Kyoichi Ono

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.

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.

Run‐down of the GABAA response under experimental ischaemia in acutely dissociated CA1 pyramidal neurones of the rat.

1. The effect of experimental ischaemia on the response to gamma‐aminobutyric acid (GABA) was assessed in acutely dissociated CA1 pyramidal neurones of rats, using the patch‐clamp technique. 2. Rapid application of 3 x 10(‐5) M GABA induced a bicuculline‐sensitive inward Cl‐ current (IGABA) at a holding potential (Vh) of ‐44 mV. The peak amplitude of IGABA showed a time‐dependent decrease (run‐down) when it was recorded with the conventional whole‐cell mode without internal ATP. The run‐down was not observed when the intracellular ATP concentration ([ATP]i) was maintained by the nystatin‐perforated recording with an intracellular Na+ concentration ([Na+]i) of 0 mM. 3. When [Na+]i was increased to more than 30 mM, the IGABA run‐down was observed even with the nystatin‐perforated recording. 4. The IGABA run‐down observed at 60 mM [Na+]i with the nystatin method was further enhanced under experimental ischaemia without changes in the reversal potential of IGABA. The enhanced run‐down was suppressed by application of the Na+,K(+)‐ATPase inhibitors, ouabain and SPAI‐1. 5. IGABA run‐down during ischaemia was also accompanied by an outward holding current and a concomitant increase in intracellular free Ca2+ concentration ([Ca2+]i) in 48.5% of the neurones. The outward current was a Ca(2+)‐activated K+ current, which was blocked by 3 x 10(‐7) M charybdotoxin. 6. In the inside‐out mode of the single‐channel analysis, GABA activated three subconductance states with conductances of 33.4, 22.7 and 15.2 pS. Reduction of ATP concentration from 2 to 0 mM on the intracellular side suppressed the channel activities, while an increase in Ca2+ concentration from 0.7 x 10(‐9) to 1.1 x 10(‐6) M had no effect. 7. These results suggest that ischaemia induces the run‐down of the postsynaptic GABA response at the GABAA receptor level, and that this run‐down is triggered by a decrease in [ATP]i.

Delayed activation of large-conductance Ca2+-activated K channels in hippocampal neurons of the rat.

We applied a fast concentration jump system to produce step changes in Ca2+ concentration [( Ca2+]i) on the cytoplasmic side of the inside-out membrane patch, excised from isolated rat hippocampal pyramidal neurons, and examined the time course of the activation phase of the large-conductance K channel (the BK channel; approximately 266 pS) after a step rise in [Ca2+]i. Diffusion of Ca2+ from the electrode tip to the cytoplasmic surface of the patch was estimated to be almost completed in 10 ms. After a step increase in [Ca2+]i from 0.04 to 3.2-1,000 microM, the activation of the K channel started after a clear latency of 280-18 ms and proceeded along a sigmoidal function. This was in sharp contrast with the rapid deactivation that began without delay and that was completed within 50 ms. The latency in activation was not accounted for by the binding of Ca2+ to EGTA in unstirred layers in the patch, since this binding was reported to be slow, taking up to seconds at physiological pH. Calmodulin (1 microM) did not affect the delay, the activation rate, or the steady-state current level. The calmodulin inhibitors W-7 and W-5 caused flickering of the single-channel current. These results indicate a delayed activation of the BK channel after a step rise in [Ca2+]i, suggesting that the BK current does not contribute to the repolarization of the action potential. Calmodulin is probably not involved in the activation process of the channel.

Background conductance attributable to spontaneous opening of muscarinic K+ channels in rabbit sino‐atrial node cells.

