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Department of Physiology and Pharmacology, State University of New York Downstate Medical Center, Brooklyn, New York
Submitted 2 May 2006; accepted in final form 31 May 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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). In addition to IA and ID, Lüthi et al. (1996) observed that a third component, termed the slowly inactivating potassium current IK(slow), also contributed to the transient potassium current. The different components of the transient potassium current are distinguished by the rate at which they inactivate and by their pharmacological properties.
The transient potassium current activated near the resting membrane potential of the cell (–50 mV) typically decays with a multiexponential time course. The component with the fastest decay (time constant of 10–50 ms) is the A-current (IA) and is blocked by 4-aminopyridine (4-AP, 1–5 mM). A second component, the D-current (ID), decays with a slower time course and was first observed in the hippocampal cells by Storm (1988). Storm described that the decay time course of ID consisted of multiple exponential components. ID has been distinguished from IA based on the higher sensitivity to 4-AP (30–100 µM 4-AP completely blocks ID; Storm 1988, 1990; Wu and Barish 1992) and on the sensitivity of the current to dendrotoxin (1 µM dendrotoxin blocks ID but not IA; Storm 1990; Wu and Barish 1992). In CA3 pyramidal cells, Lüthi et al. (1996) showed that the ID component of the transient potassium current decayed with a time constant of about 750 ms. In addition, they proposed that a distinct slowly inactivating potassium current, termed IK(slow), accounted for the slowest decaying component of the transient potassium current. IK(slow) inactivates with a time constant of several seconds (ranging from 2 to 15 s) and can be distinguished from ID by its insensitivity to 0.1–5 mM 4-AP (Lüthi et al. 1996). At present, the properties of the slowly inactivating outward current have not been extensively studied. Because of this lack of characterization, its role in the control of hippocampal neuronal excitability is unclear and its status as a current sustained by a voltage-dependent conductance is not fully acknowledged. We now report that the slowest decaying component of the transient outward current is blocked by intracellular QX-314, providing additional evidence that the current is a distinct component of the transient outward current.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Transverse hippocampal slices (about 300 µm thick) were prepared from adult guinea pigs as previously described (Bianchi et al. 1999). Brains were rapidly removed from the skull of anesthetized animals according to approved procedure by the IACUC of SUNY Downstate Medical Center. One hippocampus was dissected out in ice-cold solution containing (in mM): NaCl 124.0, NaHCO3 26.0, KCl 2.5, MgCl2 8.0, CaCl2 0.5, and D-glucose 10.0. The middle portion of the hippocampus was glued to the stage of a Lancer Vibratome 1000 (Vibratome, St. Louis, MO) and sliced. Slices were stored on a mesh in a beaker containing artificial cerebrospinal fluid (aCSF) of the following composition (in mM): NaCl 124.0, NaHCO3 26.0, KCl 5.0, MgCl2 1.6, CaCl2 2.0, and D-glucose 10.0, pH 7.4. The solution was continuously gassed with a 95% O2-5% CO2 mixture and the temperature, after a 30-min period at 35°C, was kept around 24°C. After 
1 h of recovery from the dissection, one slice at a time was placed submerged in a coverslip-bottomed recording chamber (Luigs and Neumann, Ratingen, Germany) and superfused with aCSF at 2–3 ml/min, at 31–32°C. The slice in the chamber was held down by nylon threads glued to a platinum ring. The chamber was on a stationary stage with mounted micromanipulators that were moved through electromechanical remote controls (Luigs and Neumann). The chamber was under an upright microscope equipped with water-immersed objectives and IR-DIC microscopy (BX50WI, Olympus, Middlebush, NJ). A solid-state camera (Cohu, Electronics Division, San Diego, CA) connected to a video monitor was used to image the slice and the neurons from the microscope.
