1Bloorview Epilepsy Research Laboratory, Division of Cellular and Molecular Biology, Toronto Western Research Institute; and Departments of 2Physiology, Medicine ( 3Neurosurgery, 4Neurology), and 5Pharmaceutical Science, University of Toronto, University Health Network, Toronto, Ontario M5T 2S8, Canada
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ABSTRACT |
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Pelletier, Marc R.,
Peter A. Pahapill,
Peter S. Pennefather, and
Peter L. Carlen.
Analysis of Single KATP Channels in Mammalian Dentate
Gyrus Granule Cells.
J. Neurophysiol. 84: 2291-2301, 2000.
ATP-sensitive potassium
(KATP) channels are heteromultimer complexes of
subunits from members of the inwardly rectifying
K+ channel and the ATP-binding cassette protein
superfamilies. KATP channels couple metabolic
state to membrane excitability, are distributed widely, and participate
in a variety of physiological functions. Understood best in pancreatic
cells, where their activation inhibits insulin release,
KATP channels have been implicated also in
postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a
brain region with a high density of KATP channels and is relatively resistant to ischemia/reperfusion-induced cell death.
Therefore we were interested in describing the characteristics of
single KATP channels in DG granule cells. We
recorded single KATP channels in 59/105
cell-attached patches from DG granule cells in acutely prepared
hippocampal slices. Single-channel openings had an
EK close to 0 mV (symmetrical
K+) and were organized in bursts with a duration
of 19.3 ± 1.6 (SE) ms and a frequency of 3.5 ± 0.8 Hz, a
unitary slope conductance of 27 pS, and a low, voltage-independent,
probability of opening (Popen,
0.04 ± 0.01). Open and closed dwell-time histograms were fitted
best with one (
open = 1.3 ± 0.2 ms) and
the sum of two (
closed,fast = 2.6 ± 0.9 ms,
closed,slow = 302.7 ± 67.7 ms) exponentials, respectively, consistent with a kinetic model having at
least a single open and two closed states. The
Popen was reduced ostensibly to zero
by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 µM,11/14
patches) and tolbutamide (20 µM, 4/6; 100 µM, 4/4 patches). The
blocking dynamics for glybenclamide included transition to a
subconductance state (43.3 ± 2.6% of control
Iopen channel). Unlike glybenclamide,
the blockade produced by tolbutamide was reversible. In 5/5 patches,
application of diazoxide (100 µM) increased significantly
Popen (0.12 ± 0.02), which was
attributable to a twofold increase in the frequency of bursts (8.3 ± 2.0 Hz). Diazoxide was without effect on
open and
closed,fast
but decreased significantly
closed,slow
(24.4 ± 2.6 ms). We observed similar effects in 6/7 patches after
exposure to hypoxia/hypoglycemia, which increased significantly
Popen (0.09 ± 0.03) and the
frequency of bursts (7.1 ± 1.7 Hz) and decreased significantly
closed,slow (29.5 ± 1.8 ms). We have
presented convergent evidence consistent with single
KATP channel activity in DG granule cells. The
subunit composition of KATP channels native to DG
granule cells is not known; however, the characteristics of the channel
activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.
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INTRODUCTION |
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ATP-sensitive K+
(KATP) channels are sensors of cellular metabolic
activity. Information concerning the metabolic state is transduced into
electrical information, which can influence the excitability of the
cell membrane: an increase in the ATP/ADP ratio closes
KATP channels, which produces membrane
depolarization (for reviews, see Ashcroft and Ashcroft
1990; Babenko et al. 1998
; Bryan and
Aguilar-Bryan 1997
; Davies et al. 1991
;
Quayle et al. 1997
; Seino 1999
). The
distribution of KATP channels is widespread, and
in peripheral tissues, they are present on cardiac myocytes (Noma 1983
), pancreatic
cells (Cook and Hales
1984
), vascular smooth muscle (Zhang and Bolton
1996
), and skeletal muscle (Spruce et al. 1985
).
Their distribution in the brain is also widespread, having been
identified in striatal cholinergic interneurons (Lee et al.
1998
), midbrain dopaminergic neurons (Guatteo et al.
1998
), CA1 pyramidal neurons, interneurons of the stratum
radiatum, glia, and dentate gyrus granule cells (Zawar et al.
1999
), globus pallidus and ventral pallidum (Gehlert et
al. 1991
), substantia nigra (Mourre et al.
