GABAB-receptor-activated K+ current in voltage-clamped CA3 pyramidal cells in hippocampal cultures.

GABAB receptors are a subclass of receptors for gamma-amino-n-butyric acid (GABA) that are also activated by the antispastic drug beta-p-chlorophenyl-GABA (baclofen). One effect of baclofen is to inhibit excitatory transmission from CA3 to CA1 hippocampal pyramidal cells. To identify the ionic mechanism of GABAB-receptor-mediated depression, we have studied the effect of baclofen and GABA on ionic currents in voltage-clamped CA3 pyramidal cell somata in rat hippocampal slice cultures. Baclofen (10 microM) induced an inwardly rectifying outward current that reversed at -74 +/- 4.3 mV (mean +/- SD). This appeared to be a K+ current since (i) its reversal potential showed the expected shift when extracellular K+ concentration was changed and (ii) it was blocked by external Ba2+ or internal Cs+. The action of baclofen was closely imitated by GABA after the GABAA-mediated Cl- current had been abolished with pitrazepin (10 microM); under these conditions, GABA (100 microM) also produced an inwardly rectifying, Ba2+-sensitive current with a reversal potential identical to that of the baclofen-induced current. When outward currents were blocked with internal Cs+, the residual inward voltage-dependent Ca2+ current was not changed by baclofen. It is concluded that the primary effect of GABAB-receptor activation in these neurones is to increase K+ permeability rather than to reduce Ca2+ permeability.     

Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.

Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic synapses. The alpha 1 subunit has been found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. In contrast, the alpha 2 subunit was located only in a subset of synapses on the somata and dendrites, but in most synapses on axon initial segments innervated by axo-axonic cells. The results demonstrate that molecular specialization in the composition of postsynaptic GABAA receptor subunits parallels GABAergic cell specialization in targeting synapses to a specific domain of postsynaptic cortical neurons.     

Morphine and opioid peptides reduce inhibitory synaptic potentials in hippocampal pyramidal cells in vitro without alteration of membrane potential.

We used intracellular recording in the hippocampal slice in vitro to characterize further the mechanisms behind the unusual excitatory action of opiates and opioid peptides on hippocampal pyramidal cells in vivo. No significant effect on resting membrane potential, input resistance, or action potential size in cortical area 1 (CA1) pyramidal cells was observed with morphine sulfate, beta-endorphin, [Met5]enkephalin, or [D-Ala2, D-Leu5]enkephalin at 1-50 microM. However, in all cells studied, these agents markedly reduced the size of inhibitory postsynaptic potentials generated by stimulation of the stratum radiatum or alveus. Excitatory postsynaptic potentials were also diminished in many of these cells. The effects of the opioids were antagonized by naloxone. These results are consistent with excitation of pyramidal neurons by a disinhibitory mechanism.     

The ultrarapid and the transient outward K(+) current in human atrial fibrillation. Their possible role in postoperative atrial fibrillation

Atrial fibrillation (AF) causes distinct changes in atrial conduction, characterized as electrical remodeling. Experimental data on the possible significance of alterations of specific K(+)outward currents in this process are still limited in human AF. The ultra-rapid delayed rectifier current (I(Kur)) has not been studied in AF with respect to its sensitivity to 4-Aminopyridine (4-AP). To clarify the role of (1) the 4-AP sensitive I(Kur)current, compared to recordings without using 4-AP (I(Kur*)), and (2) the transient outward current (I(to)) in changes of atrial repolarization associated with AF, whole cell voltage-clamp recordings were obtained from atrial myocytes of patients undergoing elective cardiac surgery, with and without a history of atrial fibrillation (AF/non-AF). Further, a possible relation between experimental data and postoperative AF was studied. In AF patients, I(Kur*)was reduced by 40% [5.00+/-0.32 pA/pF (non-AF) and 2.91+/-0. 45 pA/pF (AF) at +50 mV, P<0.0001, n=22/11], I(Kur)by 55% [3.81+/-0. 30 pA/pF (non-AF) and 1.71+/-0.20 pA/pF (AF) at +50 mV, P<0.0001, n=22/11]. The mean amplitude of I(Kur)was significantly smaller than I(Kur*). Consistently, I(to)was reduced by 44% [11.57+/-0.77 pA/pF (non-AF) and 6.51+/-1.31 pA/pF (AF), P<0.01, n=25/11]. In 48% of non-AF patients, postoperative AF was detected. The corresponding voltage-clamp recordings showed a trend to reduced I(Kur*)and I(Kur)currents, although it did not reach statistical significance. The consistent reduction of all three K(+)currents investigated due to the presence of AF indicates an important association of abnormalities in cellular repolarization with the onset and the self-sustaining nature of human AF.     

