Potential Of Half-maximal Inactivation Research Articles

Inhibitory effects of antidepressant fluoxetine on cloned Kv2.1 potassium channel expressed in HEK293 cells

It is well demonstrated that antidepressant fluoxetine has significant inhibitory effects on voltage-gated potassium channels. So far, the concise regulation of fluoxetine on Kv2.1, the predominant delayed rectifier potassium channel subtype in the central nervous system, are rarely reported. Here patch-clamp recording was used to investigate the inhibitory effects of fluoxetine on Kv2.1 potassium channels stably expressed in HEK293 cells. The results showed fluoxetine dose-dependently suppressed Kv2.1 currents with an IC50 of 51.3 μM. At the test potential positive to +50 mV, fluoxetine 50 μM voltage-dependently suppressed Kv2.1 currents with an electrical distance δ of 0.28. Moreover, fluoxetine 50 μM did not affect the activation process of Kv2.1, but reduced the decay time constant τinact and obviously accelerated the inactivation process of Kv2.1 and left-shifted the half-maximal inactivation potential of Kv2.1 potassium channel by 9.8 mV. Fluoxetine 50 μM notably delayed the recovery process of Kv2.1 from inactivation with increased time constants. In addition, fluoxetine 50 μM use-dependently inhibited Kv2.1 currents at different frequencies. In conclusion, the inhibition of Kv2.1 by fluoxetine was concentration-dependent, voltage-dependent and use-dependent. The accelerated steady-state inactivation of Kv2.1 channels induced by fluoxetine might be ascribed to the delay of the recovery process of Kv2.1.

Cytoskeleton disruption affects Kv2.1 channel function and its modulation by PIP2.

Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.

Potassium Currents Activated by Depolarization in Odontoblasts.

Increased intracellular free Ca2+ concentrations elicit plasma membrane depolarization, which leads to the activation of K+ currents. However, the precise properties of K+ currents activated by depolarization in odontoblasts remain to be elucidated. The present study identified biophysical and pharmacological characteristics of time-dependent and voltage-activated K+ currents in freshly dissociated rat odontoblasts using patch-clamp recordings in a whole-cell configuration. Using a holding potential of −70 mV, outwardly rectifying time- and voltage-dependent currents were activated by depolarizing voltage. To record pure K+ conductance, we substituted Cl− in both the extracellular and intracellular solutions with gluconate−. Under these conditions, observation of K+ concentration changes in the extracellular solution showed that reversal potentials of tail currents shifted according to the K+ equilibrium potential. The activation kinetics of outward K+ currents were relatively slow and depended on the membrane potential. Kinetics of steady-state inactivation were fitted by a Boltzmann function. The half-maximal inactivation potential was −38 mV. Tetraethylammonium chloride, 4-aminopyridine, and α-dendrotoxin inhibited outward currents in odontoblasts in a concentration-dependent manner, suggesting that rat odontoblasts express the α-subunit of the time- and voltage-dependent K+ channel (Kv) subtypes Kv1.1, 1.2, and/or 1.6. We further examined the effects of Kv activity on mineralization by alizarin red and von Kossa staining. Continuous application of tetraethylammonium chloride to human odontoblasts grown in a mineralization medium over a 21-day period exhibited a dose-dependent decrease in mineralization efficiency compared to cells without tetraethylammonium chloride. This suggests that odontoblasts functionally express voltage-dependent K+ channels that play important roles in dentin formation.

Voltage-dependent Sodium Channels and Calcium-activated Potassium Channels in Human Odontoblasts In Vitro

IntroductionTransmembrane ionic signaling regulates many cellular processes in both physiological and pathologic settings. In this study, the biophysical properties of voltage-dependent Na+ channels in odontoblasts derived from human dental pulp (HOB cells) were investigated together with the effect of bradykinin on intracellular Ca2+ signaling and expression of Ca2+-activated K+ channels. MethodsIonic channel activity was characterized by using whole-cell patch-clamp recording and fura-2 fluorescence. ResultsMean resting membrane potential in the HOB cells was −38 mV. Depolarizing steps from a holding potential of −80 mV activated transient voltage-dependent inward currents with rapid activation/inactivation properties. At a holding potential of −50 mV, no inward current was recorded. Fast-activation kinetics exhibited dependence on membrane potential, whereas fast-inactivation kinetics did not. Steady-state inactivation was described by a Boltzmann function with a half-maximal inactivation potential of −70 mV, indicating that whereas the channels were completely inactivated at physiological resting membrane potential, they could be activated when the cells were hyperpolarized. Inward currents disappeared in Na+-free extracellular solution. Bradykinin activated intracellular Ca2+-releasing and influx pathways. When the HOB cells were clamped at a holding potential of −50 mV, outward currents were recorded at positive potentials, indicating sensitivity to inhibitors of intermediate-conductance Ca2+-activated K+ channels. ConclusionsHuman odontoblasts expressed voltage-dependent Na+ channels, bradykinin receptors, and Ca2+-activated K+ channels, which play an important role in driving cellular functions by channel-receptor signal interaction and membrane potential regulation.