1991), and pituitary (Bernardi et al. 1993
).
Activation of KATP channels has been linked to a
variety of physiological functions including maintenance of resting
membrane potential and inhibition of insulin secretion in pancreatic
cells (Cook et al. 1988
), inhibition of dopamine
release (Zhu et al. 1999
), volatile anesthetic-induced
coronary arteriole dilation (Zhou et al. 1998
), both
termination and re-initiation of seizure activity (Klöcker
et al. 1996
) and postischemia cardioprotection (Bernardo
et al. 1999
) and neuroprotection (Lauritzen et al.
1997
; Reshef et al. 1998
; Takaba et al.
1997
; Wind et al. 1997
), and resistance against
the chronic metabolic stress-induced neurodegeneration observed in
Parkinson's and Huntington's diseases (Beal 1996
; Hanna and Bhatia 1997
).
Several studies have demonstrated that KATP
channel activation is protective against experimental
ischemia/reperfusion-induced damage (Guatteo et al.
1998; Heurteaux et al. 1993
; Wind et al. 1997
); this supports further the hypothesis that activation of KATP channels exists as a potentially important
therapeutic intervention for myocardial and cerebral ischemia
(Cason et al. 1995
; Grover 1997
).
The CA1 area of the hippocampus is a region of the brain that is
particularly vulnerable to ischemia/reperfusion-induced damage
(Smith et al. 1984
). Interestingly, the dentate gyrus
(DG), which is the site of the first synapse of the hippocampal
trisynaptic circuit and thus can exert a powerful influence on the
transfer of information in the hippocampus, possesses a higher density of KATP channels (Karschin et al.
1997
) and is an area in the brain that is relatively resistant
to ischemia/reperfusion-induced damage (Krnjevi
and
Ben-Ari 1989
). Therefore we were interested in describing the
characteristics of single KATP channels in DG granule cells. In this study, we have characterized the pharmacology and the kinetics of single KATP channels recorded
in cell-attached patches from DG granule cells in acutely prepared rat
brain slices.
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METHODS |
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Slice preparation and electrophysiology
Male Wistar rats (15-25 days of age) were anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ) and then decapitated. The brain was removed rapidly and placed for approximately 1 min in ice-cold, oxygenated (95% O2-5% CO2), artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 3.5 KCl, 2 CaCl2, 2 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose. A block of brain containing the hippocampus was fixed to an aluminum chuck using cyanoacrylate glue, and then coronal hippocampal slices (400 µm) were prepared with a Vibratome. After an incubation period of at least 1 h, slices were transferred to an interface-type chamber and perfused (1-2 ml/min) with oxygenated ACSF containing (in mM) 120 NaCl, 5 KCl, 2 CaCl2, 2 MgSO4, 26 NaHCO3, 10 glucose, 10 TEA, 2 CsCl, 1 4-aminopyridine (4-AP), and 0.0005 TTX. Experiments were conducted at room temperature (20-22°C).
Single KATP channel activity was recorded using
an Axopatch 200B amplifier in voltage-clamp mode (patch configuration)
in cell-attached patches from DG granule cells with thick-walled (0.66 mm) borosilicate glass pipettes (World Precision Instruments; Sarasota,
FL) that were pulled on a vertical electrode puller (Narishige, PP-83).
Pipettes had resistances of 6-8 M when filled with a solution
containing (in mM) 140 KMeSO4, 2 CaCl2, 0.0002 charybdotoxin (CTX), 10 TEA, 2 CsCl, 1 4-AP, and 0.01% BSA (300 mOsm, pH 7.3 with KOH). In accordance
with the instructions supplied by the manufacturer (Alomone Labs,
Jerusalem, Israel), CTX was prepared first as a stock solution (1 µM), which contained (in mM) 100 NaCl, 10 Tris, 1 EDTA, and 0.1%
BSA. Pipettes were positioned and lowered manually into the middle of
the granule cell layer using a hydraulic micromanipulator (Narishige;
WR-60). The electrode holder port was connected via tubing to a
three-way stopcock, a 3-ml syringe, and a sphygmomanometer. Constant
positive pressure (10 mmHg) was maintained during the incremental
descent into the DG cell body layer. When a cell was approached,
application, followed by the release, of negative pressure (40-60
mmHg) routinely produced high-resistance seals between the pipette and
the cell membrane ranging from 6 to 10 G
.
