Single potassium channels in neuropile glial cells of the leech central nervous system

We performed patch-clamp experiments to identify distinct K⁺ channels underlying the high K⁺ conductance and K⁺ uptake mechanism of the neuropile glial cell membrane on the single-channel level. In the soma membrane four different types of K⁺ channels were characterized, which were found to be distributed in clusters. Since no other types of K⁺ channels were observed, these appear to be the complete repertoire of K⁺ channels expressed in the soma region of this cell type. The outward rectifying 42 pS K⁺ channel could markedly contribute to the high K⁺ conductance and the maintenance of the membrane potential, since it shows the highest open probability of all channels. The channel gating occurred in bursts and patch excision decreased the open probability. The outward rectifying 74 pS K⁺ channel was rarely active in the cell-attached configuration; however, patch excision enhanced its open probability considerably. This type of channel may be involved in neuron-glial crosstalk, since it is activated by both depolarizations and increases in the intracellular Ca²⁺ concentration, which are known to be induced by neurotransmitter release following the activation of neurons. The 40 pS and 83 pS K⁺ channels showed inward rectifying properties, suggesting their involvement in the regulation of the extracellular K⁺ content. The 40 pS K⁺ channel could only be observed in the inside-out configuration. The 83 pS channel was activated following patch excision. At membrane potentials more negative than −60 mV, flickering events indicated voltage-dependent gating.     

Macroscopic and single-channel chloride currents in neuropile glial cells of the leech central nervous system

In patch-clamp experiments we characterized four Cl⁻ channels (42 pS, 70 pS, 80 pS and 229 pS) underlying the large Cl⁻ conductance of leech neuropile glial cells. They differed with respect to their gating, their rectification and their activity in the cell-attached configuration, showed the selectivity sequence I⁻>Br⁻≥Cl⁻>F⁻ and were impermeable to SO₄²⁻. The four channels were blocked by NPPB, DPC, niflumic acid and DIDS and exhibited either three or four sublevel states. The outward rectifying 42 pS, 70 pS and 80 pS Cl⁻ channels were classified as intermediate conductance Cl⁻ channels and they could contribute to the high Cl⁻ conductance of the glial membrane, which stabilizes the glial membrane potential. The inward rectifying 229 pS Cl⁻ channel is very similar to vertebrate high conductance Cl⁻ channels, which are assumed to be part of an emergency system that is activated under pathophysiological conditions. In voltage-clamp experiments we calculated that the Cl⁻ conductance amounts to one-third of the total membrane conductance. Reduction of this Cl⁻ conductance by Cl⁻ channel inhibitors markedly depolarized the glial cell membrane. These prominent depolarizations depended on Na⁺ influx and in most cases the glial cells failed to regulate their membrane potential following wash-out of the inhibitors.     

Electrophysiological measurements of volume changes in leech neuropile glial cells

Double-barrelled microelectrodes, sensitive to quaternary ammonium ions, were used for simultaneous measurements of the intracellular free concentrations of choline ([Ch]i) or tetramethylammonium ([TMA]i) as well as membrane potential (Em) in neuropile glial cells of the leech, Hirudo medicinalis. Bath application of Ch or TMA (5 mM, 1 min) resulted in a transient membrane depolarization accompanied by a long-lasting (0.5-1 h) intracellular accumulation of these compounds to levels of between 5 and 15 mM. Changes in [Ch]i or [TMA]i were used for the calculation of changes in relative cell volume. Elevation of the extracellular K+ concentration [( K+]e) from 4 to 9, 15, 21, 27.5, or 40 mM elicited a membrane depolarization and a reversible cell swelling by about 7.5, 14, 18.5, 27 and 50%, whereas reduction of [K+]e to 1.5 mM as well as bath application of serotonin (5-HT) produced a membrane hyperpolarization and a concomitant shrinkage by about 6 and 14.3%, respectively. The measured alterations in cell volume were compared with calculated data based on the assumption of an osmotic equilibrium disturbed by potential-dependent changes of the intracellular Cl- concentration. The results indicate, that K(+)- and serotonin-induced changes in the cell volume of the neuropile glial cells are due to passive KCl and water fluxes.     