Effects of thienopyridines and thienopyrimidinones on L-type calcium current in isolated cardiomyocytes

Thienopyridines (ticlopidine, clopidogrel) are frequently used drugs in antiplatelet therapy and have been shown to exert a more pronounced negative inotropic effect than thienopyrimidinones. We hypothesized that these differences are due to a differential impact of thienopyridines and thienopyrimidinones on L-type calcium current at the single-cell level. The effects of thienopyridines and thienopyrimidinones were studied on L-type calcium current and action potential parameters with the whole-cell patch-clamp technique in isolated myocytes from guinea pig ventricle and human atrial appendage. Ticlopidine showed the greatest impact on the L-type calcium current in guinea pig myocytes. It significantly reduced L-type calcium current density as well as shifted half maximal inactivation potential to more negative potentials compared to clopidogrel (at 30μmol/L) and to all thienopyrimidinones (30 and 100μmol/L). Clopidogrel significantly reduced the L-type calcium current density as well as shifted the half maximal inactivation potential to more negative potentials compared to all thienopyrimidinones at 100μmol/L only. In contrast, thienopyrimidinones did not affect L-type calcium current properties. The significant different effects of thienopyridines and thienopyrimidinones could also be demonstrated in human atrial myocytes. The more pronounced negative inotropic effect of thienopyridines is well explained by our results demonstrating a differential impairment of L-type calcium current by thienopyridines and thienopyrimidinones. L-type calcium current impairment by thienopyridines may be of special relevance for patients with cardiac diseases characterized by ionic remodelling.

K+ Currents Activated by Depolarization in Cardiac Fibroblasts

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).

Relation Between Bicarbonate Concentration and Voltage Dependence of Sodium Currents in Freshly Isolated CA1 Neurons of the Rat

It recently has been shown that whole cell calcium and sodium currents are modulated by CO(2)/HCO(3)(-)-buffered saline. While the bicarbonate ion, but not CO(2), has been proven to modulate calcium currents, this information is lacking for sodium currents. Furthermore, it is not known whether the strength of modulation dependents on the bicarbonate concentration or whether it is an all-or-nothing phenomenon. To answer these questions, we used the whole cell voltage-clamp technique on freshly isolated hippocampal CA1 neurons from the rat. A voltage step from -130 to -20 mV elicited a sodium current with an amplitude of -5.1 +/- 0.5 nA (mean +/- SE, n = 17) when cells were superfused with HEPES-buffered saline. The amplitude of this current increased during a subsequent superfusion with solutions containing increasing amounts of bicarbonate and CO(2) (%CO(2)/mM HCO(3)(-): 2.5/5.6; 5.0/18; 10/37), with a maximal increment in 10% CO(2)/37 mM HCO(3)(-) of -6.9 +/- 0.8 nA. The increase in amplitude was associated with a linear negative shift (slope: -0.7 mV/mM HCO(3)(-)) of the potential of half-maximal activation (DeltaV(h,a): -19.4 +/- 1.8 mV in 10% CO(2)) but not with an alteration in the maximal conductance (g(max): HEPES: 203.1 +/- 21.0 nS and 10% CO(2)/37 mM HCO(3)(-): 207.3 +/- 21.3 nS). In addition, the potential of half-maximal inactivation (V(h,i)) shifted to more negative potentials (slope: -0.6 mV/mM HCO(3)(-)) with increasing amounts of bicarbonate and CO(2) (HEPES: -53.6 +/- 11.8 mV; 10% CO(2)/37 mM HCO(3)(-): -69.8 +/- 2.1 mV), making the amplitude of the current highly sensitive for small potential changes at resting membrane potential. The same negative shift in voltage dependence arose when cells were exposed to solutions with different amounts of bicarbonate (5.6; 18; 26 mM) but constant CO(2) (5%) with slope rates of -0.5 mV/mM HCO(3)(-) for V(h,a) and -0.5 mV/mM HCO(3)(-) for V(h,i). Again, there was no correlation between bicarbonate concentration and the size of g(max). When currents were evoked in solutions containing a constant concentration (18 mM) of bicarbonate but different amounts of CO(2) (2.5; 5.0 10%), no significant changes have been observed. The present data demonstrate that bicarbonate ions, and not CO(2), modulate voltage-gated sodium currents in a concentration-dependent manner. Because the amplitude of the sodium current becomes highly sensitive to membrane potential changes concomitant with increased bicarbonate amounts, this may be critical for the excitability of the neuronal network in situations (like metabolic acidosis, respiratoric alkalosis and hypercapnia) in which the concentration of this ion can alter.

