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61.
A. S. Amin A. O. Verkerk Z. A. Bhuiyan A. A. M. Wilde H. L. Tan 《Acta physiologica (Oxford, England)》2005,185(4):291-301
Aim: Brugada syndrome is an inherited cardiac disease with an increased risk of sudden cardiac death. Thus far Brugada syndrome has been linked only to mutations in SCN5A, the gene encoding the α‐subunit of cardiac Na+ channel. In this study, a novel SCN5A gene mutation (D1714G) is reported, which has been found in a 57‐year‐old male patient. Since the mutation is located in a segment of the ion‐conducting pore of the cardiac Na+ channel, which putatively determines ion selectivity, it may affect ion selectivity properties. Methods: HEK‐293 cells were transfected with wild‐type (WT) or D1714G α‐subunit and β‐subunit cDNA. Whole‐cell configuration of the patch‐clamp technique was used to study biophysical properties at room temperature (21 °C) and physiological temperature (36 °C). This study represents the first measurements of human Na+ channel kinetics at 36 °C. Ion selectivity, current density, and gating properties of WT and D1714G channel were studied. Results: D1714G channel yielded nearly 80% reduction of Na+ current density at 21 and 36 °C. At both temperatures, no significant changes were observed in V1/2 values and slope factors for voltage‐dependent activation and inactivation. At 36 °C, but not at 21 °C, D1714G channel exhibited more slow inactivation compared with WT channel. Ion selectivity properties were not affected by the mutation at both temperatures, as assessed by either current or permeability ratio. Conclusion: This study shows no changes in ion selectivity properties of D1714G channel. However, the profoundly decreased current density associated with the D1714G mutation may explain the Brugada syndrome phenotype in our patient. 相似文献
62.
P. M. T. Deen Søren Nielsen René J. M. Bindels Carel H. van Os 《Pflügers Archiv : European journal of physiology》1997,433(6):780-787
Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop.
In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow,
we stably transfected LLC-PK1 and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK1 clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy
showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane,
including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant
marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability,
which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl2. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell
type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds.
Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997 相似文献
63.
W. -M. Weber K. M. Liebold F. W. Reifarth U. Uhr W. Clauss 《Pflügers Archiv : European journal of physiology》1995,429(6):820-824
Removal of Ca2+ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl– efflux, which could be strongly inhibited by the Cl– channel blockers flufenamic acid and niflumic acid. Removal of Mg2+ or Ba2+ from the solutions had the same effects as removing Ca2+. The reversal potential of –12 mV also indicated that Cl– channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca2+ concentration of about 20 M showed that quite low doses of extracellular Ca2+ profoundly influence the electrical properties of the oocyte membrane. 相似文献
64.
F. Barros Donato del Camino Luis A. Pardo Teresa Palomero Teresa Giráldez Pilar de la Peña D. del Camino 《Pflügers Archiv : European journal of physiology》1997,435(1):119-129
Reduction of an inwardly rectifying K+ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical
activity increases in GH3 rat anterior pituitary cells. However, the existence of an inwardly rectifying K+ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target
in pituitary cells, including GH3 cells. In this report, an inwardly rectifying component of K+ current is indeed demonstrated in perforated-patch voltage-clamped GH3 cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of
current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane
potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active.
Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous
outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according
to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the
possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current.
Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K+ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is
probably mediated by a HERG-like K+ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production.
Thus, an inwardly-rectifying K+ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH3 cells.
Received: 15 May 1997 / Received after revision and accepted: 24 July 1997 相似文献
65.
Ma HP Zhou ZH Liang YY Saxena S Warnock DG 《Pflügers Archiv : European journal of physiology》2004,449(1):96-105
Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl– for gluconate– shifted the reversal potential, while Cl– channel blockers, 4,4-diisothiocyanostibene-2,2-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPS (a poorly hydrolyzable analog of ATP), 2,3-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 M suramin (a P2 receptor antagonist), and was partially blocked by 100 M pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca2+ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca2+-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors. 相似文献
66.
黄皮酰胺促钾通道开放 总被引:1,自引:0,他引:1
一种新发现的具有促智作用的药物——黄皮酰胺能抑制去甲肾上腺素(NE)或KCl引起的血管平滑肌收缩。本研究旨在应用膜片钳(patch clamp)技术探讨黄皮酰胺对Wistar大鼠尾动脉平滑肌细胞膜钾离子通道的作用。单个平滑肌细胞用酶法分离,以细胞封接方式记录离子通道活动。在细胞池内注入2μM黄皮酰胺后,钾离子通道活动明显增强。用本实验室开发的计算机软件(patch clamp analysis system,Version 1.0)计算分析通道活动的特征参数。 相似文献
67.
