Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle.

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as "store-operated" cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an L-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl- currents, we found that CPA rapidly activated a membrane conductance (I(CPA)) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of I(CPA) at physiological membrane potentials (i.e., -60 mV) that was accompanied by a shift in the reversal potential for I(CPA) toward more negative membrane potentials. In addition, I(CPA) was markedly inhibited by 10 microM Gd3+ and La3+ but was largely insensitive to 1 microM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.     

Modulation of cardiac Na(+) current by gadolinium, a blocker of stretch-induced arrhythmias

Gd(3+) blocks stretch-activated channels and suppresses stretch-induced arrhythmias. We used whole cell voltage clamp to examine whether effects on Na(+) channels might contribute to the antiarrhythmic efficacy of Gd(3+). Gd(3+) inhibited Na(+) current (I(Na)) in rabbit ventricle (IC(50) = 48 microM at -35 mV, holding potential -120 mV), and block increased at more negative test potentials. Gd(3+) made the threshold for I(Na) more positive and reduced the maximum conductance. Gd(3+) (50 microM) shifted the midpoints for activation and inactivation of I(Na) 7.9 and 5.7 mV positive but did not alter the slope factor for either relationship. Activation and inactivation kinetics were slowed in a manner that could not be explained solely by altered surface potential. Paradoxically, Gd(3+) increased I(Na) under certain conditions. With membrane potential held at -75 mV, Gd(3+) still shifted threshold for activation positive, but I(Na) increased positive to -40 mV, causing the current-voltage curves to cross over. When availability initially was low, increased availability induced by Gd(3+) dominated the response at test potentials positive to -40 mV. The results indicate that Gd(3+) has complex effects on cardiac Na(+) channels. Independent of holding potential, Gd(3+) is a potent I(Na) blocker near threshold potential, and inhibition of I(Na) by Gd(3+) is likely to contribute to suppression of stretch-induced arrhythmias.     

Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.     

Is there an A-type K+ current in guinea pig ventricular myocytes?

A unique transient outward K(+) current (I(to)) has been described to result from the removal of extracellular Ca(2+) from ventricular myocytes of the guinea pig (15). This study addressed the question of whether this current represented K(+)-selective I(to) or the efflux of K(+) via L-type Ca(2+) channels. This outward current was inhibited by Cd(2+), Ni(2+), Co(2+), and La(3+) as well as by nifedipine. All of these compounds were equally effective inhibitors of the L-type Ca(2+) current. The current was not inhibited by 4-aminopyridine. Apparent inhibition of the outward current by extracellular Ca(2+) was shown to result from the displacement of the reversal potential of cation flux through L-type Ca(2+) channels. The current was found not to be K(+) selective but also permeant to Cs(+). The voltage dependence of inactivation of the outward current was identical to that of the L-type Ca(2+) current. It is concluded that extracellular Ca(2+) does not mask an A-type K(+) current in guinea pig ventricular myocytes.     

Interaction of SK(Ca) channels and L-type Ca(2+) channels in catecholamine secretion in the rat adrenal gland

We elucidated the interaction of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channels and L-type Ca(2+) channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10-300 microM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SK(Ca) channel blocker apamin (1 microM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca(2+) channel blocker nifedipine (3 microM) or Ca(2+)-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca(2+) channel activator Bay k 8644 (1 microM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SK(Ca) channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca(2+) channels and thereby attenuates adrenal catecholamine secretion.     

Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics

Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q-type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.     

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.     

Calcium currents in hair cells isolated from semicircular canals of the frog

L-type and R-type Ca(2+) currents were detected in frog semicircular canal hair cells. The former was noninactivating and nifedipine-sensitive (5 microM); the latter, partially inactivated, was resistant to omega-conotoxin GVIA (5 microM), omega-conotoxin MVIIC (5 microM), and omega-agatoxin IVA (0.4 microM), but was sensitive to mibefradil (10 microM). Both currents were sensitive to Ni(2+) and Cd(2+) (>10 microM). In some cells the L-type current amplitude increased almost twofold upon repetitive stimulation, whereas the R-type current remained unaffected. Eventually, run-down occurred for both currents, but was prevented by the protease inhibitor calpastatin. The R-type current peak component ran down first, without changing its plateau, suggesting that two channel types generate the R-type current. This peak component appeared at -40 mV, reached a maximal value at -30 mV, and became undetectable for voltages > or =0 mV, suggestive of a novel transient current: its inactivation was indeed reversibly removed when Ba(2+) was the charge carrier. The L-type current and the R-type current plateau were appreciable at -60 mV and peaked at -20 mV: the former current did not reverse for voltages up to +60 mV, the latter reversed between +30 and +60 mV due to an outward Cs(+) current flowing through the same Ca(2+) channel. The physiological role of these currents on hair cell function is discussed.     

Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra

Rabbit urethral smooth muscle cells were studied at 37 degrees C by using the amphotericin B perforated-patch configuration of the patch-clamp technique, using Cs(+)-rich pipette solutions. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca(2+) currents, were recorded. Fitting steady-state inactivation curves for the L current with a Boltzmann equation yielded a V(1/2) of -41 +/- 3 mV. In contrast, the T current inactivated with a V(1/2) of -76 +/- 2 mV. The L currents were reduced by nifedipine (IC(50) = 225 +/- 84 nM), Ni(2+) (IC(50) = 324 +/- 74 microM), and mibefradil (IC(50) = 2.6 +/- 1.1 microM) but were enhanced when external Ca(2+) was substituted with Ba(2+). The T current was little affected by nifedipine at concentrations <300 nM but was increased in amplitude when external Ca(2+) was substituted with Ba(2+). Both Ni(2+) and mibefradil reduced the T current with an IC(50) = 7 +/- 1 microM and approximately 40 nM, respectively. Spontaneous electrical activity recorded with intracellular electrodes from strips of rabbit urethra consisted of complexes comprising a series of spikes superimposed on a slow spontaneous depolarization (SD). Inhibition of T current reduced the frequency of these SDs but had no effect on either the number of spikes per complex or the amplitude of the spikes. In contrast, application of nifedipine failed to significantly alter the frequency of the SD but reduced the number and amplitude of the spikes in each complex.     

The protein kinase inhibitor, staurosporine, inhibits L-type Ca2+ current in rabbit atrial myocytes

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.     

Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Agonist specific L-type Ca(2+)-current stimulation in ventricular myocytes by a novel steroid-like compound

In cardiac muscle the amplitude of Ca(2+) transients can be increased by enhancing Ca(2+) influx. Among the processes leading to increased Ca(2+) influx, agonists of the L-type Ca(2+)-channel can play an important role. Known pharmacological Ca(2+)-channel agonists act on different binding sites on the channel protein, which may lead not only to enhanced peak currents, but also to distinct changes in other biophysical characteristics of the current. In this study, membrane currents were recorded with the patch-clamp technique in the whole-cell configuration in guinea pig isolated ventricular myocytes in combination with confocal fluorescence Ca(2+) imaging techniques and a variety of pharmacological tools. Testing a new positive inotropic steroid-like compound, we found that it increased the L-type Ca(2+)-current by 2.5-fold by shifting the voltage-dependence of activation by 20.2 mV towards negative potentials. The dose-response relationship revealed two vastly different affinities (EC(50(high-affinity))=4.5+/-1.7 nM, EC(50(low-affinity))=8.0+/-1.1 microM) exhibiting differential pharmacological interactions with three classes of Ca(2+)-current antagonists, suggesting more than one binding site on the channel protein. Therefore, we identified and characterized a novel positive inotropic compound (F90927) as a member of a new class of Ca(2+)-channel agonists exhibiting unique features, which set it apart from other presently known L-type Ca(2+)-channel agonists.     

R-type calcium channels in myenteric neurons of guinea pig small intestine

Currents carried by L-, N-, and P/Q-type calcium channels do not account for the total calcium current in myenteric neurons. This study identified all calcium channels expressed by guinea pig small intestinal myenteric neurons maintained in primary culture. Calcium currents were recorded using whole cell techniques. Depolarizations (holding potential = -70 mV) elicited inward currents that were blocked by CdCl(2) (100 microM). Combined application of nifedipine (blocks L-type channels), Omega-conotoxin GVIA (blocks N-type channels), and Omega-agatoxin IVA (blocks P/Q-type channels) inhibited calcium currents by 56%. Subsequent addition of the R-type calcium channel antagonists, NiCl(2) (50 microM) or SNX-482 (0.1 microM), abolished the residual calcium current. NiCl(2) or SNX-482 alone inhibited calcium currents by 46%. The activation threshold for R-type calcium currents was -30 mV, the half-activation voltage was -5.2 +/- 5 mV, and the voltage sensitivity was 17 +/- 3 mV. R-type currents activated fully in 10 ms at 10 mV. R-type calcium currents inactivated in 1 s at 10 mV, and they inactivated (voltage sensitivity of 16 +/- 1 mV) with a half-inactivation voltage of -76 +/- 5 mV. These studies have accounted for all of the calcium channels in myenteric neurons. The data indicate that R-type calcium channels make the largest contribution to the total calcium current in myenteric neurons. The relatively positive half-activation voltage and rapid activation kinetics suggest that R-type channels could contribute to calcium entry during somal action potentials or during action potential-induced neurotransmitter release.     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