It has been well documented that there is a reduction over time,
referred to as rundown, of single KATP channel
activity when electrophysiological recordings are made from excised
patches, a phenomenon that has been generally attributable to the
disruption, or loss, of intracellular regulatory elements
(Quayle et al. 1997). Therefore we recorded single
KATP channel activity in the cell-attached configuration, which preserves the integrity of the intracellular milieu. In the cell-attached configuration, a positive pipette potential (Vp) causes the
transmembrane potential (Vm) to become more negative or hyperpolarized. Our pipette solution contained 140 mM
KMeSO4, and assuming an intracellular
concentration of 140 mM K+, the reversal
potential for K+
(EK) predicted by the Nernst equation
will be 0 mV. The single-channel openings we recorded reversed polarity
when the Vp was equal to approximately
70 mV, which served as an estimation of the resting membrane
potential (RMP). To test this directly, we performed whole cell
voltage-clamp recordings of DG granule cells (n = 6) with pipettes containing (in mM) 140 KMeSO4, 10 HEPES, 2 MgATP, and 0.1 EGTA (270 mOsm, pH 7.3 with KOH). By measuring
the zero-current membrane potential and the current response produced
by a hyperpolarizing voltage step (
10 mV, 200-ms duration), we
obtained values of
64.8 ± 1.7 (SE) mV and 112.2 ± 3.6 M
for the RMP and the input resistance, respectively.
Data were acquired at a sampling frequency of 5 kHz, recorded on video
tape (VR-10; Instrutech), filtered off-line at 2 kHz with an eight-pole
Bessel filter (Frequency Devices, Haverhill, MA), then digitized
(Axotape 2.02). Records were first idealized, then analyzed, using the
Fetchan and the pSTAT programs, respectively, of pCLAMP version 6.0.3 software (Axon Instruments, Foster City, CA). All records were
inspected visually followed by either manual or automatic event
detection using the 50% current amplitude as the threshold. Analyses
were performed on continuous records of single-channel activity ranging
in duration from 20 to 120 s. Amplitude histograms were fitted
with Gaussian distributions using the Simplex least-squares method.
Bursts of single-channel openings were defined as groups of channel
openings separated by closed periods five times longer than the time
constant for the brief closed times (Rowe et al. 1996).
This value served as the test step value and the burst delimiter in the
analysis of interburst interval and burst duration, respectively. Burst
duration was also assessed manually from both the digitized and the
idealized records, which produced similar results. The optimal
interburst interval was determined by plotting the number of channel
closings per burst versus time (Sigurdson et al. 1987
).
Open and closed dwell-time histograms were plotted with logarithmic
time axes, which were scaled to minimize vacant bins, then fitted using
the maximum-likelihood-estimation method (Sigworth and Sine
1987
). Inspection of the residuals revealed that they were
typically distributed symmetrically above and below zero and were of
small value. To promote the quality of fit, fitting limits were
selected that did not include those bins with large residual values.
The significance of kinetic models of increasing order was determined by assessing the logarithm of the likelihood ratio. The estimation of
kinetic parameters was based on approximately 9,800 (control, 11 patches), 5,400 (diazoxide, 5 patches), and 7,640 (hypoxia/hypoglycemia, 6 patches) single-channel events. Statistical
comparisons were made with the Student's t-test.
Differences were considered significant if P < 0.05. Data
are presented as means ± SE.
Drugs
Glybenclamide (Sigma-Aldrich Canada, Oakville, Ontario),
tolbutamide (Sigma-Aldrich Canada), and diazoxide (Tocris Cookson, Ballwin, MI) were dissolved in dimethyl sulfoxide, stored frozen as
aliquots of a stock solution [(in mM) 10, 100, and 100, respectively], then diluted in ACSF to the experimental concentration
and bath applied. These drugs reach their targets by partitioning into the lipid phase of the membrane (Kozlowski et al. 1989)
and have been demonstrated previously to be effective in cell-attached patches (Sturgess et al. 1988
; Trube et al.
1986
).
Hypoxia/hypoglycemia
To induce metabolic stress, we used a conventional
hypoxia-hypoglycemia protocol, which comprised exposing the slices for 5 min to ACSF that was bubbled with 95% N2-5%
CO2 and that had sucrose substituted for glucose
(Duffy and MacVicar 1996; Perez Velazquez et al.
1997
).