External ions and membrane potential of leech neuropile glial cells

The ionic mechanism of a membrane effect of 5-hydroxytryptamine (5-HT) on neuropile glial (NG) cells in ganglia of the medicinal leech was investigated with conventional single-barrelled microelectrodes. Control experiments were made with double-barrelled ion-selective microelectrodes. 5-Hydroxytryptamine hyperpolarized the NG-cell membrane and increased the conductance considerably. Methysergide, a potent 5-HT antagonist, blocked the 5-HT-induced hyperpolarization completely. When leech ganglia were superfused with physiological bathing media free of 5-HT, the NG-cell membrane conductance returned to the original value, but the membrane potential recovered only partially from the hyperpolarization in most experiments. In glial membranes artificially depolarized by means of constant-current injection, the amplitude of the 5-HT response increased. The amplitude decreased with membrane hyperpolarization and reversed at —73 mV, close to the potassium equilibrium potential. The reversal potential changed by 52 mV when the extracellular potassium concentration was altered by a factor of 10. We conclude that 5-HT increases the potassium conductance of NG-cell membranes.     

Electrophysiological characterization of a nicotinic acetylcholine receptor on leech neuropile glial cells

Ion-selective double-barrelled microelectrodes were used to measure the activities of intracellular K+, Na+, Cl-, and H+ (aiK, aiNa, aiCl, pHi) and membrane potential (Em) in neuropile glial cells as well as extracellular K+ activity (aeK) in the neuropile of the leech, Hirudo medicinalis, during bath application of carbachol. As measured with conventional single-barrelled microelectrodes, acetylcholine (ACh), nicotine, carbachol, tetramethylammonium (TMA), and choline elicited concentration-dependent (10(-6)-5 X 10(-3) M) transient membrane depolarizations of up to 60 mV amplitude whereas muscarine (10(-6)-10(-3) M) did not affect Em. alpha-Bungarotoxin (10(-7) M), decamethonium (10(-5) M), d-tubocurarine (5 X 10(-5) M), and strychnine (5 X 10(-5) M) blocked the carbachol depolarization by about 90%. Atropine (5 X 10(-5) M) blocked the response by about 75%, whereas hexamethonium was only effective at millimolar concentrations. Average baseline levels of aeK in the neuropile and of aiK, aiNa, and aiCl in the neuropile glial cells were about 3, 70, 10, and 7 mM, respectively. During the carbachol depolarization aeK and aiNa transiently increased, whereas aiK decreased. In contrast, a rise of aiK and a fall of aiNa were observed during glial depolarizations in solutions with elevated K+ concentration. aiCl increased during both the carbachol- and the K+-induced depolarization. During carbachol, pHi transiently fell by about 0.2 units from its average baseline level of 6.9, whereas an alkalinization of small amplitude was observed in high-K+ solutions. Bath-applied choline, TMA, and decamethonium rapidly accumulated in the neuropile glial cells as intracellularly monitored with double-barrelled microelectrodes filled with Corning K+ exchanger resin, which is highly selective for these agents. The results suggest that leech neuropile glial cells have a nicotinic ACh receptor coupled to a cation channel. It is hypothesized that this channel might also be permeable to choline, TMA, and decamethonium.     