Calcium currents in acutely isolated stellate and pyramidal neurons of rat entorhinal cortex

Calcium currents were studied in morphologically identified pyramidal and stellate neurons acutely isolated from layer II/III of rat entorhinal cortex, using the whole-cell patch-clamp configuration. The peak amplitude of high-voltage activated current (HVA) measured at +10 mV was not different in both neuron populations with 0.94±0.08 nA for pyramidal and 1.03±0.08 nA for stellate cells. Stellate neurons had a larger capacitance (14.4±1.1 pF) than pyramidal neurons (9.6±0.8 pF), indicating a 50% larger cell surface. Most striking was the difference between the current density in stellate (79±8 pA/pF) versus pyramidal neurons (113±13 pA/pF). The potential of half maximal inactivation was not different: −37±2 mV (pyramidals) and −37±3 mV (stellates). Half of the cells contained a low-voltage activated calcium current (LVA) with a peak amplitude that was twice as large in stellate as in pyramidal neurons (0.21±0.04 nA resp. 0.11±0.03 nA; at −50 mV). In contrast to the HVA component, the current density of the LVA component was not different between cell types (13±3 pA/pF vs. 13±2 pA/pF). This implies that the relative abundance of LVA and HVA currents in stellate and pyramidal neurons is different which could result in different firing characteristics. The potential of half maximal LVA inactivation was −88±4 mV (pyramidals) and −85±3 mV (stellates). The slope of the voltage dependent steady state inactivation was steeper in stellate (7±1 mV) than in pyramidal cells (10±2 mV).

Sodium currents in isolated rat CA1 neurons after kindling epileptogenesis

Cellular excitability of CA1 neurons from a kindled focus in the rat hippocampus is persistently increased. The changes in the underlying voltage-dependent sodium current were characterized under whole-cell voltage-clamp conditions. We compared sodium currents in acutely isolated CA1 neurons from kindled rats with those in matched controls, one day and five weeks after cessation of kindling stimulations. The sodium current in CA1 neurons was tetrodotoxin sensitive and inactivated completely with two time-constants. In 97 cells from control rats, the current evoked at −20 mV consisted of a fast-inactivating component of 3.8±0.2 nA which decayed with a time-constant of 1.0±0.1 ms, and a slow-inactivating component of 1.2±0.1 nA with a time-constant of 3.6±0.1 ms. The potential of half-maximal inactivation was −72.2±1.0 mV for the fast-inactivating component and −63.2±1.0 mV for the slow-inactivating component. The time-constant of recovery at −80 mV was 14.1±0.4 ms for the fast-inactivating component and 9.3±0.4 ms for the slow-inactivating component. One day after kindling, the voltage dependence of inactivation of the slow-inactivating and the fast-inactivating component was shifted in the depolarizing direction (3.2±1.3 and 3.0±1.3 mV, respectively). The voltage dependence of recovery from inactivation was shifted in the same direction. Five weeks after kindling, the shift in voltage dependence of inactivation was (3.3±1.2 and 2.9±1.2 mV, respectively) and was accompanied by a 20% increase in sodium current amplitude. The voltage-dependent activation was not different after kindling. The changes in sodium current inactivation will increase the number of channels available for activation and may enhance the maximum firing rate. This implies that the changes in sodium current inactivation will contribute to the enhanced excitability of pyramidal neurons observed after kindling.

Effects of phenytoin, carbamazepine, and gabapentin on calcium channels in hippocampal granule cells from patients with temporal lobe epilepsy.