A Ca(2)+ signaling pathway regulates a K(+) channel for low-K response in Arabidopsis

Nutrient sensing is critical for plant adaptation to the environment. Because of extensive farming and erosion, low content of mineral nutrients such as potassium (K(+)) in soils becomes a limiting factor for plant growth. In response to low-K conditions, plants enhance their capability of K(+) uptake through an unknown signaling mechanism. Here we report the identification of a Ca(2+)-dependent pathway for low-K response in Arabidopsis. We are not aware of any other example of a molecular pathway for a nutrient response in plants. Earlier genetic analyses revealed three genes encoding two Ca(2+) sensors (CBL1 and CBL9) and their target protein kinase (CIPK23) to be critical for plant growth on low-K media and for stomatal regulation, indicating that these calcium signaling components participate in the low-K response and turgor regulation. In this study, we show that the protein kinase CIPK23 interacted with, and phosphorylated, a voltage-gated inward K(+) channel (AKT1) required for K(+) acquisition in Arabidopsis. In the Xenopus oocyte system, our studies showed that interacting calcium sensors (CBL1 and CBL9) together with target kinase CIPK23, but not either component alone, activated the AKT1 channel in a Ca(2+)-dependent manner, connecting the Ca(2+) signal to enhanced K(+) uptake through activation of a K(+) channel. Disruption of both CBL1 and CBL9 or CIPK23 gene in Arabidopsis reduced the AKT1 activity in the mutant roots, confirming that the Ca(2+)-CBL-CIPK pathway functions to orchestrate transporting activities in planta according to external K(+) availability.     

Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors

The principal inhibitory neurotransmitter in the mammalian brain, gamma-aminobutyric acid (GABA), is thought to regulate memory processes by activating transient inhibitory postsynaptic currents. Here we describe a nonsynaptic, tonic form of inhibition in mouse CA1 pyramidal neurons that is generated by a distinct subpopulation of GABA type A receptors (GABA(A)Rs). This tonic inhibitory conductance is predominantly mediated by alpha5 subunit-containing GABA(A)Rs (alpha5GABA(A)Rs) that have different pharmacological and kinetic properties compared to postsynaptic receptors. GABA(A)Rs that mediate the tonic conductance are well suited to detect low, persistent, ambient concentrations of GABA in the extracellular space because they are highly sensitive to GABA and desensitize slowly. Moreover, the tonic current is highly sensitive to enhancement by amnestic drugs. Given the restricted expression of alpha5GABA(A)Rs to the hippocampus and the association between reduced alpha5GABA(A)R function and improved memory performance in behavioral studies, our results suggest that tonic inhibition mediated by alpha5GABA(A)Rs in hippocampal pyramidal neurons plays a key role in cognitive processes.     

Inversion of the intracellular Na(+)/K(+) ratio blocks apoptosis in vascular smooth muscle cells by induction of RNA synthesis