钾通道阻断剂4-氨基吡啶诱导海马CA1锥体神经元钙瞬变

目的 研究4-氨基吡啶(4-AP)诱导的急性脑片海马CA1锥体神经元钙瞬变现象,探讨钾通道功能与钙瞬变的关系及可能机制.方法 荧光探针标记正常大鼠急性脑片海马神经元.共聚焦显微镜技术进行钙成像,观察不同浓度4-AP及细胞灌流液条件对神经元钙瞬变的影响.结果 低浓度(<15 mmol/L)4-AP诱导的钙瞬变峰值与剂量呈线性相关(r2=0.910,P=0.000),高浓度(20～80 mmol/L)4-AP诱导的钙瞬变峰值随浓度增高而下降.在无钙灌流液条件下,4-AP诱导的钙瞬变峰值水平下降,达峰时间延长,与含钙灌流液比较差异有统计学意义(P<0.05).结论 4-AP可诱导急性脑片海马CA1锥体神经元的钙瞬变,其机制包括细胞外钙内流与钙库钙释放.

Abstract：

Objective To investigate the calcium transient of CA1 pyramidal neurons induced by potassium blocker 4-aminopyridine (4-AP) in acute hippocampal slices to explore the relation between potassium channel function and calcium transient, and their mechanism. Methods Fluorescent probe was employed to mark the hippocampai neurons in acute brain slices of rats; confocal microscopy was used to perform calcium imaging to observe the influences of different concentrations of 4-AP and perfusate with/without calcium on calcium transient of CA1 pyramidal neurons. Results The response of [Ca2+]I to lower concentration of 4-AP (<15 mmol/L) was in a dose-dependent manner (r2=0.910, P=0.000); the higher the concentration of 4-AP (20-80 mmol/L), the lower the peak level of calcium transient. The latency and amplitude of calcium transient induced by 4-AP were obviously reduced when the extracellular condition was switched to an absence of calcium, which was significantly different as compared with that with calcium (P<0.05). Conclusion Blockade of potassium channels with 4-AP can increase [Ca2+]I in the hippocampal pyramidal neurons of acute slices. The increase of [Ca2+]1 to 4-AP could be ascribe to calcium release from intracellular stores and calcium influx from extracellular matrix.     

Effects of ATP and derivatives on neuropile glial cells of the leech central nervous system

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.     

Functions of glial cells in the retina of the honeybee drone

In the retina of the honey bee drone, Apis mellifera male, physiological interactions between glial cells and neurons (the photoreceptors) are exceptionally clear-cut and amenable to investigation. The principal glia (outer pigment cells) contribute to the homeostasis of extracellular [K+] and [Na+] by 1) spatial buffering of K+ and 2) net uptake of K+ and Cl-. The glia supply carbohydrate metabolic substrate to the neurons; only the glia take up and phosphorylate glucose. Neuronal activity 1) modifies glycogen metabolism in the glia, and 2) can be signalled to the glia in the absence of elevated extracellular [K+].     

Effects of serotonin and carbachol on glial and neuronal rubidium uptake in leech CNS

Effects of serotonin (5-HT) and carbachol on Rb uptake (used as a K marker) in leech neuron and glia were studied by electron probe microanalysis (EPMA). Hirudo medicinalis ganglia were perfused 60 s in 4 mM Rb substituted normal leech Ringer's with and without 5-HT (dosage range 5–500 μM) or carbachol (range 10–1000 μM), quench frozen, cryosectioned, and subjected to EPMA to determine elemental mass fractions and cell water content. Both 5-HT and carbachol altered leech neuron and glial cell elemental distribution and water content. In glial cells, a dose-dependent increase in Rb uptake was observed following 5-HT (control: 26 ± 2 μM; 5 μM: 47± 4; 50 μM: 62 ± 4; 500μM: 82±11 mmol/kg dry wt. ± S.E.M.) and carbachol (10 μM: 35±3; 100 μM: 52±3; 1000 μM: 68±3 mmol/kg dry w wt. ± S.E.M.). In neurons, 5-HT and carbachol had small effects. 5-HT decreased glial and neuronal cell water content. Carbachol decreased neuronal (but not glial) water content by approximately the same amount (mean decrease 9%) regardless of dose. Both 5-HT and carbachol affected glial cell K-accumulating properties, providing evidence that certain neurotransmitters may modulate invertebrate glial cells' K clearance function.     