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RESULTS |
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Immediately after switching to the patch configuration and
Vp was equal to 0 mV, we observed
outwardly directed single-channel openings. A representative example of
the single-channel openings is presented in Fig.
1A. Amplitude histograms were
fit best with the sum of two Gaussians and showed that single separate
closed and open states could be distinguished clearly for this channel, which is illustrated in Fig. 1B. The amplitude of the
single-channel outward current decreased progressively as
Vp became more negative until no
channel openings were observed when Vp
was between 60 and
70 mV. When Vp
was more negative than
70 mV, single-channel openings were reversed
in polarity; this is consistent with values for RMP and
EK equal to approximately
70 and 0 mV, respectively. Patches typically contained a single active channel,
and we recorded similar single-channel activity in 59/105 patches.
Representative records of single-channel openings in a patch at
magnitudes of Vm ranging from
90 to
+10 mV are presented in Fig. 1C.
|
We did not observe any indication of current rundown during the
experiments, and in one patch, single-channel activity was recorded for
80 min, the longest time recorded. Linear regression analysis of the
current/voltage relation produced a unitary slope conductance of 27 pS,
which is presented in Fig. 1D. The probability of channel
opening (Popen) when
Vm was equal to 70 mV was 0.04 ± 0.01, which was not different when compared with the
Popen when Vm was equal to +10 mV (0.038 ± 0.01; 5 patches), suggesting that channel activation was
voltage-independent.
Single-channel openings were organized into well-delineated bursts,
which were apparent from visual inspection of both the digitized and
the idealized records. To characterize the burst behavior, we
determined first the interburst interval, which represents the minimum
value of a closed duration that separates the bursts. The optimal
interburst interval was 286.8 ± 13.1 ms, which is consistent with
a frequency of 3.5 ± 0.8 Hz. Burst duration ranged from 8.6 to
48.7 ms, with a mean duration of 19.3 ± 1.6 ms. At a greater gain
and faster time scale, as is presented in Fig. 1B
(inset), it can be seen that the open channel returns
briefly to the closed state within a burst, which is consistent with
two types of channel closure: brief closures within a burst and long closures that determine the interburst interval. Similar burst kinetics
for single KATP channels have been published
previously (Ashcroft et al. 1988; Kakei and Noma
1984
; Rorsman and Trube 1985
; Spruce et
al. 1985
).
Single-channel openings were blocked by sulfonylureas
One pharmacological criterion used in the identification of
KATP channels is inhibition by sulfonylureas
(Babenko et al. 1998). Tolbutamide and glybenclamide are
first- and second-generation sulfonylureas, respectively. Glybenclamide
is more efficacious compared with tolbutamide for producing
hypoglycemia and is used clinically in the treatment of hyperglycemia
associated with Type II diabetes mellitus. Sulfonylureas produce
hypoglycemia via a direct blockade of the sulfonylurea receptor (SUR)
associated with KATP channels located on
pancreatic
-cells, which results in an increase in the release of
insulin (Panten et al. 1989
; Schmid-Antomarchi et
al. 1987
). Glybenclamide and tolbutamide have been demonstrated
to block KATP channels in a variety of tissues
(Gopalakrishnan et al. 1999
; Katnick and Adams
1997
; Sturgess et al. 1985
; Zhang and
Bolton 1996
; Zünkler et al. 1988
) and, as
stated in the preceding text, are considered to be prototypic blockers
of KATP channels. Therefore we were interested in
assessing whether glybenclamide and tolbutamide would block the
putative single KATP channels we had recorded.
Single-channel activity was blocked in 2/6 patches by the bath application of 500 nM glybenclamide (data not shown). At a concentration of 10 µM, glybenclamide blocked single-channel opening in 11/14 patches. Associated with this blockade was the transition to a subconductance level (43.3 ± 2.6% of the control Iopen channel), lasting for 2-3 min, prior to the complete blockade of channel activity, which required 13-17 min of application and which was irreversible. A representative example of the blockade of single-channel activity produced by glybenclamide is presented in Fig. 2.
|
Single-channel activity was blocked in 4/6 and 4/4 patches exposed to 20 and 100 µM tolbutamide, respectively. Unlike glybenclamide, the blockade of single-channel activity produced by tolbutamide was somewhat faster, requiring 8-14 min of application and was readily reversible, requiring 5-10 min after return to control ACSF for the channel activity to return to control levels. In the example shown in Fig. 2C, the Popen for the single-channel activity was, save for a few openings, ostensibly reduced to zero after application of tolbutamide (20 µM) for 12 min but returned to a level not different when compared with control after return to control ACSF for 7 min.
