Atsuya Yoshida

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.

Blockade by local anaesthetics of the single Ca2+-activated K+ channel in rat hippocampal neurones

1 Effects of local anaesthetics on single Ca2+‐activated K+ channels were investigated using the inside‐out configuration of the patch‐clamp technique in single pyramidal neurones, which were freshly dissociated from rat hippocampus by use of proteolytic enzymes. 2 No significant effect was observed when 2 mm benzocaine was applied on either side of the membrane patch, or when 3 mm lignocaine or QX‐314 was applied to the external surface of the membrane. 3 Lignocaine 1 mm, applied to the internal surface, slightly reduced the amplitude of the single K+ channel current. When applied to the internal surface, QX‐314 reduced the amplitude of the K+ channel current, accompanied by an increase in noise in the open channel current, suggesting a fast flickering block. The blocking effect of QX‐314 on the outward current increased with depolarization, suggesting a binding site for the drug at an electrical distance of about 0.5 across the membrane field. 4 The open time histogram showed one exponential component and the closed time histogram showed at least two components. The mean open time of the outward current was increased when the amplitude was reduced by the drugs. 5 The ionized form of the local anaesthetics had a similar action on the Ca2+‐activated K+ channels to that on Na+ channels, that is, they enter into the channel from the cytoplasmic side to induce open channel block. The blocking kinetics, however, might be so fast that they were beyond the frequency response of our recording apparatus, thus the recorded current amplitude was decreased. In contrast the K+ channel was not accessible via hydrophobic pathways for the neutral form, which is also known to block the sodium channel.

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)

Blockade by trifluoperazine of a Ca2+-activated K+ channel in rat hippocampal pyramidal neurons

The effects of trifluoperazine, a phenothiazine derivative, on the large-conductance Ca(2+)-activated K+ channel (BKCa) in dissociated rat hippocampal pyramidal neurons were examined using the inside-out configuration of the patch-clamp technique. The BKCa was activated by 12.6 microM Ca2+ on the internal surface of the membrane patch. The single channel conductance of the BKCa was 244 +/- 17.5 pS (n = 10) in symmetrical solutions of 150 mM K+. Trifluoperazine, applied on the internal surface of the membrane, decreased the open probability of the channel without changing the single channel conductance. The reduction in the open probability was well described by a block of the open state of the channel in a simple sequential model. The apparent dissociation constant (KD) for the reduction was calculated to be 1.4 microM and the Hill coefficient 0.69 at +20 mV. The inhibition was voltage dependent, being more pronounced at depolarized voltages. The voltage dependence enabled us to estimate that the binding site for the agent in the channel lies about half way across the membrane electrical field. It is concluded that trifluoperazine blocks the open state of the BKCa, which is known to provide an outward current for repolarization and afterhyperpolarization of the neuronal action potential. This may result in a decrease in spike intervals during burst firing of neurons.