This study examines the involvement of RNA and protein synthesis in the modulation of apoptosis in vascular smooth muscle cells (VSMC) by intracellular monovalent cations. In VSMC transfected with E1A adenovirus (VSMC-E1A), inversion of the [Na(+)](i)/[K(+)](i) ratio by an inhibitor of the Na(+),K(+) pump, ouabain, prevented the development of apoptosis triggered by serum withdrawal. Inhibition of apoptosis by ouabain was abolished by inhibitors of RNA and protein synthesis, actinomycin D, and cycloheximide, respectively. In VSMC-E1A, incubation with ouabain for 4 and 24 hours augmented RNA synthesis by 20% to 50% and 3-fold to 4-fold, respectively. In quiescent VSMC, the effect of ouabain and serum on RNA synthesis was additive. Ouabain did not affect the level of phosphorylation of ERK, JNK, and p38 MAP kinases and blocked apoptosis independent of the presence of the MAPK kinase inhibitors PD98059 and SB 202190. Equimolar substitution of NaCl with KCl in the incubation medium abolished the effect of ouabain on intracellular Na(+) and K(+) concentration, apoptosis, and RNA synthesis. Thus, our results demonstrate that the antiapoptotic effect of the inverted [Na(+)](i)/[K(+)](i) ratio is mediated by MAPK-independent induction of de novo synthesis of RNA species encoding inhibitor(s) of programmed cell death.     

Inhibition of calcium influx and calcium current by gamma-aminobutyric acid in single synaptic terminals.

Inhibition of Ca influx and Ca current by gamma-aminobutyric acid (GABA) was studied in single synaptic terminals of isolated retinal bipolar neurons. Measurements of intracellular Ca concentration [( Ca]i) using the fluorescent Ca indicator fura-2 showed that GABA potently inhibited Ca influx into the terminal elicited by high extracellular K concentration ([K]o). This inhibition was attributed to GABA type A (GABAA) receptor-activated chloride ion conductance that prevented bipolar neurons from depolarizing sufficiently to activate the Ca current, even in response to increased [K]o. Patch-clamp recordings of the Ca current revealed a second effect of GABA: GTP-dependent inhibition of the Ca current. This inhibition was not mediated by GABAA receptors, but baclofen, which binds to the GABA type B (GABAB) receptor and is known to inhibit the Ca current in other systems, was not able to mimic the action of GABA. This suggests the involvement of a different type of GABAB-like receptor in the inhibition of Ca current by GABA. GABA did not cause an overall suppression of the Ca current; rather, the voltage-dependence of Ca-channel activation was shifted to more depolarized potentials. Thus, maximal inhibition of the Ca current by GABA occurred in the physiological range of potential.     

藤黄酸调控白血病K562细胞GFI-1表达及对细胞增殖和凋亡的影响

目的: 探讨藤黄酸对白血病K562细胞增殖和凋亡的影响及可能的机制。方法：体外培养K562细胞,采用CCK-8法检测不同浓度藤黄酸(0、0.2、0.4、0.8、1.2、2.0μmol/L)处理24、48、72h后K562细胞增殖率;用流式细胞术检测K562细胞的凋亡和周期;采用实时荧光定量聚合酶链反应（RT-qPCR)）和Western blot法检测独立生长因子1(GFI-1) 的mRNA及蛋白表达水平。结果： 藤黄酸可以抑制K562细胞的增殖,并呈剂量、时间依赖性;0.4μmol/L藤黄酸处理24h后的K562细胞凋亡率增加;藤黄酸对GFI-1的mRNA表达无显著影响,藤黄酸可以下调GFI-1蛋白的表达;藤黄酸及蛋白酶体抑制剂（MG132）共处理后的K562细胞GFI-1蛋白水平较藤黄酸组升高;藤黄酸及氯喹（CQ）共处理后的K562细胞GFI-1蛋白水平与藤黄酸组无统计学差异（P>0.05）。结论： 藤黄酸可以有效抑制K562细胞的增殖并诱导细胞凋亡;藤黄酸可以诱导K562细胞G0/G1期及S期阻滞;藤黄酸通过蛋白酶体途径促进GFI-1蛋白降解。     

Muscarinic and beta-adrenergic depression of the slow Ca2(+)-activated potassium conductance in hippocampal CA3 pyramidal cells is not mediated by a reduction of depolarization-induced cytosolic Ca2+ transients.

