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Calcium-activated non-selective cation channel in ventricular cells isolated from adult guinea-pig hearts.

1. A class of Ca2+‐activated non‐selective cation channel was identified in ventricular cells, which were dissociated from adult guinea‐pig hearts using collagenase. 2. Under cell‐attached conditions the patch electrode filled with a Na+‐rich solution recorded no obvious single‐channel current at the resting membrane potential. Subsequent superfusion of the ventricular cell with a Na+‐free Tyrode solution induced an inward‐going single‐channel current as well as contracture of the cell. Kinetics of this channel were not affected by varying the membrane potential. 3. Single‐channel currents showing a conductance similar to those observed in the cell‐attached patches were recorded in isolated inside‐out membrane patches when the inner side of the membrane was exposed to a free Ca2+ concentration ([Ca2+]i) higher than 0.3 microM. The slope conductance of the channel was 14.8 +/‐ 2.9 pS (mean +/‐ S.D., n = 17) at 20‐25 degrees C. 4. The reversal potential examined in the inside‐out patch was about 0 mV irrespective of the Na+‐rich, K+‐rich, Li+‐rich or Cs+‐rich solutions on either side of the membrane, thereby indicating that the channel was almost equally permeable to these cations. 5. The open probability of the channel was increased by raising [Ca2+]i with the maximum value of 0.93 +/‐ 0.17 (n = 4) at about 10 microM [Ca2+]i. The dose‐response relation was fitted to the saturation kinetics with a Hill coefficient of 3.0 and a half‐maximum concentration of 1.2 microM [Ca2+]i. 6. The gating kinetics were complex; both the open and closed time histograms showed at least two exponential components with time constants of 3.8 +/‐ 1.3 ms and 140 +/‐ 110 ms for open time and 1.8 +/‐ 1.1 ms and 14.9 +/‐ 5.3 ms for closed time (n = 4) at 10 microM [Ca2+]i. Reduction of [Ca2+]i resulted in both a decrease of the time constant of the slow component in the open time histogram and an increase of the two time constants of the closed time histogram. 7. Contribution of the channel to the whole‐cell current was discussed based on an estimation of the channel density, presumably about 0.04 approximately 0.4/microns 2. Maximum activation of the channel would produce 7.2 approximately 72 nS of membrane conductance, which would explain the reported magnitude of the Ca2+‐mediated background conductance of the single myocyte. The channel may also contribute, at least in part, to the transient inward current which develops in Ca2+‐overloaded cardiac cells.

The Mg2+ block and intrinsic gating underlying inward rectification of the K+ current in guinea‐pig cardiac myocytes.

1. The blockade by Mg2+ and intrinsic gating of the channel, which underlie the rectification of the inward rectifier K+ current, was investigated using the oil‐gap voltage clamp method in isolated guinea‐pig ventricular cells. 2. The inward rectifier K+ current was isolated by subtracting trans‐gap currents recorded at an extracellular K+ concentration ([K+]o) of 0 mM from those obtained at 14 mM [K+]o in the presence of a given concentration of intracellular Mg2+ ([Mg2+]i). The reversal potential (V0) of the difference current was near the equilibrium potential for K+ (EK). 3. On repolarization across EK, the inward rectifier K+ current showed a rapid exponential increase. The time constant decreased with increasing hyperpolarization, but it was independent of both [Mg2+]i and the preceding depolarization. 4. When the pre‐pulse potential was made progressively positive between V0‐20 and V0 + 30 mV, the amplitude of the time‐dependent component became larger and the preceding current jump decreased at any [Mg2+]i. With pre‐pulses more positive than V0 + 40 mV, the time‐dependent component started from almost the zero current level at 2 microM [Mg2+]i. At higher [Mg2+]i (350, 500 and 3000 microM), however, the time‐dependent component became smaller as the pre‐pulse potential was made more positive than V0 + 40 mV. 5. When the membrane was depolarized from a potential of full activation at 2 microM [Mg2+]i, the initial jump in the outward current was ohmic and was followed by an exponential decay. The time‐dependent component of the inward current, recorded on repolarization after increasing durations of the preceding depolarization, developed as the outward current decayed. The time constants of both processes were in good agreement. 6. At 500 microM [Mg2+]i, the outward current on depolarization was instantaneously rectified. The time‐dependent component recorded on repolarization developed with prolongation of the pre‐pulse with a time course slower than at 2 microM [Mg2+]i. The envelope time course became slower as the potential of the depolarization became more positive. 7. Lowering the temperature from 23 to 15 degrees C slowed the time‐dependent current with an apparent Q10 of about 3.5 at V0. 8. Based on the experimental data, kinetic parameters were estimated for a model of Mg2+ block, which well simulated the inward‐going rectification of the K+ current. 9. It is concluded that the instantaneous inward rectification on depolarization is due to the Mg2+ block at physiological [Mg2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

