bluuebunny/crossref_metadata_2025_split 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.