Single-channel activity was increased by diazoxide
Diazoxide is a benzothiadiazine-derived antihypertensive with
potent hyperglycemic actions, resulting primarily from the inhibition of insulin release, an effect opposite to that of sulfonylurea drugs
(Levin et al. 1975). Diazoxide has been demonstrated to activate both type-I (SUR1)- and SUR2B-based KATP
channels but to inhibit SUR2A-based cardiac muscle
KATP channels (Ashcroft and Ashcroft
1990
; Seino 1999
). In 5/5 patches, we observed
an increase in single-channel activity after bath application of diazoxide (100 µM). The activation of single-channel activity by
diazoxide was rapid and occurred with a latency ranging from 2 to 3 min. A representative example of the single-channel activation produced
by diazoxide is presented in Fig. 3.
Diazoxide increased significantly
Popen to 0.12 ± 0.02. The
duration of bursts in the presence of diazoxide was 15.7 ± 3.1 ms, which was not different when compared with control; however, we did
observe a twofold increase in the frequency of bursts to 8.3 ± 2.0 Hz, which was significantly greater when compared with control.
Additionally, the increased burst behavior appeared to be organized in
clusters with a duration of several seconds and with an intercluster
interval of 20-30 s. There were no differences attributable to
diazoxide for either the EK or the
conductance of the single-channel openings (3/3 patches).
|
Single-channel activity was increased by hypoxia/hypoglycemia
Several studies have demonstrated that KATP
channels are activated by hypoxia, which produces an initial
hyperpolarization of the Vm, often by
more than 10 mV (Fujimura et al. 1997;
Krnjevi
and Leblond 1989
) and is thought to
contribute to the blockade of synaptic transmission associated with
hypoxia (Fujiwara et al. 1987
; Hansen et al.
1982
; Mourre et al. 1989
; Yamamoto et al.
1997
). We used a conventional protocol to induce
hypoxia/hypoglycemia in brain slices. Specifically, slices were exposed
for 5 min to ACSF that was bubbled with 95%
N2-5% CO2 and that had
sucrose substituted for glucose. In 6/7 patches, we observed an
increase in single-channel activity after 2-3 min of exposure to
hypoxia/hypoglycemia. Similar to the effect produced by diazoxide,
hypoxia/hypoglycemia produced increases in both
Popen (0.09 ± 0.03) and burst
frequency (7.1 ± 1.7 Hz), which were significantly different when
compared with control. There was no difference in burst duration
(19.5 ± 3.9 ms) when compared with control. The
hypoxia/hypoglycemia-induced activation of single
KATP channels persisted for up to 30 min after
reperfusion with control ACSF, the longest time we recorded. A
representative example of the hypoxia/hypoglycemia-induced activation of single KATP channels is presented in Fig.
4.
|
There were no differences attributable to the hypoxia/hypoglycemia
challenge in either the EK or the
conductance of the single channels (4/4 patches). Application of
glybenclamide (10 µM) during the reperfusion was without effect (3/3
patches) on the hypoxia/hypoglycemia-induced activation of
KATP channels (data not shown). Although the
activation of KATP channels by various methods is
often blocked successfully by sulfonylureas (e.g., Fujimura et
al. 1997; Guatteo et al. 1998
; Lee et al.
1998
), our observation of a loss of effectiveness of sulfonylureas on metabolic stress-induced KATP
channel activity has been reported previously in ventricular myocytes
(Findlay 1993
). Because of the rapid onset (2-3
min), modification of the protocol that we used to induce
hypoxia-hypoglycemia might permit the demonstration of blockade by
sulfonylureas of the metabolic stress-induced increase in channel
activity, e.g., repeated 1-min exposures to hypoxia-hypoglycemia
alternating with 5-min episodes of return to control ACSF.