Effects of 4-aminopyridine on calcium action potentials and calcium current under voltage clamp in spinal neurons

We examined the effects of 4-aminopyridine (4-AP) on calcium conductance mechanisms in cultured mouse spinal cord neurons. At low concentrations ( 1mM), 4-AP enhanced Ca²⁺-dependent transmitter release and prolonged the duration of Ca²⁺-dependent action potentials. Voltage clamp studies indicated that 4-AP directly facilitates Ca²⁺ entry through voltage sensitive channels apart from an effect on K⁺ currents. These results may help to explain why the drug promotes Ca²⁺-dependent transmitter release at peripheral and central synapses.     

Different effects of 4-aminopyridine on regenerated cutaneous and muscular rat sciatic nerve branches

Developing and regenerated myelinated rat dorsal and ventral root fibers respond differently to the fast potassium channel blocking agent 4-aminopyridine (4-AP). To pursue this issue further, we made unilateral sciatic nerve crushes in adult rats. Sural (SN) and lateral gastrocnemius (LGN) nerve branches were collected 4–6 months later, for physiological and morphological examination. Regenerated and control nerves in Ringers solution showed generally similar compound action potential (CAP) waveforms, but CAPs of regenerated SNs and LGNs in 4-AP were markedly different. While regenerated SNs showed a prominent late CAP negativity with a “rippled” appearance and markedly compromised recovery properties, the CAP and recovery properties of regenerated LGNs were minimally changed. Light and electron microscopic examination of SN and LGN fibers failed to reveal any features obviously related to the observed physiological differences. We conclude, that the effect of 4-AP on regenerated cutaneous afferents differs from its action on regenerated muscular afferents and efferents. This physiological diversity lacks obvious structural correlates.     

Ionic conductances related to GABA action on secretory and biosynthetic activity of pars intermedia cells

We have examined the action of GABA on the electrical, secretory and synthetic activities of rat and porcine intermediate lobe (IL) cells in primary culture. Chloride and calcium currents were investigated using patch-clamp techniques. A chloride current activated by 1–100 μM isoguvacine, a specific GABA-A agonist and antagonised by bicuculline and SR 95103 was recorded at the whole cell and single channel level current. Whole cell calcium currents were investigated and shown to be reduced by 40 μM cadmium, zero external calcium and 10 μM baclofen, a specific GABA-B receptor agonist. Both GABA-B receptor activation and use of calcium deficient medium inhibited peptide release from IL cells. Finally, pro-opiomelanocortin (POMC) mRNA levels were measured using a hybridization technique. Removal of calcium from die culture medium or long-term (48 hr) incubation with 10 μM GABA or muscimol (a mixed GABA-A and GABA-B agonist) significantly reduced POMC mRNA levels.     

Effects of methylmercury on perineurial Na and Ca-dependent potentials at neuromuscular junctions of the mouse

The ability of acute application of the neurotoxicant methylmercury (MeHg) to disrupt the function of presynaptic Ca²⁺ and Na⁺ channels at intact neuromuscular junctions was examined using mouse triangularis sterni motor nerves. In Ba²⁺-containing solutions, potential changes arising from Na⁺ and Ca²⁺ channel function could be recorded from the perineurial sheath surrounding motor neurons when K⁺ channels were blocked by tetraethylammonium chloride and 3,4-diaminopyridine. MeHg (100 μM) reduced both Na⁺- and Ba²⁺-dependent components to block within 3–5 min at apparently equivalent rates. Time to block was approximately 7 min after exposure to 50 μM MeHg. In 2 of 5 preparations exposed to 50 μM MeHg, the Ca²⁺ channel-mediated component was blocked prior to the Na⁺ channel-mediated component. In the remaining three preparations, Na⁺- and Ba²⁺-dependent potentials were blocked at similar times. Following block by MeHg, neither perfusing the preparation in MeHg-free solutions nor increasing the intensity and/or duration of stimulus to the intercostal nerves resulted in recovery of Na⁺ or Ca²⁺ potentials. In the presence of K⁺ channel blockers, repetitive firing of nerves in response to a single stimulus was observed in 20–30% of the triangularis preparations; in the two preparations treated with MeHg in which repetitive firing was observed, it decreased prior to block of the stimulus-induced Na⁺/Ba²⁺ potentials. These results corroborate the results obtained in isolated synaptosomes and pheochromocytoma cells, and suggest that MeHg decreases motor nerve excitability by disrupting Na⁺ channel function and may block neurotransmitter release by disrupting Na⁺ and Ca²⁺ channel function.     

