Voltage-dependent sodium and potassium, but no calcium conductances in DDT1 MF-2 smooth muscle cells (original) (raw)
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Pfl�gers Archiv European Journal of Physiology, 1999
Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (V m) was-41±2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycolbis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) V m became significantly less negative:-13±2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of V m. Depolarizing voltage steps up to +60 mV from holding potentials of-60 mV resulted in large (1-10 nA) timeand voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above-20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca 2+-activated potassium channels (BK Ca), diminished the total current recorded at +60 mV by 45±14% (P<0.05, n=5) and 50±6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BK Ca channels present in HSV smooth muscle cells contribute to the maintenance of the V m and sustain a significant portion of the total voltage-activated, outward current. Finally, BK Ca channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity.
P2-purinoceptor-mediated membrane currents in DDT1 MF-2 smooth muscle cells
European Journal of Pharmacology, 1989
The electrophysiological response evoked by ATP was investigated in the DDT 1 MF-2 smooth muscle cell line using the microelectrode technique and the whole-cell patch clamp technique. Application of ATP (10 3 M) to the bathing solution caused a small initial depolarization of the cell membrane, followed by hyperpolarization and slow depolarization. During voltage clamping (-50 mV) a triphasic response was recorded on stimulation with ATP (10-4_10 3 M). A short-lasting inward current was followed by a transient outward current and a slowly decreasing inward current. This response was not affected by the receptor antagonists, propranolol (3 × 10-6 M), phentolamine (3 × 10-6 M), atropine (3 × 10-6 M) or theophylline (10-3 M). The ATP-induced currents were not modified by the voltage-dependent channel blocking agents, tetraethyl ammonium (3 x 10 3 M), 3,4-diaminopyridine (10-3 M), tetrodotoxin (3 × 10-7 M) or diltiazem (10 ~ M). The fast inward current was not detectable at a low ATP concentration (10-5 M). The outward current showed a reversal potential near-76 mV, which equals the potassium equilibrium potential. This current was abolished after neutralization of the potassium electrochemical gradient. The outward current was suppressed under calcium-free conditions and also in the presence of tolbutamide (10-4 M) or glipizide (5 x 10-6 M). Guanosine triphosphate (5 x 10-6 M) promoted the outward current, while this current was inhibited in the presence of guanosine diphosphate (5 × 10-6 M). Both inward currents persisted after reduction of the external sodium concentration or the internal chloride concentration and also in the absence of external magnesium or calcium. The fast inward current, however, declined after repeated stimulation of the cell with ATP under calcium-free conditions. The results indicate that, in DDT1 MF-2 smooth muscle cells, ATP activates purinoceptor-operated ion channels, represented by a fast calcium current followed by a transient potassium current, this effect being calciumand GTP-dependent, and a non-specific inward current.
British Journal of Pharmacology, 1996
1. The conventional whole-cell recording technique was used to study the effects of the chloride channel inhibitors ethacrynic acid, anthracene-9-carboxylic acid (A-9-C) and indanyloxyacetic acid (IAA) on membrane currents in rabbit portal vein smooth muscle cells at a holding potential of 0 mV. 2. Using a pipette solution that contained 1 x 10(-4) M 1,2-bis (2-aminophenoxy)-ethane-N,N,N,N,-tetraacetic acid (BAPTA) and a normal bathing solution the addition of ethacrynic acid (2 x 10(-4) M to 1 x 10(-3) M) inhibited spontaneous transient outward currents (STOCs) and evoked a concentration-dependent current at a holding potential of 0 mV. A similar current was activated by IAA (5 x 10(-4) M to 1 x 10(-3) M) but not by A-9-C (1-5 x 10(-3) M) at a holding potential of 0 mV. 3. The amplitude of the current evoked by ethacrynic acid and IAA was linearly related to potential between -30 and 0 mV and displayed outward rectification at positive potentials. The current induced by A-9-C was evident only at potentials positive to +20 mV. 4. Glibenclamide (1 x 10(-5) M) abolished the current evoked by ethacrynic acid and IAA at potentials negative to +10 mV and partially inhibited the current positive to +10 mV. The glibenclamide-insensitive current at positive potentials was completely inhibited by 1 x 10(-3) M TEA. The A-9-C-evoked current was insensitive to glibenclamide and abolished by 1 x 10(-3) M TEA. 5. The glibenclamide-sensitive current activated by ethacrynic acid was not sustained and declined to control levels in the continued presence of ethacrynic acid. However, the outwardly rectifying current recorded at +50 mV was well maintained over the same period. 6. Outwardly rectifying currents evoked by ethacrynic acid and A-9-C were observed with a pipette solution containing 1 x 10(-2) M BAPTA in cells bathed in Ca-free extracellular solution containing 5 x 10(-4) M BAPTA and 1 x 10(-5) M cyclopiazonic acid. 7. It is concluded that all three chloride-channel blockers activated an outwardly rectifying, TEA-sensitive current. Moreover, ethacrynic acid and IAA evoked an additional glibenclamide-sensitive current which was present at all potentials between -30 and +50 mV.