The ATP-sensitive K+ channel

A small conductance K+ channel, that is inactivated by ATP, was recently found in the inner membrane of rat liver mitochondria (Inoue et al., 1991). This finding clearly indicates that a variety of K+ channels, showing ATP-sensitivity, are widely distributed. ATP is an important compound in view of its participation in oxidative phosphorylation and as the source of high-energy phosphate for nearly every energy-requiring reaction in the cell. Therefore, it is easy to speculate that transducing the ATP concentration within a cell into an electrical signal is vital for most living cells. The opening of the ATP-sensitive K+ channel by a decrease in the ATP level shifts the membrane potential in a negative direction and in general depresses cell function. The closing of the channel by an increase in ATP depolarizes the membrane and enhances membrane excitability. It might be speculated that a sequence of amino acids common for the binding site of ATP is preserved and combined with different types of K+ channels, so that the gating with ATP is quite similar between different K+ channels, but the conductance properties are different. The large variability in the value of K1/2ATP in the same cells or between different tissues might be due to modulation of the reaction of ATP and the binding site. These ideas will be substantiated by clarifying the molecular structure of the ATP-sensitive K+ channel in the near future. The molecular mechanisms for the selective channel blockers, sulfonylureas, and for the K+ channel openers should also be clarified.

Distribution of the isoprenaline-induced chloride current in rabbit heart

Current density of the isoprenaline-induced chloride current (ICl) was measured in sino-atrial (SA) node cells and atrial and ventricular myocytes dissected enzymatically from the rabbit heart. In addition to the conventional voltage clamp method the whole-cell patch clamp method using nystatin was employed to avoid run-down of ICl in dialysed cells. Isoprenaline (0.3 μM) failed to induce ICl in the 20 atrial cells examined. The integrity of the β-adrenergic system was established by recording the response of the Ca2+ current in the same cell. Both isoprenaline and acetylcholine failed to affect the background membrane conductance in the 20 SA node cells studied. Myocytes isolated from the epicardial region of the left ventricular wall showed relatively higher ICl density (24.9±12.1 μS/μF) than those from the endocardial side (12.3±8.5 μS/μF). We conclude that β-receptor-operated ICl is insignificant in atrial and SA node cells.

Triple‐barrel structure of inwardly rectifying K+ channels revealed by Cs+ and Rb+ block in guinea‐pig heart cells.

1. The hypothesis that the inwardly rectifying K+ channel consists of a triple‐barrel structure was investigated. Inward currents were recorded under the blocking effects of external Cs+ or Rb+ in the cell‐attached configuration of the patch‐clamp technique using single ventricular cells enzymatically isolated from guinea‐pig hearts. 2. Cs+ (10‐100 microM) or Rb+ (20‐100 microM) added to the 150 mM‐K+ pipette solution induced rapid open‐blocked transitions in the inward open‐channel currents. In about 20% of experiments the inward current showed two intermediate current levels equally spaced between the unit amplitude and the zero‐conductance level. The current fluctuated between these four levels. In the remaining experiments no obvious sublevels were observed except spontaneous ones, whose amplitudes were not always equal to one‐third or two‐thirds of the unit amplitude. 3. In experiments showing sublevels, the probability that the open‐channel current stayed at each level was measured at various concentrations of blockers and membrane potentials. In both Cs+ and Rb+ block, the distribution of the current levels showed reasonable agreement with the binomial theorem. This finding suggests that the inwardly rectifying K+ channel is composed of three equally conductive subunits and each subunit is independently blocked by Cs+ or Rb+. 4. The dwell‐time histogram in each substate was well fitted with a single‐exponential function. On the assumption of the binomial model, the blocking (mu) and unblocking (lambda) rate for Cs+ and Rb+ were calculated. The value of mu was linearly proportional to the concentration of the blocking ion at a given membrane potential and increased with hyperpolarization (e‐fold increase with a change of ‐43.5 mV in the Cs+ block). lambda was almost independent of the concentration of the blocking ion and less dependent on the membrane potential than mu. 5. The open and blocked times were calculated in experiments showing no clear sublevels. The mean open time was almost equal to the mean dwell time at the full open level in experiments showing sublevels under the same conditions. On the other hand, the mean blocked time was about two or three times longer than the mean dwell time at the zero‐conductance level measured in experiments with sublevels. These results may suggest that the instant one of the three subunits is plugged by blocking ions, the remaining two subunits are closed by unknown mechanisms. 6. Our results support the hypothesis that the cardiac inwardly rectifying K+ channel is composed of three equally conductive subunits.