Potent blocking action of lead on voltage-activated calcium channels in human neuroblastoma cells SH-SY5Y

Human neuroblastoma cells (SH-SY5Y) have two types of voltage-activated calcium channels, which are equivalent to the N and L types. Both types of calcium channels were equally blocked by lead in a concentration-dependent and reversible manner, with Ki congruent to 1 microM. This lead concentration is in the same order of magnitude as that found in the blood of children exhibiting neuropsychological disorders. Sodium and potassium channel currents were not significantly affected by lead at a concentration of 10 microM.     

Ionic properties of IK,n in outer hair cells of guinea pig cochlea

The ionic properties of voltage-dependent K⁺ current activated at the resting membrane potential (I_K,n) of outer hair cells (OHC) isolated from the guinea pig cochlea were studied using a patch-clamp technique in a whole-cell recording mode. The reversal potential of I_K,n indicated a high selectivity for K⁺, and the relative permeability ratios for various monovalent cations were K⁺ : Rb⁺ : NH⁺₄ = 1 : 1.21 : 0.13. Decrease in extracellular Cl⁻¹ inhibited the I_K,n. I_K,n was blocked by Cs⁺ and Ba²⁺, although the inhibitory manner of Cs⁺ and Ba²⁺ were voltage-dependent and voltage-independent, respectively. By the use of puff-application method, the local application of Ba²⁺ to basolateral surface of OHC shifted the holding current level in an inward direction, whereas the application to apex and hair showed little change. Indicating that the I_K,n channels preferentially locate at the basolateral region of cell membrane.     

Effect of ethanol on calcium regulation in rat fetal hypothalamic cells in culture

The effects of acute exposure to ethanol on calcium regulation in primary cultures of rat fetal hypothalamic cells was studied with the use of the calcium indicator fura-2 and digital imaging techniques. We found that ethanol caused cytoplasmic calcium to increase in a dose-dependent and reversible manner, and these increases could be observed at pharmacologically relevant doses (34 mM). At 170 mM ethanol 65% of 1059 cells examined responded to ethanol with an increase in cytoplasmic calcium. Removing bath calcium eliminated the ethanol-induced calcium response in most cells (76% of 427 cells). In most cells exposure to thapsigargin (20 nM) had no significant effect on the ethanol-induced calcium increase (87% of 67 cells examined). The ethanol-induced calcium increase was reduced by 79+/-5% (n=110 cells) by the P/Q-type calcium channel blocker omega-agatoxin-TK (20 nM), by 51+/-10% (n=115 cells) by the N-type calcium channel blocker omega-conotoxin-GVIA (100 nM), and by 26+/-3% (n=90 cells) by the T-type calcium channel blocker flunarizine (1 microM). The L-type calcium channel blocker nifedipine (1 microM) had complex actions, sometimes inhibiting and sometimes increasing the calcium response. These results demonstrate that ethanol can directly modulate cytoplasmic calcium levels in hypothalamic cells mostly by a pathway that involves extracellular calcium and voltage-dependent calcium channels, and that this response may participate in the biological effects of acute ethanol exposure.     