Extracellular Ba(2+) blocks the cardiac transient outward K(+) current

Ba(2+) is widely used as a tool in patch-clamp studies because of its ability to block a variety of K(+) channels and to pass Ca(2+) channels. Its potential ability to block the cardiac transient outward K(+) current (I(to)) has not been clearly documented. We performed whole cell patch-clamp studies in canine ventricular and atrial myocytes. Extracellular application of Ba(2+) produced potent inhibition of I(to) with an IC(50) of approximately 40 microM. The effects were voltage independent, and the inactivation kinetics were not altered by Ba(2+). The potency of Ba(2+) was approximately 10 times higher than that of 4-aminopyridine (a selective I(to) blocker with an IC(50) of 430 microM) under identical conditions. By comparison, Ba(2+) blockade of the inward rectifier K(+) current was voltage dependent; the IC(50) was approximately 20 times lower (2.5 microM) than that for I(to) when determined at -100 mV and was comparable to I(to) as determined at -60 mV (IC(50) = 26 microM). Ba(2+) concentrations of 相似文献   

Voltage-dependent modulation of L-type calcium currents by intracellular magnesium in rat ventricular myocytes

Effects of changing cytosolic free Mg(2+) concentration on L-type Ca(2+) (I(Ca)) and Ba(2+) currents (I(Ba)) were investigated in rat ventricular myocytes voltage-clamped with pipettes containing 0.2 or 1.8mM [Mg(2+)] ([Mg(2+)](p)) buffered with 30mM citrate and 10mM ATP. Increasing [Mg(2+)](p) from 0.2 to 1.8mM reduced current amplitude and accelerated its decay under a variety of experimental conditions. To investigate the mechanism for these effects, steady-state and instantaneous current-voltage relationships were studied with two-pulse and tail current (I(T)) protocols, respectively. Increasing [Mg(2+)](p) shifted the V(M) for half inactivation by -20mV but dramatically decreased I(Ca) amplitude at all potentials tested, consistent with a change in gating kinetics that decreases channel availability. This conclusion was supported by analysis of I(T) amplitude, but these latter experiments also suggested that, in the millimolar concentration range, [Mg(2+)](p) might also inhibit permeation through open Ca(2+) channels at positive V(M).     

Voltage-operated Ca(2+) channels involved in K(+)-evoked release of vasoactive intestinal polypeptide from the rat hypothalamus

Rat brain hypothalami were exposed to various depolarizing stimuli and vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) release was measured by means of a radioimmunoassay (RIA) procedure. Under conditions of noradrenergic blockade, exposure to high K(+) (40-100 mM) produced dose-dependent increases in the VIP-LI release in a Ca(2+)-dependent manner. Exposure to veratridine (3-100 microM) also induced concentration-dependent increases in VIP-LI release, an effect that was Ca(2+)-dependent and tetrodotoxin (TTX)-sensitive. Specific ligands for the L, N, and P/Q-type voltage-operated Ca(2+) channels (VOCCs) were used to determine which channel subtypes were involved in the K(+)-evoked VIP-LI release. The L-type VOCC ligand, nifedipine (10 microM), had no effect on release. In contrast, the N-type VOCC blocker, omega-conotoxin GVIA (omega-CgTx GVIA) (0.1-100 nM), markedly reduced the K(+)-evoked response, with maximal inhibition of approximately 60+/-8%. omega-Agatoxin IVA (omega-Aga IVA) (1-50 nM), which binds P-type and, at high doses, also Q-type VOCCs, produced dose-dependent inhibition of up to 25+/-3%, while the maximal inhibition observed with the non-selective VOCCs ligand, omega-conotoxin MVIIC (omega-CmTx MVIIC) (1 nM-3 microM), amounted to 85+/-8%. These findings indicate that N and P-type Ca(2+) channels play predominant roles in the high K(+)-evoked release of VIP-LI from the rat hypothalamus.