Single myocytes were dissociated from the rabbit sino‐atrial node, and the membrane background conductance produced by spontaneous opening of the muscarinic K+ channels was investigated by recording whole‐cell and single channel currents in both normal K+ (5.4 mM) and high‐K+ (145 mM) external solutions. Increasing external K+ concentration ([K+]o) from 5.4 to 145 mM induced a large inward shift of the whole‐cell current accompanied by considerable current fluctuations at ‐50 mV. The high‐K(+)‐induced current was both K+ selective and voltage dependent, which was examined by varying [K+]o. This current was almost completely suppressed by 1‐5 mM Ba2+ or 2‐10 mM Cs+ and it was partly blocked by 10 microM atropine. In high‐K+ (145 mM) solution, 20 nM acetylcholine (ACh) further increased the K+ conductance as well as the current noise. The power density spectrum of the noise was fitted with a sum of two Lorentzian functions. The corner frequencies of both the slow (approximately 5 Hz) and fast (approximately 120 Hz) components were comparable between the noise before and during the ACh application. Internal dialysis with a non‐hydrolysable derivative of ATP, 5‐adenylylimido‐diphosphate (AMP‐PNP) or Mg(2+)‐free solution markedly decreased both the amplitude and fluctuations of the high‐K(+)‐induced current. The relation between the variance of the current fluctuations and the mean current amplitude was linear in every experiment using dialysis of AMP‐PNP or Mg(2+)‐free internal solution, or using superfusion of ACh. The slopes of these relations gave comparable single channel current amplitudes of ‐0.7 pA at ‐50 mV. These results indicate that the spontaneous opening of the muscarinic K+ channels is largely responsible for the high‐K(+)‐induced current. In the high‐K+ solution, the variance‐mean relation at ‐50 mV showed that the muscarinic K+ channel provides an inward current of 3.12 +/‐ 2.13 pA pF‐1 (n = 23), which was about 60% of the total inward background current. In the normal K+ solution, the variance‐mean relation at ‐50 mV indicated that an outward current of 6.0 +/‐ 2.0 pA (0.33 +/‐ 0.28 pA pF‐1, n = 8) was provided by the K+ channel. The single channel current amplitude was estimated to be 0.06 +/‐ 0.02 pA (n = 9). Cell‐attached recordings in the absence of ACh demonstrated sporadic and brief openings of channels identical to the ACh‐induced channels. The power density spectra of the single channel currents exhibited kinetic properties comparable with those of the whole‐cell currents.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Synergistic action of cyclic GMP on catecholamine-induced chloride current in guinea-pig ventricular cells.

1. Effects of cyclic GMP on the catecholamine‐induced chloride current (ICl) were studied using the whole‐cell patch‐clamp technique combined with internal perfusion in single ventricular myocytes dispersed from guinea‐pig heart. 2. When ICl was activated by submaximal doses of isoprenaline (0.01‐0.1 microM), adrenaline (0.5‐1 microM) and histamine (0.2‐0.5 microM), intracellular dialysis with cyclic GMP (10‐100 microM) induced an extra increase of ICl. No further increase of ICl was induced by cyclic GMP when ICl was maximally activated. In the absence of agonists, cyclic GMP failed to induce ICl. 3. The enhancement by cyclic GMP was also observed when ICl was activated by external application of 0.2‐1.0 microM‐forskolin or by internal dialysis with a pipette solution containing 50‐200 microM‐cyclic AMP. 4. In contrast to cyclic GMP, 10‐1000 microM‐dibutyryl cyclic GMP and 8‐bromo‐cyclic GMP were ineffective in modifying ICl. 5. Milrinone (1‐10 microM), a specific inhibitor of a kind of phosphodiesterase which is inhibited by cyclic GMP, also enhanced ICl activated by submaximal doses of isoprenaline. Milrinone itself did not activate ICl. 6. When ICl was enhanced by 5 microM‐milrinone, an additional application of cyclic GMP failed to increase ICl. In the presence of cyclic GMP, milrinone failed to enhance ICl. 7. The above findings on ICl are analogous to the enhancement by cyclic GMP of the beta‐adrenergic stimulation of the Ca2+ current reported in the same preparation, and support the hypothesis that in mammalian cardiac cells cyclic GMP potentiates elevation of cyclic AMP induced by beta‐adrenergic agents, and thereby increases the amplitudes of ionic currents.

Modulation of beta‐adrenergic responses of chloride and calcium currents by external cations in guinea‐pig ventricular cells.