Ionic mechanism of a hyperpolarizing glutamate effect on two identified neurons in the buccal ganglion of Aplysia

The ionic mechanism of a membrane effect of L-glutamate on two identified neurons in the buccal ganglion of Aplysia kurodai was investigated with conventional microelectrode techniques and glutamate iontophoresis. Bath-applied and iontophoresed glutamate hyperpolarized the membrane and increased the membrane conductance. The hyperpolarizing glutamate response decreased in amplitude and finally reversed its polarity by conditioning hyperpolarization. The reversal potential of the hyperpolarizing glutamate response was close to the ECl (-60 mV). The reversal potential changed by 22.4 mV when the external chloride concentration was altered by a factor of 5. The relationship between the iontophoretically applied current and the membrane conductance changes was suggestive of two glutamate molecules reacting with a single receptor site. The hyperpolarizing glutamate response was essentially unaffected by 2-amino-4-phosphonobutyric acid (2-APB), L-proline, and quinuclidinyl benzilate (QNB). It was concluded that the hyperpolarizing glutamate response was generated by an activation of Cl- conductance.     

The mechanism of calcium-independent catecholamine depleting action of monensin from clonal rat pheochromocytoma cells

Monensin, a monovalent cation ionophore, induced profound release of radiolabeled materials from clonal rat pheochromocytoma cells (PC12h) preloaded with [³H]norepinephrine (NE). The release was suppressed in the absence of external Na⁺, but was not affected at all in the absence of external Ca²⁺. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), that was monitored by means of a fluorescent Ca²⁺ indicator, Quin 2, was temporarily increased upon a depolarizing stimulus of high-K⁺, which induced the Ca²⁺-dependent release of [³H]NE from PC12h cells. On the other hand, monensin induced only a slight increase in [Ca²⁺]_i. The radiolabeled materials released by high-K⁺ treatment were mainly [³H]NE, whereas those by monensin were mainly the metabolites of [³H]NE. Pargyline, a monoamine oxidase inhibitor, suppressed both the degradation of [³H]NE,stored in PC12h cells and the monensin-induced release of radiolabeled compounds from them. Monensin decreased the content of [³H]NE in storage granules of pargyline-treated cells. Thus, it is likely that monensin expels NE from the storage vesicles to cytosol and then its metabolites by monoamine oxidase are released in a non-exocytotic manner.     

Differential effects of K(+) channel blockers on frequency-dependent action potential broadening in supraoptic neurons

Recordings were made from magnocellular neuroendocrine cells dissociated from the supraoptic nucleus of the adult guinea pig to determine the role of voltage gated K(+) channels in controlling the duration of action potentials and in mediating frequency-dependent action potential broadening exhibited by these neurons. The K(+) channel blockers charybdotoxin (ChTx), tetraethylammonium (TEA), and 4-aminopyridine (4-AP) increased the duration of individual action potentials indicating that multiple types of K(+) channel are important in controlling action potential duration. The effect of these K(+) channel blockers was almost completely reversed by simultaneous blockade of voltage gated Ca(2+) channels with Cd(2+). Frequency-dependent action potential broadening was exhibited by these neurons during trains of action potentials elicited by membrane depolarizing current pulses presented at 10 Hz but not at 1 Hz. 4-AP but not ChTx or TEA inhibited frequency-dependent action potential broadening indicating that frequency-dependent action potential broadening is dependent on increasing steady-state inactivation of A-type K(+) channels (which are blocked by 4-AP). A model of differential contributions of voltage gated K(+) channels and voltage gated Ca(2+) channels to frequency-dependent action potential broadening, in which an increase of Ca(2+) current during each successive action potential is permitted as a result of the increasing steady-state inactivation of A-type K(+) channels, is presented.     

Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells

The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl⁻ concentration were microscopically monitored using a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K⁺ with valinomycin. Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl⁻ permeability, but neither by the increased K⁺ conductance with valinomycin nor by the increased Ca²⁺ conductance with A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A. These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells; the K⁺ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl⁻ channel opening depends on calyculin A-sensitive protein phosphatase activity.