Single-channel kinetics of KATP channels in DG granule cells
Cell-attached patches typically contained only a single active
channel, which permitted the estimation of parameters for a kinetic
model. The open-state histograms could be fitted with a single
exponential, and the fit was not improved significantly using the sum
of two exponentials. The closed-state histograms in contrast required
the sum of two exponentials to obtain an adequate fit, which was not
improved significantly, by the sum of three. Representative examples of
open and closed dwell-time histograms for the three recording
conditions are presented in Fig. 5. The
kinetic analyses suggest that the single KATP
channel activity we recorded in DG granule cells can be
characterized by a kinetic model with at least a single fast open state
with a time constant (open) of 1.3 ± 0.2 ms, a fast closed state with a time constant
(
closed,fast) of 2.6 ± 0.9 ms, and a
second, much longer, closed state with a time constant
(
closed,slow) of 302.7 ± 66.7 ms. A
summary of the channel kinetics for the single
KATP channels we recorded is presented in Table
1. Activation of
KATP channels by either diazoxide or
hypoxia/hypoglycemia had no effect on either
open or
closed,fast but decreased
significantly
closed,slow by an order of
magnitude, which is consistent with the significant increase in burst
frequency we observed for these treatments.
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DISCUSSION |
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We have recorded single-channel activity in DG granule cells from acutely prepared rat brain slices with pharmacological, electrophysiological, and kinetic features consistent with what has been published previously to be characteristic of KATP channels. The single-channel activity we recorded demonstrated the following: an EK close to 0 mV, consistent with the EK predicted by the Nernst equation for symmetrical K+ solutions; a low Popen, which was voltage-independent; openings that were organized into bursts; a unitary conductance of 27 pS; blockade by the sulfonylureas, glybenclamide, and tolbutamide; and activation by both diazoxide and by hypoxia/hypoglycemia-induced metabolic stress.
Included in the latter two points is fulfillment of two pharmacological
criteria that are generally accepted as being indicative of
KATP channels (Babenko et al.
1998). As stated in the preceding text, sulfonylureas represent
a class of hypoglycemics that increase insulin release by direct
inhibition of KATP channels on pancreatic
cells. Sensitivity to sulfonylureas is determined by the SUR subunit
and differences reflect channels composed of SUR1 (high affinity) and
SUR2A,2B (low affinity) subunits. The relative potency of sulfonylureas
has been studied in detail in SUR1-based channels in
cells where
glybenclamide (Ki = 4 nM) is three
orders of magnitude more potent when compared with tolbutamide
(Ki = 4 µM) in inhibiting whole cell
currents (Zünkler et al. 1988
). The Ki for glybenclamide and tolbutamide
in pancreatic
cells has generally been reported to be in the low
nanomolar and the low micromolar range, which is two orders of
magnitude lower when compared with the
Ki for SUR2A-based channels in cardiac
muscle (Davies et al. 1991
). We selected high
concentrations of sulfonylureas to maximally block
KATP channel activity and did not investigate systematically a concentration-response relation for the blockade of
single-channel activity produced by glybenclamide and tolbutamide. The
concentrations of glybenclamide (10 µM) and tolbutamide (20, 100 µM) we used are similar to those used in previous studies attempting
to characterize KATP channels (e.g.,
Fujimura et al. 1997
; Gopalakrishnan et al.
1999
; Guatteo et al. 1998
; Jiang and Haddad 1997
; Katnick and Adams 1997
; Lee
et al. 1998
). Consistent with previous observations
(Gillis et al. 1989
; Liss et al. 1999
; Zünkler et al. 1989
), the effect produced by
glybenclamide in our experiments was slower and irreversible compared
with tolbutamide, which was faster and reversible.
In contrast to what we observed, fast block kinetics with
subconductance states, i.e., resolvable conductance levels that are
intermediate between Iclosed and
Iopen channel, have not been reported
previously for glybenclamide. Unlike tolbutamide, Hill coefficients
greater than unity, e.g., 1.8 (Zünkler et al.
1988) and 1.5 (Sturgess et al. 1986
), have been
published previously for glybenclamide, which is consistent with
ligand-receptor interactions more complex than mass action, e.g.,
cooperativity. The transient subconductance state we observed might be
attributable to ligand/receptor interactions between glybenclamide
molecules and a novel splice variant of the SUR subunit comprising
native DG granule cell KATP channels, e.g.,
transition to a conformational state that partially occludes the pore
region. Subconductance kinetics has been described for a variety of
ligand-gated ion channels and might represent a fundamental mode of ion
channel operation (Gage 1988
). Blocking reactions where
subconductance behavior has been observed in other channels include the
blockade of Na+, large-conductance
Ca2+-dependent K+ (Maxi
K+) and skeletal muscle mechanosensitive channels
by veratridine (Wang et al. 1990
), dendrotoxin
(Lucchesi and Moczydlowski 1990
), and the aminoglycoside
antibiotic, neomycin (Winegar et al. 1996
), respectively.
