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Introduction
Ca2+/calmodulin-dependent protein phosphatase 2B
(calcineurin) is widely distributed within the brain with the
highest levels in the hippocampus and
striatum[1,2]. It has been found that calcineurin regulates ion channel activities,
neurotransmitter release, synaptic plasticity, and gene
transcription through dephosphorylation of a variety of target
proteins[2,3]. Calcineurin-mediated dephosphorylation plays
important roles not only in normal neuronal functions, but
also in pathological processes in the
brain[1,4-6]. For instance, both ischemic insults and kainate-induced seizures were
demonstrated to cause Ca2+-dependent activation of
calcine-urin, which resulted in surface translocation of the Kv2.1
channel in rat cortical neurons and marked enhancement of
the delayed rectifier K+ current
(IK)in cultured rat hippocampal
neurons[5,6]. The changes were thought to be a novel
compensatory mechanism, which suppresses neuronal
hyperexcitability and excitotoxicity in the pathological
conditions.
Immunosuppressant drugs cyclosporin A (CsA) and
tacrolimus (FK506) are the specific inhibitors of calcineurin.
The drugs were found to bind to immunophilins cyclophilin
A and FKBP12, respectively, within neurons. Both the
CsA/cyclophilin A and FK506/FKBP12 complexes specifically
inhibit the phosphatase activity of
calcineurin[7]. Nowadays, the drugs are routinely used as research tools to elucidate
the functional roles of calcineurin in the brain. Thus far,
however, interests have been focused on their actions on
individual target proteins, such as ion channels, G
protein-coupled receptors, and other
proteins[2,3]. It is unclear whether and how the drugs affect the excitability of neurons in the
brain. The aim of the present study was to investigate the
actions of CsA and FK506 on the intrinsic membrane
properties of CA1 pyramidal neurons in rat hippocampal slices.
The ionic basis underlying the actions was further addressed
in acutely dissociated hippocampal neurons.
Materials and methods
Materials CsA and other reagents were purchased from
Sigma-Aldrich China. FK506 was kindly provided by the
representative of Fujisawa Pharmaceutical in
Shanghai. CsA and FK506 were dissolved in absolute ethyl alcohol to
prepare stock solutions, with a concentration of 10 mmol/L, which
were diluted to the desired concentrations before
use. The concentration of ethanol in the final dilution was less than
0.1% and had no observed effect on the membrane
properties and voltage-activated K+ currents of hippocampal CA1
pyramidal neurons.
Experiments on hippocampal slices Sprague-Dawley rats
(5_9 d old) were obtained from the Shanghai Experimental
Animal Center, Chinese Academy of Sciences (Shanghai,
China). Transverse hippocampal slices (400 µm) were cut in
ice-cold artificial cerebrospinal fluid (ACSF) using a M752
vibroslice (Campden Instruments, UK). The ACSF contained
the following (in mmol/L): 125 NaCl, 1.25 KCl, 1.25
KH2PO4, 2 CaCl2, 1
MgCl2, 26 NaHCO3, and 10 glucose, bubbled with a
gas mixture (95% O2 /5% CO2). The slices were incubated at
24_25 oC at least for 1 h, and then transferred to a submerged
recording chamber (Medical System, USA) perfused with
the ACSF at a rate of 2 mL/min at 30_32
oC. Recording electrodes (a tip resistance of 3_5
MΩ) were pulled from borosilicate glass pipettes (Sutter Instruments, USA), and filled
with a standard pipette solution containing the following (in
mmol/L): 140 KCl, 2 MgCl2, 1
CaCl2, 10 HEPES, and 10 EGTA 10 (pH 7.3 with KOH). As previously
described[8], the membrane potential of CA1 pyramidal neurons was monitored
using whole-cell current clamp recoding with an Axoclamp
2B amplifier (Axon Instruments, USA). Input resistance was
calculated as the slope of the current-voltage
(I-V) curve between the current amplitudes of -50 and +50
pA[9]. Depolarizing current pulses with a 300 ms duration were injected
to elicit a train of action potentials once every 30 s. The first
action potential in each train was used to compare the action
potential shape. Signals were filtered at 2 kHz and sampled
at frequencies of 10_40 kHz using pClamp 9.0 software via a
DigiData-1322A interface (Axon Instruments, USA). FK506-
and CsA-containing ACSF was delivered through perfusion.