Voltage gated ion channels on the somatic membrane of rat cerebellar Purkinje cells were studied in dissociated cell culture with the combination of cell-attached and whole-cell variation of patch clamp technique. The method enables us to record local somatic membrane current under an improved space clamp condition. Transient (fast-inactivating) and steady (slow inactivating) Ca channel currents, Na current, transient (fast-inactivating) and steady (slow-inactivating) K currents, were observed. Transient and steady Ca channel currents were activated at test potentials more positive than –40 mV and –20 mV, respectively (in 50 mM external Ba). The transient current inactivated with a half-decay time of 10–30 ms during maintained depolarizing pulses, while the steady current showed relatively little inactivation. Na current was activated at more positive potentials than –60 mV, and inactivated with a half-decay time of less than 5 ms. Transient and steady K outward currents were recorded at more positive potential than –20 mV and –40 mV, respectively. The transient current inactivated with a half-decay time of 2–8 ms. Ca, Na and K channels showed different patterns of distribution on the somatic membrane. Steady Ca channels tended to cluster compared with Na or K channels. 相似文献
68.
T. Danker Birgit Gaßner Hans Oberleithner Albrecht Schwab 《Pflügers Archiv : European journal of physiology》1996,433(1-2):71-76
Madin Darby canine kidney cells transformed by alkaline stress (MDCK-F cells) constitutively migrate at a rate of about 1
μm·min–1. Migration depends on the intermittent activity of a Ca2+-stimulated, 53-pS K+ channel (KCa channel) that is inhibitable by charybdotoxin. In the present study we examined whether this intermittent KCa channel activity results in a significant K+ loss across the plasma membrane. K+ efflux from MDCK-F cells should result in a transient increase of extracellular K+ ([K+]e) in the close vicinity of a migrating cell. However, due to the rapid diffusion of K+ ions into the virtually infinite extracellular space, such a transient increase in [K+]e was too small to be detected by conventional K+-selective electrodes. Therefore, we developed a ”shielded ion-sensitive microelectrode” (SIM) that limited diffusion to a
small compartment, formed by a shielding pipette which surrounded the tip of the K+-sensitive microelectrode. The SIM improved the signal to noise ratio by a factor of at least three, thus transient increases
of [K+]e in the vicinity of MDCK-F cells became detectable. They occurred at a rate of 1.3 min–1. The cell releases 40 fmol K+ during each burst of intermittent KCa channel activity, which corresponds to about 15% of the total cellular K+ content. Since transmembrane K+ loss must be accompanied by anion loss and therefore leads to a decrease of cell volume, these findings support the hypothesis
that intermittent volume changes are a prerequisite for the migration of MDCK-F cells.
Received: 15 April 1996 / Received after revision: 18 June 1996 / Accepted: 23 July 1996 相似文献
69.
Takao Shioya Hiroko Matsuda Akinori Noma 《Pflügers Archiv : European journal of physiology》1993,422(5):427-435
The present patch-clamp study shows that external Mg2+, Ca2+ and Sr2+ decrease the unit amplitude of inward current through the inward-rectifier K+ channel in a concentration-dependent manner. Sr2+ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr2+ binding site () is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg2+ or Ca2+ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg2+ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs+ block (=1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs+ and Sr2+ bind. 相似文献
70.
Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig 总被引:2,自引:0,他引:2
Masaru Hirano Y. Imaizumi Katsuhiko Muraki A. Yamada Minoru Watanabe 《Pflügers Archiv : European journal of physiology》1998,435(5):645-653
Three major ionic currents, Ca2+-dependent K+ current (I
K-Ca), delayed rectifier type K+ current (I
kd) and Ca2+ current (I
Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary
bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I
K-Ca and I
Ca at 0 mV (IC50 values were 4.2 and 5.6 μM, respectively), but did not affect I
Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I
caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I
K-Ca during depolarization, STOCs and I
caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca2+-dependent K+ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the
channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic
Ca2+ concentration was increased. These results indicate that RuR blocks BK and Ca2+ channels in urinary bladder smooth muscle cells. The decrease in I
K-Ca, STOCs and I
caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca2+ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR
with the Ca2+-binding sites of the channel protein.
Received: 15 October 1997 / Received after revision and accepted: 25 November 1997 相似文献