The single-channel conductance of KATP channels
(symmetrical high K+) reported previously are
wide ranging and can be divided into small/medium (20-100 pS)- and
large-conductance (more than 100 pS) channels. Based on this
classification, the KATP channels, we recorded in
DG granule cells reside at the lower end of the conductance range for
small/medium-conductance channels. The majority of the
KATP channels described thus far have been
small/medium-conductance channels and have been recorded in vascular
smooth muscle including rabbit portal vein (25 pS) (Kamouchi and
Kitamura 1994) and pig coronary artery (35 pS) (Dart and
Standen 1995
), cardiac muscle (80 pS) (Findlay
1988
; Noma 1983
), and pancreatic
cells
(50-65 pS) (Ashcroft and Kakei 1989
; Cook and
Hales 1984
; Misler et al. 1986
).
Large-conductance channels have been recorded in ventromedial hypothalamic neurons (150 pS) (Ashford et al. 1990
) and
rabbit renal arterioles (258 pS) (Lorenz et al. 1992
).
The single KATP channels we recorded had a low
open-state probability (Popen of
0.04 ± 0.01), which was voltage independent. These observations
are consistent with what has been generally reported for
KATP channels (Dart and Standen
1995; Davies et al. 1991
; Qin et al.
1989
; Rorsman and Trube 1985
; Zhang and
Bolton 1995
). Voltage-dependent channel activity, which
increases with membrane depolarization, has also been reported
(Hunter and Giebisch 1988
; Spruce et al.
1985
; Sturgess et al. 1987
). The activity of
KATP channels has been generally described as
being relatively insensitive to Ca2+
(Ashcroft and Ashcroft 1990
); however, a type of
KATP channel associated with epithelial cells is
activated by micromolar concentrations of
Ca2+ (Hunter and Giebisch
1988
). We did not assess the
Ca2+-dependence of the KATP
channels we recorded.
In comparison to the members of the inwardly rectifying
K+ channel (Kir) 2.0, 3.0, and 4.0 subfamilies
(Doupnick et al. 1995), which show strong inward
rectification, members of the Kir6.0 subfamily confer only weak inward
rectification, which requires depolarization to potentials greater than
+50 mV (Inagaki et al. 1995b
). Similar to other
Kir-based channels, the rectification of KATP
channels is mediated by a voltage- and a
K+-gradient-dependent block by internal ions such
as Na+ and Mg2+
(Horie et al. 1987
; Woll et al. 1989
). We
did not observe inward rectification of the single-channel openings we
recorded. The most depolarized potential at which we recorded
single-channel activity was +30 mV, which may have been insufficient to
produce much rectification. Additionally, recording conditions with
symmetrical high concentrations of K+ typically
produce a near linear unitary current/voltage relation (Quayle
et al. 1997
).
Subunit composition of native KATP channels
KATP channels are heteromultimer complexes
of an ion channel and a receptor that are structurally unrelated (for
reviews, see Babenko et al. 1998; Bryan and
Aguilar-Bryan 1997
; Seino 1999
). Subunits
comprising functional KATP channels are members
of the Kir and the ATP-binding cassette protein (SUR) superfamilies, which are associated as tetramers with a 1:1 stoichiometry. The Kir
tetramer forms the pore region and determines the channel conductance
(Kir6.1-based channels possess smaller unitary conductance), whereas,
the SUR tetramer determines the sensitivity to nucleotides and to
sulfonylurea drugs (Inagaki et al. 1996
). The recent
cloning of Kir6.0 and of SUR has led to the identification of Kir6.1, Kir6.2 (Inagaki et al. 1995a
,b
; Sakura et al.
1995
) and SUR1 (high affinity), SUR2 (Aguilar-Bryan et
al. 1995
; Chutkow et al. 1996
; Inagaki et
al. 1996
; Isomoto et al. 1996
) subunits,
respectively. Additionally, two splice variants of SUR2 (also referred
to as SUR2A) have been identified and are referred to as SUR2B and
SUR2C (Ashcroft and Gribble 1998
).