The anticonvulsants phenytoin (PHT), carbamazepine (CBZ), and gabapentin (GBP) are commonly used in the treatment of temporal lobe epilepsy. Ca2+ current modulation has been proposed to contribute to the antiepileptic activity of these drugs. The purpose of this study was to determine the effects of these anticonvulsants on voltage-dependent calcium channels in pathologically altered neurons from patients with chronic temporal lobe epilepsy. Acutely isolated human hippocampal granule cells were examined by using the whole-cell configuration of the patch-clamp technique. PHT and CBZ produced a reversible, concentration-dependent inhibition of high-voltage-activated (HVA) Ca2+ currents without affecting voltage-dependent activation. The concentration-response curves of PHT and CBZ indicated maximal inhibition of 35 and 65%, respectively, with half-maximal inhibition being obtained at 89 and 244 microM, respectively. At therapeutic cerebrospinal fluid (CSF) concentrations, HVA currents were not significantly altered by PHT and CBZ. However, PHT but not CBZ showed a reduction of HVA currents of 16% at a therapeutic whole-brain concentration of 80 microM. In contrast to CBZ, PHT produced a small hyperpolarizing shift in the voltage dependence of steady-state inactivation. PHT, 80 microM, shifted the potential of half-maximal inactivation by -3.1 +/- 0.5 mV (p < 0.05). GBP, which was recently found to bind to the alpha2delta subunit of a neuronal Ca2+ channel, showed no modulation of Ca2+ conductances. These results suggest that, in contrast to GBP and CBZ, modulation of postsynaptic Ca2+ channels can contribute to the anticonvulsant action of PHT in human hippocampal granule cells.

Inhibition of voltage-dependent Ca 2+ channels of porcine tracheal smooth muscle by the novel Ca 2+ channel antagonist RWJ-22108

1. 1. We compared electrophysiological effects of the bronchoselective Ca 2+ channel antagonist RWJ-22108 on voltage-dependent Ca 2+ channels (VDCs) of porcine tracheal smooth muscle cells to the effects of nicardipine and verapamil. 2. 2. Each of the three Ca 2+ channel antagonists tested inhibited inward Ca 2+ currents ( I Ca ) measured by whole-cell patch clamp techniques. Inhibition was dose-dependent with ∼50% inhibition of peak I Ca at +20 mV obtained with 3 × 10 −6M RWJ-22108, 3 × 10 −7M nicardipine, or 10 −5M verapamil. 3. 3. Both RWJ-22108 (3 × 10 −6M) and nicardipine (3 × 10 −7M) shifted the voltage dependence of steady-state inactivation to more negative potentials; however, the change in the potential of half-maximal inactivation induced by RWJ-22108 (−18 mV) was significantly greater than that induced by nicardipine (−12 mV). Verapamil did not alter the voltage dependence of inactivation. 4. 4. We conclude that inhibition of VDCs by RWJ-22108 is qualitatively similar to that by nicardipine but with a greater stabilizing effect on the inactivated channel state.

Multiple second messenger routes enhance two high-voltage-activated calcium currents in molluscan neuroendocrine cells.

Two types of high-voltage-activated calcium currents were identified in whole-cell voltage-clamp recordings of the neuroendocrine caudodorsal cells, which control egg-laying in the freshwater snail Lymnaea stagnalis. The currents were: (i) a rapidly inactivating high-voltage-activated current, with an activation threshold of -40 mV and maximal amplitude at +10 mV; and (ii) a slowly inactivating high-voltage-activated current, with a threshold of -10 mV and a peak at +30 mV. Both currents were reduced by nifedipine and verapamil, but not by omega-conotoxin GVIA, suggesting that they belong to the L-type family of calcium currents. The voltage-dependence of inactivation of the rapidly inactivating high-voltage-activated current was bell-shaped. Time-constants of inactivation ranged from 10 to 25 ms. Steady-state inactivation was characterized by a potential of half maximal inactivation of -21.7 +/- 3.4 mV and a slope factor of 8.1 +/- 1.7 mV. The voltage-dependence of inactivation of the slowly inactivating high-voltage-activated current was S-shaped. Time-constants of inactivation increased with depolarization up to a maximum of 300 ms. The steady-state inactivation parameters were a potential of half maximal inactivation of +6.8 +/- 2.2 mV and a slope factor of 6.0 +/- 1.1 mV. The membrane-permeable analog of cAMP, 8-chlorophenylthio-cyclic AMP, predominantly increased the slowly inactivating high-voltage-activated current, and shifted its voltage-dependence of activation and inactivation 10 mV to the left. The rapidly inactivating high-voltage-activated current was slightly increased by 8-chlorophenylthio-cyclic AMP. 8-Bromo-cyclic GMP and the phorbol ester, 12-O-tetradecanoyl-13-phorbol acetate, had qualitatively similar effects. Both agents enhanced the rapidly inactivating current and, to a lesser degree, the slowly inactivating current, without affecting their voltage-dependence. The cyclic AMP-dependent protein kinase inhibitor, Walsh inhibitor peptide, antagonized the stimulating effect of 8-chlorophenylthio-cyclic AMP. The broad-spectrum protein kinase inhibitor 1-(5-isoquino-linylsulfonyl)-2-methyl-piperazine (H-7) strongly attenuated the effects of 8-chlorophenylthio-cyclic AMP, 8-bromo-cyclic GMP and 12-O-tetradecanoyl-13-phorbol acetate, suggesting that all treatments increase both types of high-voltage-activated calcium currents through phosphorylation of the channel-complex.