Experiments on dissociated hippocampal neurons
Transverse hippocampal slices (500
µmol/L) were cut in oxygenated ice-cold dissociation solution containing the following
(in mmol/L): 82 Na2SO4, 30
K2SO4, 5 MgCl2, 10 HEPES, and 10
glucose (pH 7.3). The slices were incubated in the solution
containing protease XXIII (3 g/L) for 8 min at 32
oC and then placed in the solution containing trypsin inhibitor type II-S
(1 g/L) and bovine serum albumin (1 g/L) under an oxygen
atmosphere at 24_25 oC. The slices remained viable at least
for 5_6 h. When neurons were needed, the CA1 regions
were dissected from 3_4 slices and triturated using a series
of fire-polished Pasteur pipettes with decreasing tip
diameters. Dissociated neurons were placed in a recording
dish and superfused with a standard external solution
containing the following (in mmol/L): 135 NaCl, 5 KCl, 1
CaCl2, 2 MgCl2, 10 HEPES, 10 glucose, and 0.001 tetrodotoxin (pH 7.3
with NaOH) at 24_25 oC. Whole-cell voltage-clamp
recording was made from large pyramidal-shaped neurons using
an Axopatch 200A amplifier (Axon Instruments,
USA). Voltage protocols were provided by pClamp 9.0
software. Series resistance was compensated by
75%_85%. Linear leak and residual capacitance currents were subtracted online using
a P/4 protocol.
CsA-containing external solution was delivered to the
neuron using RSC-100 rapid solution changer with a 9-tube
head (BioLogic, France). For the intracellular dialysis, CsA
in the pipette solution was diffused into the recorded
neuron immediately after the patch membrane
ruptured[10]. The low
Ca2+ external solution was similar to the standard
external solution, except that the concentration of
CaCl2 was reduced to 0.25 mmol/L and nifedipine (5 µmol/L) was
included. The composition of the 0
Ca2+ pipette solution was similar to that of the standard one, but
CaCl2 was replaced with equimolar
MgCl2.
Data analysis Data are presented as mean±SEM. The
duration of the action potential was measured at
half-maximal spike amplitude (half-height width). The amplitude of
IK was measured at 300 ms latency. For analyzing the
voltage-dependence of steady-state activation or inactivation of the
IK, normalized conductance or current was plotted against
the membrane potential and fitted to the Boltzmann equation:
Y=1/(1+exp[{V-V1/2}/
k]), where Y is the normalized conductance or current,
V is the test potential,
V1/2 is the voltage at half-maximal activation or inactivation, and
k is the slope factor. The time course of the recovery of
IK from inactivation was fitted with a mono-exponential function:
I/Imax=A*(1-exp[-Δt
/t]), where Imax is the maximal current amplitude,
I is the current after a recovery period of
Δt, t is the time constant, and A is the amplitude coefficient.
Statistical significance was assessed using a Student's
paired or unpaired t-test or ANOVA, as appropriate.
P<0.05 was considered significant. All analyses were performed
using the Prism 3.0 software.
Results
Effects of FK506 and CsA on intrinsic membrane
pro-perties of hippocampal neurons Perfusion with CsA (100
µmol/L) or FK506 (50 µmol/L) for 10 min did not significantly
alter the membrane potential and input resistance of the
neurons tested (Figure 1). The pooled data from the groups of
neurons are presented in Table 1. The drugs did not cause a
significant change in the firing pattern of CA1 pyramidal
neurons in response to depolarizing current pulses
(n=7 for CsA and n=6 for FK506; Figure 2A, 2B).
However, a closer examination revealed that both the
drugs caused moderate but consistent broadening of the
ac-tion potential without affecting its upstroke and amplitude
(Figure 2C). As shown in Table 1, the half-height width of
the action potential was increased by 18%±5%
(n=7, P<0.05) during perfusion with CsA (100 µmol/L), and by 19%±3%
(n=6, P<0.05) during perfusion with FK506 (50 µmol/L).
