) such as UDP. In contrast, Kir6.2 was activated upon excision of patch membrane. When Kir6.2 underwent rundown, UDP reactivated the channel.

In order to eliminate UDP dependence from Kir6.1, it was necessary to replace both N‐ and C‐termini; chimera 2‐1‐2 opened in UDP‐free conditions. With Kir6.2, substitution of the N‐terminus with that of Kir6.1 conferred UDP dependence on chimeras 1‐2‐2 and 1‐2‐1. Chimera 2‐2‐1 opened in UDP‐free conditions, but UDP potentiated the channel activity by > 20‐fold.

The kinetics of UDP‐dependent activation were significantly different between Kir6.1 and Kir6.2. Kir6.1 maximally activated by UDP was sensitive to intracellular ATP, although its ATP sensitivity was significantly lower than that of Kir6.2 measured in identical conditions. The kinetics of UDP‐dependent activation and ATP sensitivity could be transferred between Kir6.1 and Kir6.2 only when both N‐ and C‐termini were replaced. We therefore concluded that nucleotide‐dependent gating was regulated by the N‐ and C‐terminal domains irrespective of the transmembrane domains.
Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes
Authors: Callamaras Nick , Sun Xiao‐Ping , Ivorra Isabel , Parker Ian
Published: September 2004

The mechanisms underlying hemispheric asymmetry of the inositol 1,4,5‐trisphosphate (InsP3)‐calcium signalling pathway in Xenopus oocytes were examined by fluorescence imaging of calcium signals and recording calcium‐activated Cl− currents (ICl,Ca) evoked by intracellular calcium injections and photorelease of InsP3.

The maximal ICl,Ca evoked by strong photorelease of InsP3 was 8 times greater in the animal than the vegetal hemisphere, but the average threshold amounts of InsP3 required to evoke detectable currents were similar in each hemisphere.

Currents evoked by injections of calcium were about 2.5 times greater near the animal pole than near the vegetal pole, whereas fluorescence signals evoked by injections were similar in each hemisphere.

Calcium waves were evoked by photolysis flashes of similar strengths in both hemispheres of albino oocytes, but peak calcium levels evoked by supramaximal stimuli were 70 % greater in the animal hemisphere.

Elementary calcium release events (puffs) in the animal hemisphere had amplitudes about double that in the vegetal hemisphere, and more often involved coupled release from adjacent sites. Calcium release sites were more closely packed in the animal hemisphere, with a mean spacing of about 1.5 μm compared with 2.25 μm in the vegetal hemisphere.

The larger amplitude of currents mediated by InsP3 in the animal hemisphere, therefore, involves an increased flux of calcium at individual release units, a more dense packing of release units and a higher density of Cl− channels.