Patch-clamp recordings
Visually identified CA3 pyramidal cells were recorded in whole cell voltage clamp using glass pipettes (World Precision Instruments, Sarasota, FL) with resistances in the range of 3–6 M
. Voltage commands and membrane current recordings were carried out with a patch-clamp amplifier (EPC-7, HEKA Instruments, Southboro, MA). The signals, which were stored on an Intel-based computer running pCLAMP software (Molecular Devices, Sunnyvale, CA), were simultaneously displayed on an oscilloscope (DSO 400, Gould Instruments, Valley View, OH) and recorded on a chart (TA240, Gould Instruments). Recordings were sampled at 3 kHz and digitized at 0.1–1 kHz. The access resistance of the whole cell recordings included in this study was 8–26 M and did not vary more than 5% throughout the experiment. The solution used to fill the recording pipette contained (in mM): 119.0 K+ gluconate, 13.0 KCl, 10.0 HEPES, 5.0 NaCl, 2.0 CsCl, 2.0 Mg-ATP, 2.0 EGTA, and pH was adjusted to 7.3 with KOH. In some experiments (Fig. 6A), KCH3SO3 replaced K+ gluconate. For intracellular application of Cs+ (Fig. 6B) the pipette solution contained (in mM): 119.0 CsCH3SO3, 10.0 HEPES, 5.0 NaCl, 15.0 CsCl, 2.0 Mg-ATP, 2.0 EGTA (pH adjusted to 7.3 with CsOH). For intracellular application of QX-314 (Figs. 7 and 8), 5.0 mM of QX-314 was added to the K+ gluconate pipette solution.
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Pharmacological agents were added to the perfusing solution at the indicated final concentrations. The Mn2+/low Ca2+-containing solution had the same composition as the aCSF except for 0.2 mM CaCl2 and added 0.5 or 1 mM MnCl2. All chemicals were purchased from Sigma–Aldrich (St. Louis, MO).
Data analysis and statistics
Usually, to elicit slowly inactivating outward currents, cells were first hyperpolarized to about –100 mV for 3–5 s and then depolarized to voltages up to –30 mV and kept at the depolarized levels for 16 s. The slowly decaying currents recorded at the depolarized potentials were fitted with single exponential functions starting at 2 s after the beginning of the depolarization to minimize the contribution of the capacitive current, of fast inward currents (e.g., activation of Ca2+ currents and deactivation of Ih), and of inactivating outward currents (IA and ID) with decay faster than that of IK(slow). When the tail of the slowly decaying current was recorded (Figs. 3 and 4), the fitting monoexponential function started 2 s after stepping from 0 mV to the voltage at which the current was measured. The best-fitting function (R > 0.9) for each current record was selected. The term a in the single exponential function y = y0 + ae–bx is the value of the fit at the beginning of the depolarization minus the baseline (dashed line in Fig. 1Ab) and was taken as the amplitude of the slowly decaying outward current (Amplitude in Fig. 1Ab). The time constant of decay was calculated as 
= 1/b. SigmaPlot 8 (Systat Software, Point Richmond, CA) was used for fits and plots. Statistical comparisons of data were carried out with the indicated tests and the level of significance was set at P = 0.05 (GB STAT, Dynamic Microsystems, Silver Spring, MD).
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RESULTS |
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Activation and inactivation properties of the slowly inactivating outward current in CA3 pyramidal cells
Figure 1Aa shows typical current responses to a series of depolarizations from a conditioning potential of –100 mV obtained in the presence of TTX (1 µM). Step depolarizations to –60 mV and beyond elicited a transient outward current. The current decayed to the baseline at the holding potential of –50 mV. The time course of decay of the slowest component of the transient outward current (the slowly inactivating outward current) could be well described by a single exponential fit for the data starting at 2 s after the onset of the depolarization. This fitting procedure should minimize the contributions of the faster inactivating components, including IA and ID, to the time constant of the slowly decaying current. On average, the transient outward current elicited at the holding potential of –50 mV had a mean decay time constant of 3.439 ± 0.507 s (n = 8). The amplitude of the slowly inactivating outward current was determined by extrapolating the exponential curve fitted for the decay of the current to the onset of the depolarization. The current–voltage (I–V) relationship shows that the slowly inactivating outward current had a threshold of about –60 mV (Fig. 1Ac). Additional depolarizations beyond –60 mV elicited a steep rise in the amplitude of the current. Depolarizations were not made to exceed –30 mV because K+ currents other than the transient ones were activated beyond this level (Numann et al. 1987; Storm 1990).
The voltage dependency of the inactivation of the slowly inactivating outward current was examined. Cells were held at –45 mV. Hyperpolarizing prepulses to –105 mV in 15 mV increments were used to activate the current at the holding potential (Fig. 1, Ba and Bb). The I–V plot (Fig. 1Bc) shows that the current amplitude increased with increases in the level of the hyperpolarizing prepulses. The plot indicates that the slowly inactivating current was completely inactivated at about –45 mV and that the inactivation was fully removed at about –105 mV.