A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells
The Journal of physiology, 1991
1. Voltage- and time-dependent outward currents were recorded from relaxed enzymatically isolated smooth muscle cells from the rabbit left descending coronary artery using a single pipette voltage clamp technique. The calcium-activated potassium current was blocked by inclusion of EGTA in the pipette solution and CdCl2 in the extracellular bath. 2. Outward currents were elicited with depolarizing voltage steps to potentials positive to -20 mV. Long (5 s) voltage steps revealed slow inactivation of the current with a time constant of nearly 3 s at +60 mV. Potassium was identified as the predominant charge carrier by reversal potential measurements in potassium substitution experiments. 3. The results of kinetic analyses compared favourably with the Hodgkin-Huxley model for a delayed rectifier with some deviations. The sigmoid current onset was best fitted by raising the activation variable (n) to the second power. Deactivation tail currents were consistently found to be comprised of ...
Voltage-Dependent Calcium Channel in Intestinal and Vascular Smooth Muscle Cells
Annals of the New York Academy of Sciences, 1988
The calcium channels in a variety of cell types have been studied using single channel recording methods. In neurons, three types have been identified;' in cardiac muscle, two.' There is still some uncertainty, however, as to how these channel types contribute to the transient and more sustained components of the whole-cell current. There is evidence that the shape of the inward current in rat sensory neurons can be modified by guanosine triphosphate (GTP) analogues implying that G protein binds to the channels and alters their kinetics (see Reference 3). which may indicate that calcium channel types can be interconverted under certain conditions. In vascular smooth muscle, evidence for two channel types has been obtained." It is not certain as yet, however, that the additional component of more rapidly inactivating inward current that appears when holding a t-80 mV, rather than-30 mV, is contributed solely by the activity of a T-type channel and that the more slowly inactivating component of inward current is due only to the activity of high-threshold L-type channels as has been claimed or Stepping from negative (say-70 mV) holding potentials to positive potentials should activate current through both Tand L-type channels. If the L-type channel is distinctly more sensitive to dihydropyridine-type (DHP) calcium entry blocking drugs than are T channels as has been claimed'~~ (but see References 6 and 7) in all cells where an additional inward current component appears upon holding at-70 mV rather than-30 mV, an equal component of inward current resistant to DHP drug blockade should be found. What is more generally reported is the ability to identify separate cells, some with a DHP-drug-resistant transient inward current component and others with more sustained, DHP-sensitive, c~rrent.~.'~.'' Clear biphasic concentration-effect curves for the effect of DHP drug on inward current a t a test potential where both T and L channels are activated have not yet been described; the two phases would correspond to progressive effects first on the L and then on the T channels as the concentration of DHP drug was increased. Since both channels had been held at the same potential prior to stepping to the test potential, complications associated with increased voltage-dependent binding of DHP drug at more positive holding potentials would be avoided in the interpretation of such records. The inward current has been studied in single cells of rat u t e r u~, '~* '~ urinary bladder'4.15 and longitudinal muscle of guinea pig,'"'' and rabbit inte~tine.'~**~ Half activation of inward current has been reported to be-14 mV in bladder," about-12 mV in uterus," and-27 mV in guinea pig ileum using bariumI6 with half inactivation
Pfl�gers Archiv European Journal of Physiology, 1993
A slow outward current, activated during depolarization, which induced contraction in whole-cell patch-clamped rat skeletal muscle cells in primary culture [10], was extensively characterized in the present study. This current, Io, was simultaneously recorded with the contraction as a slow outward current during the test pulse, and a slow outward bell-shaped tail after repolarization. Io never appeared below the threshold potential for contraction, and the tail amplitude displayed a similar evolution with peak contraction amplitude as a function of membrane potential. This feature is consistent with the fact that Io was suppressed when contraction was blocked by 5 gM nifedipine [10], and it suggests :that Io was dependent on calcium released during contraction. This was confirmed by the fact that the presence of 10 mM EGTA in the patch pipette prevented the development of both contraction and Io, and that Io could be activated during caffeine-induced contractures without applying depolarizations. Io could be carried by K + or Cs + ions, but not by Na +. The pharmacology of Io was different from that of Ca2+-dependent BK and SK channels, since it was resistant to tetraethylammonium (135 mM), charybdotoxin (25 nM) and apamin (50 riM). Io was also insensitive to 4-aminopyridine (1 raM) but blocked by 5 mM Ba 2+ without change to contraction. It was concluded that rat cultured myoballs exhibit a Cs + permeation through an atypical K + channel type, which is activated by the calcium released during contraction.