1. The catecholamine‐induced Cl‐ current and the Ca2+ current were recorded in the single ventricular cells of guinea‐pig hearts, using the whole‐cell patch clamp technique combined with internal perfusion. Dependence of the beta‐adrenergic responses on external monovalent cations was investigated. The Cl‐ current was recognized by measuring the reversal potential of the agonist‐induced current. 2. The amplitude of the Cl‐ current, activated by 1 microM adrenaline or 0.01‐0.1 microM isoprenaline, was decreased when the external Na+ concentration ([Na+]o) was reduced by replacement with Tris+. The conductance of the catecholamine‐induced Cl‐ current was proportional to the logarithm of the [Na+]o over a range of 15‐140 mM. When the conductance was plotted against the concentration of Tris+, a dose‐dependent inhibition of the Cl‐ response by Tris+ was suggested with a half‐maximum concentration of 95 mM. 3. The inhibitory effect of the Na+ substitute TEA+ on the Cl‐ current was not affected by either increasing the buffer for the internal Ca2+ (10 mM BAPTA) or for the pH (50 mM HEPES). 4. In the relationship between agonist concentration and the Cl‐ conductance, the half‐maximum concentration (K1/2) of isoprenaline was 0.013 microM in the control Na+ solution, and was shifted to 0.07, 0.08, 0.1 and 0.3 microM in the Li+, Cs+, TEA+ and Tris+ external solutions, respectively. The maximum slope conductance was not significantly affected, except for a slight depression on the Tris+ solution. When the current was induced by adrenaline, qualitatively the same finding was obtained; K1/2 was 0.15 and 3.2 microM in the Na+ and Tris+ solutions, respectively. 5. As a substitute for the external Na+, sucrose seemed to be inert. The activation of the inward Cl‐ current was conserved in the 300 mM sucrose solution ([Cl‐]o = 8 mM) with a K1/2 value of 0.015 microM isoprenaline. 6. The Cl‐ current, when activated by either an external application of forskolin (0.2‐10 microM) or an internal perfusion of cyclic AMP (100‐500 microM), was not affected by replacing external Na+ with other cations. Activation of the Cl‐ current by 0.2‐5 microM histamine was also insensitive to a substitution of Na+. These findings indicate that the inhibition by the Na+ substitute is at a point before the activation of GTP‐binding protein. 7. The effects of Na+ substitution were not affected by varying the Na+ concentration (0‐115 mM) in the internal solution, excluding an involvement of a change in the [Na+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage- and time-dependent block of If by Sr2+ in rabbit sino-atrial node cells

The hyperpolarization-activated cation current (If) was recorded in single myocytes dissociated from the rabbit sino-atrial node and the Sr2+-mediated block of If examined. In the presence of 0.1–10 mM Sr2+, the activation phase of If was followed by a slower decay during hyperpolarization. In the steady state I/V diagram, the Sr2+ block progressed with increasing hyperpolarization. Ba2+ also blocked If, but no time dependency could be observed. The blocking effect of Ca2+ was weak, and Mg2+ had little effect on If. The relationship between extracellular Sr2+ concentration [Sr2+]o and the block was described by a Hill coefficient of 1. The half-saturating [Sr2+]o were 2.0, 1.6, 1.1 and 0.65 mM at −90, −100, −110 and −120 mV, respectively. The rapid application of Sr2+ during the full activation of If using the jet-stream method induced an exponential decline of If. The reciprocal time constants were linearly correlated to [Sr2+]o, suggesting 1∶1 binding stoichiometry. The fractional electrical distance for the binding site was approximately 0.5 from the external side of the channel. Based on the multiple closed and open states for the If channel, a mathematical model for the Sr2+ block was constructed in which the time course of If in the presence of Sr2+ was described by the sum of three exponential functions. Fitting the model to the original traces revealed blocking and unblocking rates similar to those obtained from the jet-stream method. At −110 mV, the blocking rate was 410 M−1s−1 while the unblocking rate was 0.16 s−1. These values are the smallest so far reported for time- and voltage-dependent blockade of ionic channels.

Kinetic analysis of glutamate-induced chloride current in Aplysia neurones: a 'concentration clamp' study.

L-Glutamate (Glu) applied by the concentration clamp technique to isolated neurones of Aplysia induced a chloride current (ICl) by activating a single population of the channel. The concentration-response curve for the peak ICl gave a dissociation constant of 1.3 x 10(-4) M and a Hill coefficient of 1.8. The current-voltage relationship was linear in the voltage range examined (-60 to +10 mV). The activation phase of the ICl followed a single-exponential time course and desensitization was complete with a double-exponential time course. The time constants for activation and desensitization decreased with increasing concentrations of Glu but were voltage-independent. The process of recovery from desensitization was also double-exponential. The single-channel conductance estimated by ensemble noise analysis was 50 +/- 4.7 pS (n = 4). These results suggest that the Glu receptor-Cl channel complex in Aplysia neurones consists of a single population with two binding sites for the agonist.