Pharmacological and ionic properties of the slowly inactivating outward current
The transient outward current was elicited at a holding potential of –30 mV after a hyperpolarization to –70 mV (Fig. 2Aa). The amplitudes of the slowly inactivating outward currents activated in TTX (Fig. 2, Aa, Ba, and Ca) were compared with those obtained after addition of Cs+ (1 mM; Fig. 2Ab), or 4-AP (1 mM; Fig. 2Bb) to the perfusate, or after switching the perfusion to a solution containing Cs+ (1 mM), Mn2+ (0.5 mM), and low Ca2+ (0.2 mM; Fig. 2Cb). Summary data provided in Fig. 2D show that these broad-spectrum potassium channel blockers did not significantly affect the amplitude of the slowly decaying outward current.
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The sensitivity of the slowly inactivating outward current to K+ channel blockers was further tested at different membrane potentials with a voltage protocol similar to that used for the measurement of the current reversal potential (Fig. 4Aa, inset). The current amplitudes before (Fig. 4, Aa and B, hollow circles) and after addition of 4-AP (5 mM; Fig. 4, Ab and B, filled circles, and 4C, filled histogram) were similar in the range –90 to –45 mV. In contrast, subsequent addition of tetraethylammonium (TEA, 50 mM; Fig. 4, Ac and B, gray squares) significantly inhibited the slowly inactivating current (by nearly 24% at –45 mV; Fig. 4C, gray histogram).
The hyperpolarizing pulse applied before the depolarization to fully deactivate the slowly inactivating K+ current also turned on the hyperpolarization-activated inward current (Ih; Fig. 5Aa). We tested a possible contribution of Ih deactivation to the slowly inactivating outward current. The currents elicited by a hyperpolarization to –90 mV followed by a depolarization to –40 mV in the presence of TTX (1 µM) were recorded before (Fig. 5Aa) and after (Fig. 5Ab) application of the selective Ih blocker ZD7288 (100 µM). ZD7288 clearly blocked Ih but did not significantly alter the amplitude of the slowly inactivating outward current (Fig. 5, A and B).
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Figure 7 shows that the slowly inactivating outward current was suppressed by the inclusion of QX-314 (5 mM), a quaternary derivative of the local anesthetic lidocaine, to the intracellular recording K+ gluconate solution (QX-314 solution). A depolarization from –100 to –30 mV delivered soon after the break-in of a CA3 neuron in the whole cell configuration using the QX-314 solution elicited a transient outward current (Fig. 7A, 2 min). With time, the amplitude of the transient outward current decreased (Fig. 7, A and B). Suppression was most obvious for the slowest decaying component, i.e., the slowly inactivating outward current, with the longest decaying time constant. The gradual suppression of the current probably reflected the progressive diffusion of the recording pipette solution into the intracellular compartment because recordings of the slowly inactivating outward current were stable in K+ gluconate solution (e.g., Figs. 1–5, and 8A). The suppression of the slowly inactivating outward current by the QX-314 solution was seen at all membrane potentials examined (Fig. 8, A and C, filled circles). The QX-314 solution also blocked the slowly inactivating outward current in the presence of extracellular TTX (1 µM), Cs+ (1 mM), Mn2+ (0.5 mM), low Ca2+ (0.2 mM), 4-AP (5 mM), and TEA (50 mM; Fig. 8, B and C, filled squares).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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). The slowly inactivating outward current had a reversal potential close to the equilibrium potential for K+, was resistant to extracellular Cs+, was partially suppressed by TEA, and was blocked by intracellular Cs+. Furthermore, the current was not suppressed by the extracellular Mn2+/low Ca2+ solution, distinguishing it from the Ca2+-dependent, slow decaying outward current observed in dissociated CA1 pyramidal cells (Numann et al. 1987). The lack of effect of the Mn2+/low Ca2+ perfusing solution on the amplitude of the slowly inactivating K+ current suggests that the current is not gated by intracellular calcium. Although the Mn2+/low Ca2+ solution may not have blocked all transmembrane Ca2+ entry by the voltage-gated Ca2+ channels, it should have suppressed a significant portion of such Ca2+ entry. The finding that the time course of the slowly inactivating K+ current was not altered by the Mn2+/low Ca2+ solution makes it unlikely that the current is dependent on Ca2+ entry because a commonly noted property of Ca2+-dependent K+ currents is their sensitivity to suppression of voltage-gated Ca2+ currents (Storm 1990). The results also indicate that intracellular QX-314 (5 mM) is an effective blocker of the slowly inactivating outward current (Figs. 7 and 8).