Characteristics of Na+ current in Schwann cells cultured from frog sciatic nerve

Characteristics of voltage-dependent currents in cultured frog Schwann cells were investigated by the whole-cell clamp technique. An inward current was detectable at a membrane potential level more positive than -50 mV and reached a maximum value at about -10 mV, while no rectifying channel was present. The inward current was carried by Na+ ions, because the extrapolated reversal potential of the current agreed with the calculated ENa, and the current was sensitive to tetrodotoxin. The membrane potential for half-maximal inactivation was -82 mV. The inactivation curve indicated that more than 90% of the Na+ channels were inactivated at the resting membrane potential, suggesting that the cultured frog Schwann cells could not generate an action potential under physiological conditions. The time constant for the inactivation at a maximum current was 5.3 ms (-10 mV, 13 degrees C). The electrophysiological characteristics of the Na+ current in the cultured frog Schwann cells were compared with those in other tissues. This Na+ current was quantitatively different from that observed in the amphibian node of Ranvier but was similar to that in the mammalian Schwann or glial cells, especially in the more hyperpolarized half-maximal inactivation potential and in the slower inactivation time course.

Potential-dependent inward currents in single isolated smooth muscle cells of the rat ileum.

1. Calcium (ICa) and sodium (INa) currents were studied in single smooth muscle cells freshly isolated from both the newborn (1-3 days old) and adult rat ileum, using the patch-clamp technique (whole-cell configuration). 2. Under conditions when INa was blocked, two components of ICa, low-voltage activated or ICa,low and high-voltage activated or ICa,high, were observed in the newborn rat ileal cells. ICa,high and ICa,low have differing voltage ranges of activation and steady-state inactivation and time courses of recovery from inactivation. Potential dependence of ICa,low was much steeper and shifted toward negative membrane potential than that for ICa,high (slope factors and the potential of half-maximal inactivation were 13.6 and -60.6 and 8.8 and -49 mV for ICa,low and ICa,high, correspondingly). 3. Nifedipine at the high concentration of 30 microM exerted no effect on ICa,low and only slightly suppressed ICa,high, decreasing its peak to 0.81 +/- 0.04 (n = 7) at the holding potential of -80 mV and to 0.66 +/- 0.05 (n = 3) at -50 mV. ICa,high was suppressed significantly by Cd2+ ions, while ICa,low was more sensitive to Ni2+ ions. 4. Results presented here suggest that the properties of high-voltage-activated (HVA) Ca2+ channels in the rat small intestine are quite different to those described for L-type Ca2+ channels found in other smooth muscles. It is proposed that HVA Ca2+ channels are similar to N-type Ca2+ channels. 5. Comparison of Ca2+ currents in newborn and adult rat ileal cells showed that the contribution of ICa,low to the net Ca2+ current was negligible in adults, whereas the properties of HVA Ca2+ channels were similar in the neonatal and adult animals. 6. INa, studied in nominally Ca(2+)-free physiological salt solution, activated in the voltage range between -50 and -40 mV and reached its peak at -10 mV. INa was blocked in a dose-dependent manner by TTX with an apparent dissociation constant of 4.5 nM. 7. INa decay was monoexponential in the voltage range studied and its time constant decreased monotonically with membrane depolarization from 4.7 +/- 0.2 ms (n = 6) at -30 mV to 0.51 +/- 0.03 ms (n = 7) at 20 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Dopamine D2 receptor stimulation differentially affects voltage‐activated calcium channels in rat pituitary melanotropic cells.