External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells.

We have observed a novel class of calcium-activated potassium channel which is activated by physiological levels of intracellular ATP. These KCa,ATP channels are found on smooth muscle cells isolated from the pulmonary artery. Since their activation by ATP is Mg2+ dependent and is poorly evoked by nonor slowly-hydrolyzed ATP analogues, we conclude that it involves phosphorylation. We suggest that in hypoxia a reduction of intracellular ATP may reduce KCa,ATP channel activity and thereby tend to depolarize the cells. This effect would increase Ca2+ entry through voltage-activated Ca2+ channels and contribute to vasoconstriction.

Beta‐adrenergic and muscarinic regulation of the chloride current in guinea‐pig ventricular cells.

1. Single guinea‐pig ventricular cells were voltage clamped using the patch clamp method combined with the pipette‐perfusion technique. The voltage‐dependent current systems were mostly blocked, and the background membrane conductance was measured by applying ramp pulses. 2. beta‐Adrenergic effectors and related substances such as adrenaline, isoprenaline, forskolin or internal application of cyclic AMP induced a current component which showed a reversal potential near the expected Cl‐ equilibrium potential as well as an outward rectification in the I‐V relation. It is suggested that the activation of this Cl‐ current was due to phosphorylation of the channel protein or related structure by the cyclic AMP‐dependent protein kinase. Coincidentally with the activation of the Cl‐ current, the membrane capacitance of the cell decreased reversibly. 3. Acetylcholine (ACh) depressed the responses induced by beta‐adrenergic stimulation and forskolin, but failed to interfere with the one induced by cyclic AMP. 4. The dose dependence of the Cl‐ current activation by isoprenaline or forskolin was fitted by the Hill equation, with a coefficient of 1.9 and a half‐maximum concentration K 1/2 = 13 nM for isoprenaline, and with a Hill coefficient of 3 and a K 1/2 = 1.2 microM for forskolin. In the presence of 5.5 microM‐ACh the dose‐response relation shifted to higher doses; K 1/2 was 65 nM for isoprenaline and 3.6 microM for forskolin. 5. Washing out ACh in the presence of isoprenaline frequently caused transient overshoots of the response. When a saturating concentration of isoprenaline was used, this rebound was not observed. 6. The internal application of cyclic GMP enhanced the response of the Cl‐ current induced by isoprenaline or adrenaline. 7. When cyclic AMP was applied internally, the response was small in most cells. When the cell was superfused with 20 microM‐IBMX (3‐isobutyl‐1‐methylxanthine), the Cl‐ current was consistently induced by the application of cyclic AMP. It is suggested that phosphodiesterase activity strongly buffered the influx of cyclic AMP through the patch pipette tip. 8. We suggest that the compensatory interaction between the beta‐adrenergic stimulation and the muscarinic inhibition is at the membrane level, most probably via GTP‐binding proteins in activating adenylate cyclase.

Conductance properties of the Na(+)‐activated K+ channel in guinea‐pig ventricular cells.

1. The Na(+)‐activated K+ channel current was recorded from inside‐out membrane patches excised from single ventricular cells of guinea‐pig hearts. 2. The single channel current‐voltage relations showed inward‐going rectification with an asymptotic conductance of 180‐210 pS for the inward current at 150 mM [K+]o, when [K+]i was changed between 5.4 and 150 mM. The reversal potential indicated the PNa/PK of about 0.02. 3. The amplitude of outward current was reduced by increasing [Mg2+]i or [Na+]i, but no obvious blocking noise was recorded. The outward current, which remained shortly after quick removal of both [Na+]i and [Mg2+]i, revealed an ohmic conductance of the K+ channel. 4. The [Mg2+]i and [Na+]i block was increased e‐fold by depolarizing the membrane by 49 mV, while the inward current was not blocked. 5. The Na(+)‐activated K+ channel showed frequent subconductance levels. The variance‐mean analysis resolved at least ten major sublevels. The density distribution of the sublevels were measured by composing the conventional amplitude histogram, excluding clear closed state currents, and then dividing the histogram into five segments. The probability of staying in each segment (Pn) was almost always voltage independent, and the grand averages were P1 = 9.5 +/‐ 5.9%, P2 = 6.3 +/‐ 2.1%, P3 = 4.2 +/‐ 1.8%, P4 = 7.8 +/‐ 2.5%, and P5 = 39.3 +/‐ 5.6%, from the lowest segment, respectively. 6. The values of Pn in partially blocked conditions by Na+ and Mg2+ (outward current) were not clearly different from those without any channel block (inward current). The values of Pn, measured before and after applying Ba2+ in the pipette, were also very similar. 7. The above findings indicate that the inward‐going rectification of the Na(+)‐activated K+ channel is due to the Na+ and Mg2+ block. The subconductance of the channel is not due to any channel block by Na+ or Mg2+, but may be attributable to multiple open states of a single‐barrel channel, which has a large conductance. The channel may be blocked from any open conformation with an equal probability and with very fast kinetics.