QX-314 is known to be an effective blocker of the voltage-dependent Na+ current (Connors and Prince 1982; Isaac and Wheal 1993). In addition, the agent is effective against the hyperpolarization-activated cation current Iq (Perkins and Wong 1995), the Ca2+-activated K+ current (Oda et al. 1992), the K+ leak current (Perkins and Wong 1995; Segal 1988; for review see Kindler and Yost 2005), the G-protein–coupled K+ current (Andrade 1991; Nathan et al. 1990), and Ca2+ currents (Talbot and Sayer 1996) in hippocampal cells. Thus the QX-314 action on the slowly inactivating outward current is not unique and its use as a pharmacological tool against this current is limited. Further studies on the dose–response properties of QX-314 action against each of the above-mentioned ionic channels will be required to facilitate the use of the agent as a selective blocker.
There are at least two known mechanisms whereby QX-314 suppresses the currents mentioned above. The agent directly interacts with the channel protein and blocks ion flow (e.g., Na+ channel; Ragsdale et al. 1994) or, for G-protein–gated channels, QX-314 interferes with the G-protein function (e.g., G-protein–coupled inward rectifier K+ channel; Hollmann et al. 2001). Our results do not provide indications on how QX-314 blocked the slowly inactivating potassium current. Furthermore, the data do not eliminate the possibility that the current is perpetually gated by G-protein activation by transmitters such as adenosine (Alzheimer and ten Bruggencate 1991; Ikeuchi et al. 1996). However, this seems unlikely in view of the finding that the Mn2+/low Ca2+ perfusing solution, which suppresses Ca2+-dependent transmitter release, did not affect the amplitude of the slowly inactivating K+ current (Fig. 2, C and D).
The identification of QX-314 as an effective blocker of the slowly inactivating outward current is useful in two ways. First, it adds to the pharmacological identity of the relatively unexplored current. Second, it provides support for a voltage-dependent conductance as a mechanism underlying the current. The slowly inactivating outward current was observed only after prolonged hyperpolarizing prepulses. Such voltage perturbation can cause shifts in transmembrane ionic concentration gradients resulting in voltage-independent current flow. This issue has been raised with respect to the generation mechanisms of IK(slow) (Lüthi et al. 1996). The blockade of the slowly inactivating outward current by QX-314 suggests that ionic shifts during the prolonged hyperpolarizing prepulse per se cannot account for the generation of the current.
Recent studies show that the IA and ID components of the transient outward current are targets for synaptic and pharmacological modulation. Modulation of IA has been emphasized as a mechanism for synaptic plasticity in CA1 pyramidal cells (Ramakers and Storm 2002; Watanabe et al. 2002; Yuan et al. 2002). Activation of cannabinoid receptors reduces both IA and ID (Mu et al. 1999). In addition, Wu and Barish (1999) showed that metabotropic glutamate receptor (mGluR) activation suppressed ID by increasing the rate of inactivation. A similar effect of mGluR activation on IK(slow) has also been described (Lüthi et al. 1996). However, the data regarding a specific action of mGluR stimulation on IK(slow) may be complicated by the fact that mGluR agonists have multiple effects on hippocampal cells. In particular, the activation of a slow inward current (Chuang et al. 2001) can emerge to overlap with the time course of IK(slow) thereby causing an apparent accelerated decay of IK(slow). QX-314 may be applied in this case to separate the overlapping actions of mGluR activation and to evaluate its specific action on the slowly inactivating outward current.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Bianchi, Department of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203 (E-mail: rbianchi{at}downstate.edu)
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REFERENCES |
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Andrade R. Blockade of neurotransmitter-activated K+ conductance by QX-314 in the rat hippocampus. Eur J Pharmacol 199: 259–262, 1991.[CrossRef][ISI][Medline]
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Ikeuchi Y, Nishizaki T, Mori M, and Okada Y. Adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor in hippocampal neurons. Eur J Pharmacol 304: 191–199, 1996.[CrossRef][ISI][Medline]
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, n = 5; , n = 7; 
, n = 5; 
, n = 4). Current was significantly suppressed by QX-314 at voltages > –50 mV in both experimental conditions (2-way ANOVA, P < 0.001; post hoc Newman–Keuls test: 
