L .-W. Gong et al. Brain Research 884 2000 147 –154
149
of 260 mV for at least 1 min. The composition of the flow unitary conductance, and sensitivity to extracellular TEA
21
solution that bathed the intracellular face of the patch and intracellular Ca
. The channels recorded were selec-
1
membrane after excision was in mM: 140 KCl, 10 NaCl; tive for K , as its channel current–voltage relations
21
10 HEPES. Free Ca concentrations of 0.01, 0.1, 0.5 and
reverse at 3.1762.3 mV n 518, very close to the equilib- 2 mM were obtained by adding a total of 55.7, 279, 435
rium potential for potassium ion E , with symmetrical
K 1
and 485 mM of CaCl , respectively, to a 500 mM-EGTA K
140 140 mM in both the pipette and bathing
2
Sigma solution [29]. For a solution with a desired free solution. The reversal potential shifted to 262.765.9 mV
1
calcium of 5 mM or higher, EGTA was omitted and CaCl n 517 with low concentration of 5 mM K
in pipette,
2 1
was added as necessary. Solutions were adjusted to a final indicating a high selectivity for K . Activity of BK
Ca
pH of 7.40 with KOH. Pipette solution consisted of either channels recorded was affected by the concentration of
21
the bathed solution to give symmetrical charge-carrier calcium ion at the intracellular side, [Ca
] , of excised
i 1
1
distribution, or low K solution 5 mM K
and 2 mM membrane patches Fig. 2B. Whereas less than micromo-
21 1
21
Ca to further examine channel selectivity for K .
lar concentrations of [Ca ] were sufficient to activate
i
MgCl at 0.5 mM was routinely added to the pipette
BK channels from the inner membrane surface, 100 mM
2 Ca
21
solution purely for the convenience of easier seal forma- Ca
could not activate the channels when applied only at tion in the absence of other divalent ions. All experimental
the extracellular membrane surface of excised membrane solutions were made using deionized water.
patches, and this was the case for all membrane potentials Sensitivity of the channel recorded to the external
tested from 250 mV to 150 mV. Current–voltage tetraethylammonium TEA, Sigma was examined by
relations showed that the channels recorded had high using inside-out configuration when TEA was included in
unitary conductance 245.44619.14 pS in symmetrical
1
the pipette as described previously [26]. 140 140 mM K
in inside-out patch; n 515. BK
Ca
channel in CA1 neuron was blocked by low concentration of external 0.5 mM TEA n 523 and was less sensitive to
2.4. Data collection and analysis 5 mM TEA n 521 applied to the internal side of the
patches. These points served as keys to the identification of The single-channel currents were recorded using a
the channels recorded as BK channels.
Ca
Nihon Kohden CEZ-2300 patch clamp amplifier, with the In addition to BK
channels, the membrane patches
Ca
current filtered 23 dB, four-pole Bessel filter at 1 kHz. typically contained other channels with smaller conduct-
Data were digitized at sampling rates of 10 kHz using a ance than that of BK
channels. These channels with
Ca
TL-125 kHz interface Scientific Solutions. The analysis smaller conductance will not be considered in this paper,
routines used PCLAMP version 5.5.1, Axon Instruments and we have typically selected records in which they are
to determine distributions for channel amplitudes, and not obviously present.
open and closed times. A 50 threshold criterion was used to determine the durations of open and closed events.