CsA selectively inhibits the
IK in hippocampal neurons FK506-induced spike broadening might be ascribed to its
inhibition on the
IK[11]. However, the mechanism underlying
CsA-induced spike broadening remains unclear. Thus, the
effects of CsA on voltage-activated K+ currents were
investigated in dissociated hippocampal neurons. As shown in
Figure 3A, the external application of CsA (100 µmol/L)
inhibited the IK, but did not affect the fast transient
K+ current (IA). The inhibition of
IK by CsA developed rapidly and reached a level of nearly 80% of the maximum inhibition within
20 s (Figure 3B). Moreover, the current only partially
recovered upon washing out for 2 min. Comparing the
I-V relationship of IK in the control and in the presence of CsA
reveals that the drug does not change the threshold for the
activation of the K+ current, but markedly reduces its
amplitude over the entire activation range (Figure 3C). In the
presence of CsA (100 µmol/L), the relative amplitudes of the
IK
(I/I0) elicited with depolarizing steps to 0, +20, +40, and
+60 mV were 70.9%±6.1%, 76.4%±4.3%, 76.7%±2.8%, and
73.1%±3.3%, respectively (n=5,
P>0.05, ANOVA), suggesting that the inhibition was voltage-independent. The
threshold concentration of CsA was 10 µmol/L (Figure 3D). At 30
and 100 µmol/L, the drug inhibited the K+
current by 25.5%±1.9% and 34.6%±4.8% (n=5 for each), respectively. At
concentrations above 100 µmol/L, CsA was not completely
dissolved.
Effects of CsA on the kinetic properties of the
IK in hippocampal neurons The external application of CsA did not
significantly alter the voltage-dependence both of the
steady-state activation and inactivation of the
IK (Figure 4A, 4B). In the presence of CsA (100 µmol/L), the value of
V1/2 of activation was changed from -0.4±4.3 mV to -2.5±2.9 mV
(n=6, P>0.05), whereas the value of slope factor k of activation was
almost identical (from 16.5±1.2 to 16.0±0.7,
n=6, P>0.05). The value of
V1/2 of inactivation was changed from -87.4±2.4 mV
to -92.9±1.2 mV (n=5, P>0.05), whereas the value of slope
factor k of inactivation changed from -11.6±0.6 to -12.7±2.0
(n=5, P>0.05). The external application of CsA (100 µmol/L)
also did not alter the time course of recovery of the
IK from inactivation (Figure 4C). In the control and in the presence
of CsA, the time constant of recovery was 301.9±8.9 ms and
314.9±16.4 ms, respectively (n=5,
P>0.05).
Lack of effect of intracellular dialysis of CsA on the
IK in hippocampal neurons CsA is membrane
permeable[3]. If externally-applied CsA had inhibited the
IK through the inhibition of calcineurin within neurons, the intracellular
dialysis of CsA would have caused similar inhibition. The
concentration of CsA for the intracellular dialysis was 100
µmol/L, which inhibited the K+ current by approximately 35%, when
applied externally (Figure 3D). After the patch membrane
was ruptured, the relative amplitudes of the
IK (I/I0
) in the neurons dialyzed with CsA were nearly identical to the
respective control values throughout the recording period of
10 min (Figure 5A). Instead, the intracellular dialysis of a
blocker of the IK, tetraethylammonium (TEA; 5 mmol/L)
caused progressive inhibition on the K+ current (Figure 5B).
At 10 min after the patch membrane was ruptured, the value
of I/I0 in the neurons dialyzed with TEA (49.5%±9.7%,
n=5) was significantly different from that in the control group
(88.6%±2.3%, n=6, P<0.05). The result suggests that the
inhibition of the IK by CsA should not be caused by the inhibition
of calcineurin.