Combined intracellular and microfluorometric recording techniques were used to evaluate whether the inhibition by cholinergic or adrenergic transmitters of the Ca2(+)-activated potassium current (IAHP) in hippocampal CA3 pyramidal cells was mediated by an alteration of depolarization-induced change in cytosolic free Ca2+ concentration [(Ca2+]i). Low concentrations of isoproterenol (1-10 microM) and muscarine (0.25-1 microM) reversibly abolished IAHP without affecting concomitant Ca2+ transients or the steady-state [Ca2+]i. Only after application of higher concentrations of muscarine, [Ca2+]i increased; in the presence of potassium channel blockers, muscarine depressed Ca2+ currents and concomitant Ca2+ transients. These observations provide direct evidence that the inhibition of IAHP by isoproterenol and muscarine are not mediated by an alteration of Ca2+ dynamics.     

Stimulation of arachidonic acid release and inhibition of mitogenesis by cloned genes for muscarinic receptor subtypes stably expressed in A9 L cells.

A family of genes encoding four distinct muscarinic receptors (designated m1-m4) has been cloned and stably expressed in A9 L cells. When the m1 and m3 receptors were stimulated with carbachol, there was a rapid rise of liberated arachidonic acid, inositol phosphates, and cAMP, while m2 and m4 receptor stimulation had no detectable stimulation of these second messengers. Pretreatment with phorbol 12-myristate 13-acetate (PMA) caused a marked acceleration and amplification of m1 and m3 receptor-mediated arachidonic acid release. In contrast, m1- and m3-mediated inositol phosphate formation was inhibited by the same PMA pretreatment. Arachidonic acid release was unaffected by manipulations of cAMP levels. Arachidonic acid production was inhibited by calcium-free medium and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8; an inhibitor of cytosolic calcium mobilization) yet was unaffected by verapamil, a calcium-channel blocker. These experiments show that arachidonic acid release induced by the m1 and m3 receptors is regulated independently of phospholipase C and cAMP accumulation. Carbachol stimulation of the m1 and m3 cAMP accumulation. Carbachol stimulation of the m1 and m3 receptors also markedly decreased mitogenesis as measured by thymidine incorporation. The m1 receptor-mediated inhibition of mitogenesis could be partially blocked by indomethacin, a cyclooxygenase inhibitor. The inhibition of mitogenesis could be mimicked by cAMP elevation.     

Serotonin stimulates phospholipase A2 and the release of arachidonic acid in hippocampal neurons by a type 2 serotonin receptor that is independent of inositolphospholipid hydrolysis.

Serotonin (5-HT) stimulated the release of arachidonic acid in hippocampal neurons cocultured with glial cells but not in glial cultures alone. Similar results were observed for the 5-HT-stimulated release of inositol phosphates. These results suggest a neural but not glial origin of both responses. Pharmacological studies suggested that release of arachidonic acid and inositol phosphates was mediated by a type 2 5-HT (5-HT2) receptor. 5-HT-stimulated release of arachidonic acid was also detected in cortical neurons, which contain high levels of 5-HT2 receptors, but not striatum, spinal cord, or cerebellar granule cells, which have very low levels or are devoid of 5-HT2 receptors. The phorbol ester phorbol 12-myristate 13-acetate augmented the 5-HT-stimulated release of arachidonic acid but inhibited the 5-HT-stimulated release of inositol phosphates. 5-HT-stimulated release of arachidonic acid, but not inositol phosphates, was dependent on extracellular calcium. 5-HT stimulated the release of [3H]lysophosphatidylcholine from [3H]choline-labeled cells with no increase in the release of [3H]choline or phospho[3H]choline. These data suggest that 5-HT stimulated the release of arachidonic acid in hippocampal neurons through the activation of phospholipase A2, independent of the activation of phospholipase C.     

Cross-talk between CD8(+) and CD4(+) T cells in experimental cutaneous leishmaniasis: CD8(+) T cells are required for optimal IFN-gamma production by CD4(+) T cells

Although the importance of CD8(+) T cells for vaccination and immunity to reinfection with Leishmania parasites is well established, their role in primary infections is disputed. In the present study we further characterized the role of CD8(+) T cells in primary L. major infections. We used two groups of L. major infected BALB/c mice: both groups were immunomanipulated to heal and in one group CD8(+) T cells were depleted throughout the course of infection. Our results show that the reversal of healing caused by the absence of CD8(+) T cells did not alter the proliferation of CD4(+) T cells, however, the frequency of CD4(+) T cells expressing IFN-gamma as well as the levels of this cytokine were clearly reduced. These lower levels of IFN-gamma correlated with a higher parasite load. Our results show that transient depletion of CD4(+) T cells allows the establishment of an equilibrium between CD4(+) and CD8(+) T cells and allows CD8(+) T cell activation and effector functions to develop. In addition, our results suggest that cross-talk between CD4(+) and CD8(+) T cells is crucial for the host defence against L. major.     