Logarithmic distributions of open and closed durations 3.1. Comparison of channel unitary conductance before
were exponentially fitted with the use of the least-square and after ischemia
algorithm method. The ignored level for detecting events was limited to 300 ms. Channel open probability P is
Fig. 1A shows original traces of single-channel current
o
obtained by dividing NP by N [43], and NP was defined from CA1 neurons before and after ischemia. Amplitude
o o
as: NP 5 oht 1 2t 1 2t 1 ? ? ? 1 nt j, where N is the histograms constructed from Fig. 1A exhibited that single
o 1
2 3
n
channel number, t , t , t are the ratios of open time to channel current was 4.89 pA in control neuron Fig. 1B
1 2
n
total time of measurement for each channel at each of the and 5.94 pA in post-ischemic neuron Fig. 1C at 2 mM
21
current levels [28]. Single channel data were obtained at [Ca
] with membrane potential of 120 mV. Unitary
i
room temperature 20–228C. The data in text are ex- current amplitude increased across the entire membrane
pressed as mean6S.D. and Student’s t-test was used for voltage range tested in both neurons from two groups. To
statistical analysis. compare unitary conductance of BK
channels between
Ca
two groups, amplitudes of the single-channel currents were measured at a number of different membrane potentials
and unitary conductance of BK channels was determined
Ca
3. Results by fitting a regression line through the data Fig. 1D. The
mean slope conductance of BK channels in post-is-
Ca
Successful recordings were made in 217 membrane chemic neurons 291.35611.48 pS, n 515 was signifi-
patches of CA1 pyramidal neurons obtained from 116 cantly higher than that in control neurons 245.44619.14
1
adult rats. Properties of BK channels in adult CA1
pS, n 515 P ,0.01 in symmetrical 140 140 mM K ,
Ca
pyramidal neurons were similar to those in other prepara- and the difference in unitary conductance of BK
chan-
Ca 1
tions: voltage dependence, high selectivity to K , high nels showed no voltage dependence.
150 L
Fig. 1. Comparisons of conductance and open probability P of BK channels in CA1 neurons before and after ischemia. A Traces of currents recorded
o Ca
showing a higher amplitude in neurons after ischemia lower as compared to control upper with membrane voltage held at 120 mV or 220 mV and 2
21
mM [Ca ] . Outward and inward currents evoked are shown as downward and upward deflections, respectively. The dotted lines indicate the current level
i
at which all channels were closed; dashes on the sides of the records denote unitary current amplitudes. Note also that a larger open probability P after
o
ischemia with two active BK channels in post-ischemic neuron and one in control neuron. B and C Amplitude histograms constructed from A
Ca
showing that single-channel current in post-ischemic neurons 5.93 pA, C was higher than that in control 4.96 pA, B. D Plots of amplitude I of BK
Ca
channels against membrane potential V in control d and post-ischemic m neurons showing a higher unitary conductance of BK channels after
Ca
ischemia as compared with control.
21
3.2. Comparison of voltage dependence and Ca 120 mV, BK
channels in both control and post-
Ca
sensitivity of BK channels before and after ischemia
ischemic neurons exhibited long-lasting openings while
Ca
multiple openings were frequently observed in neurons Fig. 1A shows that activity of BK
channels in post- after ischemia; at negative membrane potential 220 mV,
Ca
ischemic neurons lower trace was greater than that in more openings were seen in patches from post-ischemic
control neurons upper trace at fixed membrane potential than from control neurons. A larger open probability of
21
BK channels may be due to being more sensitive to
with 2 mM [Ca ] . At depolarized membrane potential
Ca i
L .-W. Gong et al. Brain Research 884 2000 147 –154
151
21
[Ca ] and or a sharper voltage dependence of BK
i Ca
channels in post-ischemic neurons. To analyze the differ- ences in voltage dependence of BK
channels between
Ca
two groups, individual P –V curves were fitted by the
o
Boltzmann equation
P 5 P hP
1 exp[1
o o,max
o,max 21
K V 2V ]j with 2 mM [Ca
] . The equation was
1 2 i
transformed into the logarithmic form, V 5 V 1 K 3
1 2
lnhP [P 1 2 P ]j, where K is the membrane depolar-
o o,max
o
ization for an e-fold increase in P , and V is the patch
o 1 2
potential at which P is one-half of the maximum P
o o
P . V
and K could be obtained by plotting lnhP
o,max 1 2
o
[P 1 2 P ]j against voltage Fig. 2A. The values of
o,max o
V and K were 2.662.1 mV and 17.060.7 mV n 510 for
1 2
CA1 neurons in control and 212.361.7 mV and 16.661.1 mV n 510 for CA1 neurons after ischemia, respectively.
Statistical analysis showed that the K value was not significantly different P .0.05 between two groups while
the V
was more negative P ,0.01 after ischemia when
1 2
compared with control.