Inhibition of the IK by CsA in low
Ca2+ conditions To further rule out the involvement of calcineurin in the
inhibition of the IK by CsA, we examined whether the inhibition
could occur under low Ca2+ conditions that block the basal
activity of calcineurin. The conditions were achieved with a
0 Ca2+ pipette solution containing EGTA (10 mmol/L) and a
low Ca2+ external solution that contained
Ca2+ (0.25 mmol/L) and nifedipine (5 µmol/L) to minimize the influx of
Ca2+ [17]. We found that the effect of CsA (100 µmol/L) on the
IK examined under low
Ca2+ conditions was almost identical to that
examined under the control conditions (Figure 5C). The value
of I/I0 under low
Ca2+ conditions was 75.2%±6.9%
(n=5), whereas that under control conditions was 70.3%±4.9%
(n=6, P=0.58 vs low
Ca2+ conditions).
Discussion
A large body of evidence has shown that CsA and FK506
exert modulatory actions on ion channels, G protein-coupled
receptors, and other target proteins in the brain through the
inhibition of calcineurin[2,3]. The present study demonstrates
for the first time that CsA (up to 100 µmol/L) and FK506
(up to 50 µmol/L) did not significantly alter the passive electrical
properties of native cortical neurons, but slowed down
repolarization of the action potential. The results differ from
that obtained in cultured cortical neurons, where the
perfusion of CsA (20 µmol/L) caused sustained depolarizing
responses with an increasing rate of spontaneous
firing[14]. The discrepancy is most likely due to the different
preparations used. In the present study, we further demonstrate
that CsA selectively inhibits the
IK in rat hippocampal neurons. Taken together with a similar result with
FK506[11], it is conceivable that the spike broadening caused by the
immunosuppressant drugs is due to the inhibition of the
IK.
An interesting finding in this study is that in addition to
acting through the inhibition of
calcineurin[5,6], CsA could exert a direct inhibition on the
IK in rat hippocampal neurons. Increasing evidence shows that FK506 could modulate the
activities of K+ channels without the involvement of
calcineurin. For instance, FK506 was found to directly
prolong the mean open time of the Ca2+-activated
K+ channel in cultured rat hippocampal
neurons[15]. FK506 was also found to prolong the duration of the action potential of rat
ventricular myocytes, which resulted from the direct inhibition
on the transient outward and the delayed rectifier
K+ currents[16,17]. In 2 recent studies, FK506 was demonstrated to
directly inhibit the IK in rat hippocampal CA1 pyramidal
neurons[11] and in the Kv1.3 channel expressed in CHO
cells[18]. Thus far, however, it is unclear whether CsA may directly
affect the K+ channels. In this study, we demonstrate that
the inhibition of the IK by CsA occurred without the
involvement of calcineurin. The intracellular dialysis of CsA was
ineffective (Figure 5A), suggesting that the inhibition of the
IK by CsA was not caused by the formed CsA/cyclophilin A
complex, or the subsequent inhibition of
calcineurin[7], but by the drug molecule itself. The inhibition persisted under
the low Ca2+ conditions (Figure 5C), which led to a low
nanomolar level of intracellular-free Ca2+
[12,18] and blocked the basal activity of
calcineurin[17].
It should be noted that CsA is much less potent than
FK506 for causing direct inhibition on the
IK. In contrast to FK506, which caused marked hyperpolarizing shifts of
steady-state activation and inactivation curves of the
IK[11], CsA at the highest concentration tested (100 µmol/L) did not
significantly alter the kinetic parameters of the
IK (Figure 4), suggesting that the mechanism of the inhibition of the
IK by CsA was different from that by FK506. The hyperpolarizing
shift of the steady-state inactivation curve by FK506 has
been proposed as the mechanism underlying its inhibition
on the Kv1.3 channel expressed in CHO
cells[18], and is probably responsible for the slowly developed inhibition on the
IK in hippocampal neurons, which accounts for approximately
60% of the maximum inhibition[11]. In contrast, the inhibition
of the IK by CsA had rapid onset, and immediately reached a
level of nearly 80% of the maximum inhibition (Figure 3B),
suggesting that CsA mainly acts as a blocker at the out mouth
of the delayed rectifier K+ channel.
In conclusion, the present study demonstrates that CsA
and FK506 do not significantly alter the passive electrical
properties of rat hippocampal pyramidal neurons, but slow
down the repolarizing phase of the action potential. Taken
together with our previous finding with FK506, it is
conceivable that the spike broadening caused by the
immunosuppressant drugs is due to the direct inhibition on the
IK.
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