Modulation of a transient K+ current in the pleural sensory neurons of Aplysia by serotonin and cAMP: implications for spike broadening.

To study the contribution of cAMP to the spike broadening produced by serotonin (5-HT) in the pleural sensory neurons of the tail withdrawal reflex, we utilized two phosphodiesterase-resistant cAMP analogs: the Sp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Sp-cAMP[S]), which activates protein kinase A, and the antagonist Rp diastereomer of cyclic adenosine 3',5'-monophosphothioate (Rp-cAMP[S]), agonist Sp-cAMP[S] was injected into the sensory neurons, it caused spike broadening comparable to that induced by 5-HT. In turn, the cAMP antagonist Rp-cAMP[S] blocked approximately 50% of the 5-HT-induced spike broadening. We next examined the K+ currents that are modulated by 5-HT and determined how these currents are affected by cAMP. Confirming Baxter and Byrne [(1989) J. Neurophysiol. 62, 665-679], we found that 5-HT modulated two currents, an S-type K+ current (IKS) as well as a transient and voltage-dependent K+ current (IKV). Rp-cAMP[S] blocked the reduction by 5-HT of the early phase of IKV in parallel with, and to the same degree (60%), as this inhibitor blocked the IKS and spike broadening. These results support the idea that in the pleural sensory neurons cAMP mediates a significant part of the spike broadening that accompanies short-term facilitation produced by 5-HT and that cAMP can produce spike broadening by modulating both IKV and IKS.     

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Previous work suggested that the structural gene for the A system transporter and the mRNA for the alpha subunit of the Na+,K(+)-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance of the alpha 1 subunit of Na+,K(+)-ATPase mRNA over that found in repressed WT. The level of Na+ per cell in alar4 was not significantly greater than that found in the WT. To further characterize the likely coregulation of both genes, we have studied the A system activity and Na+,K(+)-ATPase mRNA alpha 1-subunit levels in cells grown under various conditions that result in repression or derepression of the A system in the WT. System A activity increased up to 2-3 times the basal transport rate (repressed state) and Na+,K(+)-ATPase mRNA alpha 1-subunit levels showed a 3-fold increase after amino acid starvation (derepressed state). These changes occurred along with a decrease in intracellular Na+ levels. N-Methyl-alpha-aminoisobutyric acid and beta-alanine, previously shown to be corepressors for the A system, prevented to a similar extent A system derepression and Na+,K(+)-ATPase mRNA alpha 1-subunit accumulation. On the other hand, phenylalanine and lysine, amino acids that are not corepressors of the A system, failed to significantly prevent derepression of both genes. Hybrids between the WT and alar4 have the phenotype of the WT when grown under repressed conditions. These results give further support to the proposition that both the A system transporter and mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase are coordinately controlled by regulatory gene R1 and elevated Na+ concentrations are not involved. No Na+,K(+)-ATPase activity was detected in derepressed cells. Activity was restored by the addition of monensin. However, this activity was no greater than that obtained in repressed cells. Indications are that the reduced Na+ content in derepressed cells inhibits Na+,K(+)-ATPase activity and that conditions that favored derepression do not allow for de novo synthesis of the Na+,K(+)-ATPase.     