Acta Physiologica Scandinavica, 2001
The aim of the present study was to characterize voltage-gated Ca 2+ currents in smooth muscle cells freshly isolated from rat tail main artery in the presence of 5 mmol L ±1 external Ca 2+ . Calcium currents were identi®ed on the basis of their voltage dependencies and sensitivity to nifedipine, Ni 2+ and cinnarizine. In the majority of the cells studied, T-and L-type currents were observed, while the remaining cells showed predominantly L-type currents. In the latter group of cells, holding potential change from ±50 to either ±70 or ±90 mV increased the corresponding inward current amplitude while its voltage activation threshold remained unchanged. The steady state inactivation of L-type Ca 2+ channels showed half-maximal inactivation at ±38 mV. A Ca 2+dependent inactivation was also evident. Nifedipine (3 lmol L ±1 ) blocked L-type but not T-type Ca 2+ currents. Ni 2+ (50 lmol L ±1 ) as well as cinnarizine (1 lmol L ±1 ) suppressed the nifedipineresistant, T-type component of the currents. At higher concentrations, both Ni 2+ (0.3±1 mmol L ±1 ) and cinnarizine (10 lmol L ±1 ) blocked the net inward current. Replacement of Ca 2+ with 10 mmol L ±1 Ba 2+ signi®cantly increased the amplitude of L-type Ca 2+ currents. These results demonstrate that smooth muscle cells freshly isolated from rat tail main artery may be divided into two populations, one expressing both L-and T-type and the other only L-type Ca 2+ channels. Furthermore, this report shows that in arterial smooth muscle cells cinnarizine potently inhibited Ttype currents at low concentrations (1 lmol L ±1 ) but also blocked L-type Ca 2+ currents at higher concentrations (10 lmol L ±1 ).
AJP: Cell Physiology, 2011
Large-conductance voltage-and Ca 2ϩ -activated K ϩ channels regulate human detrusor smooth muscle function. The large-conductance voltageand Ca 2ϩ -activated K ϩ (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel ␣-,  1-, and 4-subunits. Western blot and immunocytochemistry confirmed BK channel ␣,  1, and 4 protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patchclamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K ϩ current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin-and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans. urinary bladder; patch-clamp; calcium-dependent potassium channels
Biophysical Journal, 1998
The Ca 2ϩ sensitivity of large conductance Ca 2ϩ -and voltage-activated K ϩ channels (BK V,Ca ) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BK V,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BK V,Ca channel activity with changes in cytoplasmic intracellular [Ca 2ϩ ] ([Ca 2ϩ ] i ). The latter were induced by ionomycin, an electroneutral Ca 2ϩ ionophore. At 0 mV, the Hill coefficient (n H ) and the [Ca 2ϩ ] i to attain half of the maximal BK V,Ca channel activity (Ca 50 ) were 8 and 1 M, respectively. The data suggest that this large Hill number was not a consequence of the difference between the nearmembrane [Ca 2ϩ ] ([Ca 2ϩ ] s ) and the bulk [Ca 2ϩ ] i , indicated by fura-2. High Hill numbers in the activation by Ca 2ϩ of BK V,Ca channels have been seen by different groups (e.g., filled squares in of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. . Biophys. J. 70:2640 -2651. However, such high n H has always been considered a peculiarity rather than the rule. This work shows that a high Ca 2ϩ cooperativity is the normal situation for BK V,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca 50 did not display any significant variation among different channels or cells. It was also evident that BK V,Ca channel activity could decrease in elevated [Ca 2ϩ ] i , either partially or completely. This work implies that the complete activation of BK V,Ca channels occurs with a smaller increment in [Ca 2ϩ ] s than previously expected from in vitro characterization of the Ca 2ϩ sensitivity of these channels. Additionally, it appears that the activity of BK V,Ca channels in situ does not strictly follow changes in near-membrane [Ca 2ϩ ].
Journal of General Physiology, 1986
We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by rep...