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The present study examined temporal changes in activity of large conductance, Ca2+-activated potassium (BKCa) channels in postischemic CA1 pyramidal neurons at 2, 6, 24 and 48 h after reperfusion. These changes in activity and possible cellular mechanisms were examined using the inside--out configuration of patch clamp. The unitary conductance of postischemic BKCa channels increased transiently to 119% of the control at 2 h after reperfusion, and recovered to the control level thereafter. A persistent increase in [Ca2+]i sensitivity of BKCa channels was observed in postischemic CA1 neurons with the maximal sensitivity to [Ca2+]i at 6 h after reperfusion while channel voltage- dependence showed no obvious changes. Kinetic analyses showed that the postischemic enhancement of BKCa channel activity was due to longer open times and shorter closed times as there was no significant changes in opening frequency after ischemia. Glutathione disulphide markedly increased BKCa channel activity in normal CA1 neurons, while reducing glutathione caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to [Ca2+]i in postischemic CA1 neurons. Similar modulatory effects on postischemic BKCa channels were also observed with another redox couple, DTNB and DTT, suggesting an oxidation modulation of BKCa channel function after ischemia. The present results indicate that a persistent enhancement in activity of BKCa channels, probably via oxidation of channels, in postischemic CA1 pyramidal neurons may account for the decrease in neuronal excitability and increase in fAHP after ischemia. The ischemia-induced augmentation in BKCa channel activity may be also associated with the postischemic neuronal injury.

The properties of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels were studied in rat hippocampal CA1 pyramidal neurons by using the patch-clamp technique in the excised-inside-out-patch configuration. The lowest [Ca(2+)](i) in which BK(Ca) channel activities were observed was 0.01 microM with the membrane potential of +20 mV and the [Ca(2+)](i) at which P(O) of the channel is equal to 0.5 was 2 microM. The unitary conductance of the single BK(Ca) channel was 245.4 pS with symmetrical 140 mM K(+) on both sides of the excised membrane. With a fixed [Ca(2+)](i) of 2 microM, P(O) increased e-fold with a 17.0 mV positive change in the membrane potential. Two exponentials, with time constants of 2.8 ms and 19.2 ms at the membrane potential of +120 mV with 2 microM [Ca(2+)](i), were required to describe the observed open time distribution of BK(Ca) channel, suggesting the existence of two distinct open channel states with apparently normal conductance. A BK(Ca) channel occasionally entered an apparent third open channel state with the single channel current amplitude about 45% of the normal amplitude. The properties of BK(Ca) channel, which were found in this study to be more steeply dependent on voltage and more sensitive to [Ca(2+)](i) in adult hippocampal neurons than in cultured or immature hippocampal neurons, may be responsible for the shortened duration of action potential in hippocampal CA1 pyramidal neurons of adult rat.

Redox regulation of BKCa channels was studied in CA1 pyramidal neurons of adult rat hippocampus by using inside-out configuration of patch clamp. Intracellular application of oxidizing agent 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) markedly increased activity of BKCa channels and this stimulating action persisted even after washout. In contrast, the reducing agent dithiothreitol (DTT) had no apparent effects on channel activity but could reverse the pre-exposure of DTNB-induced enhancement. The increase in channel activity produced by DTNB was due to shortened closed time as well as prolonged open time. The effects exerted by another redox couple glutathione disulphide and its reducing form were similar as DTNB and DTT. The present results indicate that BKCa channels in CA1 pyramidal neurons can be modulated by intracellular redox potential, and that augmentation of BKCa channels by oxidative stress might contribute to the postischemic electrophysiological alterations of CA1 pyramidal neurons.

Classically, ion channels are classified into 2 groups: chemical-sensitive (ligand-gated) and voltage sensitive channels. Single ATP-sensitive K(+) (K-ATP) channel currents were recorded in acutely dissociated rat neocortical neurons using patch clamp technique. A type of K-ATP channel has been found to be gated not only by intracellular ATP, but also by membrane potential (V(m)), and proved to be a novel mechanism underlying the gating of ion channels, namely bi-gating mechanism. The results also show that the K-ATP channels possess heterogeneity and diversity. These types of K-ATP channels have been identified in 40.12% of all patches, which are different in activation-threshold and voltage-sensitivity. The present experiment studied the type-3 K-ATP channel with a unitary conductance of about 80 pS in detail (n = 15). Taking account of all the available data, a variety of K-ATP channels are suggested to exist in body, and one type of them is bi-gated by both chemical substances and membrane potentials. This property of the K-ATP channels may be related to their pathophysiological function.

Using inside-out configuration of patch clamp techniques, ATP modulation of BK(Ca) channels was studied in hippocampal CA1 pyramidal neurons of adult rat. Intracellular ATP application markedly increased BK(Ca) channel activity, and this ATP-produced increase in BK(Ca) channel activity was characterized by a higher opening frequency with no changes in channel open times. In the presence of specific inhibitor against protein kinase A, H-89, ATP did not induce any increase in the channel activity. Furthermore, adding H-89 after addition of ATP reversed the modulation produced by ATP. In contrast, protein kinase C inhibitor chelerythrine exerted no apparent effects on ATP-induced channel activation. The present study suggests that BK(Ca) channels from hippocampal CA1 pyramidal neurons could be modulated by ATP via a functionally associated protein kinase A-like protein.

The present study examined temporal changes in activity of large conductance, Ca2+-activated potassium (BKCa) channels in postischemic CA1 pyramidal neurons at 2, 6, 24 and 48 h after reperfusion. These changes in activity and possible cellular mechanisms were examined using the inside--out configuration of patch clamp. The unitary conductance of postischemic BKCa channels increased transiently to 119% of the control at 2 h after reperfusion, and recovered to the control level thereafter. A persistent increase in [Ca2+]i sensitivity of BKCa channels was observed in postischemic CA1 neurons with the maximal sensitivity to [Ca2+]i at 6 h after reperfusion while channel voltage- dependence showed no obvious changes. Kinetic analyses showed that the postischemic enhancement of BKCa channel activity was due to longer open times and shorter closed times as there was no significant changes in opening frequency after ischemia. Glutathione disulphide markedly increased BKCa channel activity in normal CA1 neurons, while reducing glutathione caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to [Ca2+]i in postischemic CA1 neurons. Similar modulatory effects on postischemic BKCa channels were also observed with another redox couple, DTNB and DTT, suggesting an oxidation modulation of BKCa channel function after ischemia. The present results indicate that a persistent enhancement in activity of BKCa channels, probably via oxidation of channels, in postischemic CA1 pyramidal neurons may account for the decrease in neuronal excitability and increase in fAHP after ischemia. The ischemia-induced augmentation in BKCa channel activity may be also associated with the postischemic neuronal injury.

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca(2+)-activated potassium (BK(Ca)) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK(Ca) channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K(+) in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P(o)) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P(o) was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca(2+)](i) required to half activate BK(Ca) channels was only 1 microM in post-ischemic whereas 2 microM in control neurons, indicating an increase in [Ca(2+)](i) sensitivity after ischemia; and (4) BK(Ca) channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK(Ca) channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.