21
To determine differences in sensitivity to [Ca ] of
i
BK channels between two groups, inside-out patch
Ca 21
recordings were obtained with different Ca concen-
trations at the cytosolic surface of the membrane. In
21
constructing this plot, data with each [Ca ] were aver-
i
aged from 15 different membrane patches at fixed mem- brane potential of 120 mV. A quantitative comparison of
21
sensitivity to [Ca ] of BK
channels is illustrated in
i Ca
21
Fig. 2B, where P is plotted as a function of [Ca ] . BK
o i
Ca
channels in CA1 neurons showed a concentration-depen-
21
dent increase in P as [Ca ] was raised from 0.01 mM to
o i
100 mM. P values in post-ischemic CA1 neurons were
o
larger than those in control neurons, especially when the
21
[Ca ] was between 0.5 and 2.0 mM Fig. 2B. The
i 21
[Ca ] required to half activate P 50.5 BK
channels
i o
Ca
was 2 mM in control neurons while it was only 1 mM after ischemia.
These results indicated that a larger open probability of BK
channels from CA1 pyramidal neurons after is-
Ca 21
chemia mainly reflected more sensitivity to [Ca ] , rather
i
than voltage dependence. 3.3. Comparison of kinetics of BK
channels before
Ca
and after ischemia
21
Kinetic analysis of BK channels was obtained from
Fig. 2. Comparisons in voltage dependence and Ca sensitivity of BK
Ca
Ca
channels in control and post-ischemic CA1 neurons. A Logarithmic
patches in which single-channel activities were observed.
21
Boltzmann fittings of P –V curves for BK channels in control d and
o Ca
At a given holding voltage and [Ca ] , the open prob-
21
i
post-ischemic m neurons with 2 mM [Ca ] . After ischemia, the patch
i
ability is determined by channels’ open time and open
potential required to produce one-half of the maximum open probability
frequency. To understand which one is the major com-
P was more negative than in control but the membrane depolarization
o
ponent contributing to the differences in open probability
for an e-fold increase in P showed no significant difference, indicating
o
no obvious changes in channel voltage dependence. B Plots of channel
between two groups, kinetics of BK channels were
Ca
21
open probability P against log [Ca ] with membrane voltage held at
o i
compared between control and post-ischemic CA1 neu-
120 mV in control d and post-ischemic m neurons showed that the
rons. Fig. 3 shows dwell time histograms constructed from
21
P –[Ca ] curves shifted to the left after ischemia, indicating an increase
o i
21
21
membrane patches held at 120 mV with 2 mM [Ca ] .
in [Ca ]
sensitivity of BK channels after ischemia. Values are
i
i Ca
The distributions of open and closed times of BK
means6S.D. The number of neurons in each group was 15. P ,0.05,
Ca
†
P ,0.01 compared with control Student’s t-test.
152 L
Fig. 3. Comparisons in kinetics of BK channels before and after ischemia. Distributions for open time A and C and closed time B and D of BK
Ca Ca
channels in control A and B and post-ischemic neurons C and D. All histograms of dwell times could be well-fitted by a two-exponential function. Time constants were
t 52.07 ms and t 514.36 ms in A, t 52.28 ms and t 528.14 ms in B, t 59.51 ms and t 569.63 ms in C, and t 51.47 ms
o1 o2
c1 c2
o1 o2
c1 21
and t 521.52 ms in D, respectively. V 5 1 20 mV; [Ca ] 52 mM for A–D. After ischemia, BK
channels had a longer open time and a shorter
c2 h
i Ca
closed time as compared to control.
channels from two groups could be fitted well by a two- differences were detected in open frequency before is-
exponential function Fig. 3. It was shown clearly from chemia: 51.2621.3 events s, after ischemia: 52.8622.3
the data summarized in Table 1 that the open time of BK events s, n 515, P .0.05, indicating a major contribution
Ca
channels was longer with a shorter closed time after of open time to difference in open probability before and
ischemia as compared with control, whereas no significant after ischemia.
Table 1
a
Kinetics of BK channels in CA1 neurons before and after ischemia
Ca
Open time constants ms Closed time constants ms
t t
t t
o1 o2
c1 c2
Before ischemia 2.7662.11
19.20610.10 2.6961.62
26.89617.88
† †
†
After ischemia 11.8066.38
51.97632.29 1.4960.90
16.72611.12
a †
Values are means6S.D. The number of neurons in each group was 15. P ,0.05, P ,0.01 compared with before ischemia Student’s t-test.
L .-W. Gong et al. Brain Research 884 2000 147 –154
153
4. Discussion suggest that one more explanation for the increased