Control of the hyperpolarization‐activated cation current by external anions in rabbit sino‐atrial node cells.

1. Effects of varying concentrations of anions on the hyperpolarization‐activated current (I(f)) were studied in myocytes isolated from the rabbit sino‐atrial node. Substituting Cs+ for the intracellular K+ clearly separated I(f) from the delayed rectifier K+ current. Control properties, including gating kinetics and ion selectivity, similar to previous studies were obtained. 2. Substitution of extracellular Cl‐ with larger anions including isethionate, glutamate, acetate, and aspartate, reduced the amplitude of I(f) without changing the reversal potential. Substitution with small anions such as iodide or nitrate supported an intact I(f). These effects were reproduced in the excised outside‐out patch conformation. 3. The conductance for I(f) was a saturating function of the extracellular Cl‐ concentration ([Cl‐]o) with an equilibrium binding constant (K1/2) of 11 mM and a slope factor of about 1 when substituted with large anions. Total removal of small anions completely abolished I(f). 4. The voltage‐dependent gating of I(f) was not affected by changing ([Cl‐]o), suggesting that Cl‐ modulates conductance properties of I(f). 5. The results indicate that I(f) conductance is unique in that it is dependent on an extracellular anion (Cl‐), yet it is carried exclusively by cations, K+ and Na+. These effects are independent of any measurable voltage‐dependent gating parameters.

Cation-dependent gating of the hyperpolarization-activated cation current in the rabbit sino-atrial node cells.

1. The gating properties of the hyperpolarization‐activated cation current (I(f) or Ih) were investigated in single pacemaker cells dissociated from the rabbit sino‐atrial node. 2. The whole‐cell I(f) was recorded in the presence of different external cations. The inward I(f) was increased when external Na+ was replaced with K+, and was decreased in Li+ or Rb+ solution. In Tris+ and Cs+ solutions, the inward I(f) was negligible. The outward tail current recorded upon depolarization was largest in Li+ solution and smaller in a sequence of Na+, Tris+ and K+ solutions. In Rb+ and Cs+ solutions, only a small tail current was recorded. 3. The outward tail current had a ‘shoulder’ in Na+ solution, which was much delayed by replacing Na+ with Li+. In K+ solution, the decay of the tail current was much faster, and no obvious shoulder was recorded. The tail current was slowest in Li(+)‐rich and 0 mM K+ solution, and was progressively accelerated by adding K+ over the range from 0 to 3 mM. The tail current at 30 mM [K+]o showed only a small shoulder. A common binding site to modulate the I(f) deactivation was suggested for monovalent cations. 4. The shoulder of the I(f) tail became more evident as I(f) was activated to a larger extent either by prolonging the duration or by increasing the amplitude of the preceding hyperpolarization in both Na+ and Li+ solutions. 5. The I(f) was first activated by hyperpolarizing the membrane to ‐110 mV, and then deactivated by depolarization. The inward tail current at ‐50 mV showed a single exponential decay. At more positive potentials, the shoulder of the outward tail currents became more evident and the rate of the final decay was increased. 6. The time course of I(f) activation was well fitted with the sum of two exponential functions. Time constants of both components were not affected by the external cation (Na+, K+ or Li+) replacement. Likewise, the quasi‐steady state activation was conserved when external Na+ was replaced with Li+. 7. Two closed and three open states were assumed in a sequential state model of the I(f) channel. The cation effects were well simulated by assuming that the deactivation rate was selectively modulated. The flow of I(f) during the spontaneous action potential was calculated. The activation of I(f) started on repolarization to the maximum diastolic potential and reached a maximum in the middle of the diastolic period. Its peak amplitude was 14% of the net inward current during the diastolic period.