Overexpressed A(1) adenosine receptors reduce activation of acetylcholine-sensitive K(+) current by native muscarinic M(2) receptors in rat atrial myocytes

In adult rat atrial myocytes, muscarinic acetylcholine (ACh)-sensitive K(+) current activated by a saturating concentration of adenosine (I(K(ACh),(Ado))) via A(1) receptors (A(1)Rs) amounts to only 30% of the current activated by a saturating concentration of ACh (I(K(ACh),(ACh))) via muscarinic M(2) receptors. The half-time of activation of I(K(ACh),(Ado)) on a rapid exposure to agonist was approximately 4-fold longer than that of I(K(ACh),(ACh)). Furthermore, I(K(ACh),(Ado)) never showed fast desensitization. To study the importance of receptor density for A(1)R-I(K(ACh),(Ado)) signaling, adult atrial myocytes in vitro were transfected with cDNA encoding for rat brain A(1)R and enhanced green fluorescent protein (EGFP) as a reporter. Whole-cell current was measured on days 3 and 4 after transfection. Time-matched cells transfected with only the EGFP vector served as controls. In approximately 30% of EGFP-positive cells (group I), the density of I(K(ACh),(Ado)) was increased by 72%, and its half-time of activation was reduced. Density and kinetic properties of I(K(ACh),(ACh)) were not affected in this fraction. In approximately 70% of transfection-positive myocytes (group II), the density of I(K(ACh),(ACh)) was significantly reduced, its activation was slowed, and the fast desensitizing component was lost. Adenosine-induced currents were larger in group II than in group I, their activation rate was further increased, and a fast desensitizing component developed. These data indicate that in native myocytes the amplitude and activation kinetics of I(K(ACh),(Ado)) are limited by the expression of A(1)R. Overexpression of A(1)R negatively interferes with signal transduction via the muscarinic M(2) receptor-linked pathway, which might reflect a competition of receptors with a common pool of G proteins. Negative interference of an overexpressed receptor with physiological regulation of a target protein by a different receptor should be considered in attempts to use receptor overexpression for gene therapy.     

Paracrine hypertrophic factors from cardiac non-myocyte cells downregulate the transient outward current density and Kv4.2 K+ channel expression in cultured rat cardiomyocytes.

OBJECTIVES: Cardiac hypertrophy is characterized by a prolongation of action potential duration (APD) and a reduction of outward K+ currents, primarily the transient outward current (Ito). Since the interaction between cardiac non-myocyte cells (NMCs) and cardiomyocytes (MCs) plays a critical role during the process of myocardial hypertrophy, in the present study, we investigated the effects of NMCs on cell growth and K+ channel expression in cultured newborn rat ventricular cells. METHODS: Single MCs were isolated from day-old Wistar rat ventricles and cultured for a period of five days. The effects of NMCs were examined by MC-NMC co-culture or incubating pure MCs in NMC-conditioned growth medium (NCGM). Whole-cell voltage-clamp recording and Western blot analysis using a polyclonal antibody against rat Kv4.2 channel protein were performed. RESULTS: A marked increase in surface area and total cell protein concentration of MCs was observed in the MC-NMC co-culture. In the pure MC culture, this hypertrophic effect could be mimicked by a 72-h addition of NCGM, with a significant prolongation of APD25 (APD at 25% repolarization) and a 42% decrease in Ito density (at +30 mV). The rates of inactivation and recovery from inactivation of Ito were unchanged. In the NCGM-treated MC culture, Western blots of MC proteins also showed a 36% reduction of the Kv4.2 K+ channel protein level. In addition, the NCGM-induced MC hypertrophy was partially inhibited by anti-insulin-like growth factor-1 (IGF-1) antibody, while it revealed no effects on Ito density and Kv4.2 channel expression. CONCLUSIONS: These findings first demonstrate that some paracrine hypertrophic factors released from cardiac NMCs, although unidentified, downregulate cardiac K+ channel expression.     

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K⁺ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K⁺ currents, an inactivating outward-rectifying K⁺ current was characterized (plant A-type current: I_AP). I_AP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. I_AP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward I_AP was mainly carried by K⁺ ions. In contrast to the sustained outward-rectifying K⁺ currents, cytosolic alkaline pH was found to inhibit I_AP and extracellular K⁺ was required for I_AP activity. Furthermore, increasing cytosolic free Ca²⁺ in the physiological range strongly inhibited I_AP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K⁺ current in a higher plant cell. Regulation of I_AP by diverse factors including membrane potential, cytosolic Ca²⁺ and pH, and extracellular K⁺ and Ca²⁺ implies that the inactivating I_AP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.