Properties of Ca2+-mediated inactivation of L-type Ca channel in smooth muscle cells of the guinea-pig urinary bladder

1995 ◽  
Vol 73 (1) ◽  
pp. 27-35 ◽  
Author(s):  
M. Yoshino ◽  
Y. Matsufuji ◽  
H. Yabu
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Patch Clamp ◽  
Double Pulse ◽  
Ca Channel ◽  
Time Constants ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Pulse Experiment

The properties of Ca2+-mediated inactivation as revealed by a conventional double-pulse protocol were examined by using the whole-cell patch clamp technique. A U-shaped relationship between the conditioning potential and the Ca2+ current (ICa) inactivation was observed, with a maximum inactivation of 52 ± 4% (n = 5) at 10 mV with 0.5 mM EGTA in the patch pipettes. The maximum inactivation was reduced significantly, to 31 ± 5.7% (n = 12) and 32 ± 7.0% (n = 5), when a high concentration of EGTA (20 mM) or a more efficient Ca2+ chelator, BAPTA, was included in the patch pipettes, respectively. The same double-pulse protocol was applied under conditions where the stored Ca2+ was depleted by using caffeine or the stored Ca2+ release function was blocked by using ryanodine or procaine and heparin. No significant difference in the maximum ICa inactivation before (45%) and after (50%) application of 10 mM caffeine was observed. The maximum ICa inactivations of 48 ± 3.2% (n = 4) and 52 ± 8.4% (n = 6) were still observed after treatment of the cell with ryanodine (20 μM) or loading 10 mM procaine and 1 mg/mL heparin in the patch pipettes, respectively. These results suggest that Ca2+ mobilization from an internal Ca2+ store is not essential for the Ca2+-mediated inactivation observed in the double-pulse experiment, rather influx of Ca2+ through a voltage-dependent Ca channel seems to be important for ICa inactivation. Recovery from Ca2+-mediated inactivation could be fitted by the sum of two exponentials; the time constants of the fast and slow components were 210 and 810 ms, respectively, at a holding potential of −80 mV. By lowering the holding potential to −30 from −80 mV, the time constants of both fast and slow components were increased to 740 and 4820 ms, respectively, suggesting that the recovery of Ca2+ channels from Ca2+-mediated inactivation is voltage dependent.Key words: urinary bladder, smooth muscle, whole-cell patch clamp, Ca2+ current, double-pulse protocol, Ca2+-mediated inactivation.

scholarly journals Expression and function of KV2-containing channels in human urinary bladder smooth muscle

2012 ◽  
Vol 302 (11) ◽  
pp. C1599-C1608 ◽  
Author(s):  
Kiril L. Hristov ◽  
Muyan Chen ◽  
Serge A. Y. Afeli ◽  
Qiuping Cheng ◽  
Eric S. Rovner ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Electrical Field Stimulation ◽  
Bladder Smooth Muscle ◽  
Rt Pcr ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

The functional role of the voltage-gated K+ (KV) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of KV2.1, KV2.2, and the electrically silent KV9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of KV2.1, KV2.2, and KV4.2 homotetrameric channels and of KV2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca2+ imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of KV2.1, KV2.2, and KV9.3 (but not KV4.2) channel subunits in human isolated DSM cells. KV2.1 and KV2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced KV current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca2+ level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5–30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive KV2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Hypoxia sensitivity of a voltage-gated potassium current in porcine intrapulmonary vein smooth muscle cells

2012 ◽  
Vol 303 (5) ◽  
pp. L476-L486 ◽  
Author(s):  
Ciprian Dospinescu ◽  
Hélène Widmer ◽  
Iain Rowe ◽  
Cherry Wainwright ◽  
Stuart F. Cruickshank
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Smooth Muscle Cells ◽  
Pulmonary Vein ◽  
Muscle Cells ◽  
Bk Channel ◽  
Whole Cell ◽  
Time To Peak ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Hypoxia contracts the pulmonary vein, but the underlying cellular effectors remain unclear. Utilizing contractile studies and whole cell patch-clamp electrophysiology, we report for the first time a hypoxia-sensitive K+ current in porcine pulmonary vein smooth muscle cells (PVSMC). Hypoxia induced a transient contractile response that was 56 ± 7% of the control response (80 mM KCl). This contraction required extracellular Ca2+ and was sensitive to Ca2+ channel blockade. Blockade of K+ channels by tetraethylammonium chloride (TEA) or 4-aminopyridine (4-AP) reversibly inhibited the hypoxia-mediated contraction. Single-isolated PVSMC (typically 159.1 ± 2.3 μm long) had mean resting membrane potentials (RMP) of −36 ± 4 mV with a mean membrane capacitance of 108 ± 3.5 pF. Whole cell patch-clamp recordings identified a rapidly activating, partially inactivating K+ current ( IKH) that was hypoxia, TEA, and 4-AP sensitive. IKH was insensitive to Penitrem A or glyburide in PVSMC and had a time to peak of 14.4 ± 3.3 ms and recovered in 67 ms following inactivation at +80 mV. Peak window current was −32 mV, suggesting that IKH may contribute to PVSMC RMP. The molecular identity of the potassium channel is not clear. However, RT-PCR, using porcine pulmonary artery and vein samples, identified Kv1.5, Kv2.1, and BK, with all three being more abundant in the PV. Both artery and vein expressed STREX, a highly conserved and hypoxia-sensitive BK channel variant. Taken together, our data support the hypothesis that hypoxic inhibition of IKH would contribute to hypoxic-induced contraction in PVSMC.

Perchlorate potentiation of excitation-contraction coupling in mammalian skeletal muscles

1993 ◽  
Vol 264 (3) ◽  
pp. C559-C567 ◽  
Author(s):  
E. M. Gallant ◽  
N. S. Taus ◽  
T. F. Fletcher ◽  
L. R. Lentz ◽  
C. F. Louis ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Membrane Vesicles ◽  
Voltage Sensor ◽  
Ca Channel ◽  
Release Channel ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Ec Coupling ◽  
Whole Cell Patch Clamp ◽  
Intact Muscle

The action of perchlorate (ClO4-), an agonist of the voltage sensor in excitation-contraction (EC) coupling, has been examined using bundles of intact muscle cells, isolated membrane vesicles [sarcoplasmic reticulum (SR) and transverse tubule (TT)], and cultured myotubes. The effect of ClO4- on mechanical parameters was investigated in isolated murine limb muscles. The presence of ClO4- (5 or 10 mM) greatly increased twitch tension ( > 250%), slightly enhanced tetanic tension, and increased K contracture tension. K contracture thresholds of extensor digitorum longus (EDL, 40 mM K+) and soleus (30 mM K+) muscles were not altered by ClO4-. However, in whole cell patch clamp studies of mouse myotubes, contractile activation was shifted by approximately -10 mV by 10 mM ClO4-. To further define the site of alteration of EC coupling by ClO4-, studies were conducted with isolated porcine SR and TT vesicles and with cultured mouse myotubes. The rate constant of Ca-induced 45Ca release from SR vesicles was significantly increased by ClO4-. However, neither the affinity nor level of [3H]PN200-110 binding to TT vesicles was significantly affected by ClO4- concentrations that increased twitch tension. Furthermore, slow plasmalemmal Ca currents of myotubes recorded in the whole cell patch-clamp mode were enhanced by 10 mM ClO4-, and the current-voltage relationship was shifted approximately -7mV. Thus, in enhancing EC coupling in mammalian muscle, ClO4- may act at multiple sites including the SR Ca release channel and the TT Ca channel-voltage sensor.

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