1. Whole-cell voltage clamp recordings were made from 141 rat pituitary melanotropic cells in short-term, serum-free, primary culture. The effects of the dopamine D2 receptor agonist, LY 171555, on sodium, potassium and barium currents were investigated. 2. Application of 1 microM-LY 171555 did not affect the inward sodium and outward potassium currents. 3. Application of LY 171555 reversibly inhibited barium currents, with the strongest inhibition on the early inward current. The effect was dose dependent (IC50 = 4 x 10(-8) M), maximal inhibition of the total current was 30% and the LY 171555-induced block (1 microM) was reversibly antagonized by (+/-)sulpiride (4 microM). 4. Using barium-selective saline solutions, different types of barium current (T, N, and two L components) were identified on the basis of their voltage-dependent kinetics. Their relative amplitudes differed between cells. 5. The T-type current activated at potentials positive to -60 mV, reaching peak amplitude between -20 and -10 mV. At -30 mV, this current was inhibited up to 30% by 1 microM-LY 171555. The time constants of activation (10-3 ms) and inactivation (50-20 ms) as well as the voltage dependence of inactivation (potential of half-maximal inactivation (H), -61 mV; slope factor (S), 4.9 mV) were not affected by LY 171555 application. 6. A rapidly inactivating (time constants 100-50 ms), high threshold current component was identified as an N-type current. This current activated at command potentials positive to -30 mV and reached a maximal amplitude at +10 mV. The steady-state inactivation was described by a single Boltzmann equation with H = -65 mV and S = 11.7 mV. Application of 1 microM-LY 171555 completely suppressed this current. 7. The slowly inactivating (time constants > 1500 ms), high-threshold, L-type current displayed the same voltage dependence of activation as the N current. The voltage dependence of inactivation was modelled by the sum of two Boltzmann equations (L1: H1 = -45 mV, S1 = 13.0 mV; L2:H2 = -11 mV, S2 = 6.0 mV), indicating the existence of two L channel populations. Neither time course, nor voltage dependence of inactivation were influenced by LY 171555. However, LY 171555 induced a slow-down in the time course of activation, which necessitated the use of two time constants to model the activation kinetics. One of these (approximately 2 ms) was also observed under control conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Negative shift of cardiac Na+ channel kinetics in cell-attached patch recordings

Na+ channel kinetics were studied by recording single-channel currents in the cell-attached patch configuration of the patch-clamp technique in single ventricular cells isolated from guinea pig hearts. The inactivation time course of ensemble currents was accelerated, and the peak amplitude increased temporarily and then decreased within a few minutes after the gigaohm seal formation. After reaching a new steady state, the inactivation-voltage relation was found to have shifted to more negative potentials. The potential of half-maximal inactivation was more negative by 20-31 mV from the resting potential or between -96 and -112 mV. The voltage dependency of the channel activation also shifted. Although the cell membrane was depolarized using the whole cell patch-clamp electrode and single-channel currents were recorded with an independent cell-attached electrode, the shift of the inactivation curve was also evident. Complete removal of Ca2+ using 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution failed to prevent the shift. Increasing Ca2+ to 10 mM, however, reduced magnitude of the shift significantly. Involvement of an increased membrane fluidity and surface potential of the glass pipette to the shift is discussed.

Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig)

Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl2 and 5.4 mM KCl (35 degrees C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50 +/- 5 pF per cell, an input resistance of 200 +/- 25 kOhm X cm2 and a series resistance of 44 +/- 4 Ohm X cm2. The cells had resting potentials of -52 +/- 2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (-38 mV) with a maximal rate of 6.5 +/- 1.8 V/s to an overshoot of 22 +/- 3 mV. The AP lasted for 36 +/- 4 ms (measured between threshold and -40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (iCa, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward current iK. Ca-channel blockers (5 mM NiCl2) reduced iK suggesting that (part of) iK was Ca-activated. iK rose within about 100 ms to a peak of 40-200 muA/cm2 from which it inactivated slowly and incompletely. The inactivating iK followed a bell-shaped voltage-dependence, the noninactivating iK an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at -36 mV and a slope of 9 mV. Repolarization to -50 mV induced outward tail currents which reversed polarity at -85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion to iK during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse. iK was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)