The subunit composition of KATP channels native
to DG granule cells is not known. The electrophysiological (small
conductance), pharmacological (blockade by sulfonylureas, activation by
diazoxide), and physiological (activation by metabolic stress)
characteristics of the single-channel activity we recorded are
consistent with what has been described previously for Kir6.1/SUR1-,
SUR2B-based channels. Because both SUR1- and SUR2B-based channels are
activated by diazoxide, the use of selective K+
channel openers (KCOs) might differentiate between these two candidates. The KCOs, pinacidil, cromakilim, and nicorandil, are far
more selective for SUR2B-based channels, having little, or no, effect
in SUR1-based channels in pancreatic cells. On the other hand, both
SUR1- and SUR2B-based channels have been observed in the same cell
type, e.g., dopaminergic substantia nigra neurons (Liss et al.
1999
). To unequivocally identify the subunit composition of
native channels requires a combination of pharmacological and molecular
techniques. The KATP channels we recorded appear
to be distinct from those channels found in both rat and in human neocortical neurons where two distinct types have been described: S-KATP, small conductance (47 pS), strong inward
rectification (symmetrical K+), glybenclamide
sensitive and L-KATP: large conductance (200 pS),
weak rectification, glybenclamide sensitive, and
Ca2+ (µM) dependent (Jiang and Haddad
1997
).
It is known that different cell types display differential
susceptibility to metabolic stress such as that produced by hypoxia, that activation of KATP channels is protective
against ischemia/reperfusion-induced damage and that individual cells
may express different KATP channels (Zawar
et al. 1999). Therefore it is important to elucidate the subunit composition of the critical KATP channels
in the resistant cells. By using a combination of whole cell recording,
pharmacology, and RT-multiplex PCR, Liss et al. (1999)
reported that KATP channels in dopaminergic
midbrain neurons express both SUR1 and SUR2B subunits in combination
with a Kir6.2 subunit and importantly, the Kir6.2/SUR1 combination
confers the greatest protection against metabolic stress.
Single-channel kinetics of KATP channels
A prominent feature of KATP channel activity
is that the channel openings are organized into bursts (Ashcroft
et al. 1988; Davies et al. 1992
; Inagaki
et al. 1996
; Kamouchi and Kitamura 1994
;
Rorsman and Trube 1985
; Spruce et al.
1987
). Burst kinetics characteristic of single
KATP channels can be described in simple terms as
bursts of openings separated by longer interburst intervals (Ashcroft and Ashcroft 1990
). Differences in burst
kinetics have been attributed to the SUR subunits: SUR2A-based channels
display longer burst duration and interburst intervals when compared
with SUR1-based
-cell KATP channels
(Inagaki et al. 1996
).
Indicative of burst behavior, the closed dwell-time histograms
constructed for the single KATP channels we
recorded were fitted best with the sum of two exponentials and
consisted of a fast closed state, with a time constant
(closed,fast) of 2.6 ± 0.9 ms, which
represents the duration of the channel closings within a burst, and a
second, much longer, closed state with a time constant (
closed,slow) of 302.7 ± 66.7 ms, which
represents the duration of the much longer interburst interval. Our
analyses suggest that single KATP channel
activity in DG granule cells is consistent with a kinetic model with at
least a single fast open state, with a
open of
1.3 ± 0.2 ms, and at least two closed states. The kinetic behavior of KATP channels is complex and not
understood fully. A variety of kinetic models have been proposed
previously including models with a single open and a single closed
state (Qin et al. 1989
), at least two closed states
(Ashcroft et al. 1988
), and at least two open and three
closed states (Davies et al. 1992
; Spruce et al.
1987
). A fourth, long-duration closed state has also been
proposed (Davies et al. 1991
), which is reminiscent of
the increased burst behavior produced by diazoxide that we observed
where the increase in frequency of bursts appeared to be nested
together in clusters separated by long-duration intercluster intervals.
The long-duration closed states are difficult to resolve due primarily
to their low frequency and requires long-duration recording of channel activity.
Application of ATP to the intracellular face of an excised patch
containing KATP channels results in a reduction
of channel activity, which in cardiac myocytes has been attributed to a
prolongation of the interburst interval and to a shortening of the
burst duration (Kakei and Noma 1984). A similar
mechanism was described for the reduction of single
KATP channel activity produced by glucose in
pancreatic
cells (Ashcroft et al. 1988
).
Munemori et al. (1996)
reported that in inside-out
patches of frog ventricular myocytes, the decrease in
Popen produced by ATP was attributable solely to an increase the interburst interval. The activation of single
KATP channels in DG granule cells produced by
diazoxide and hypoxia/hypoglycemia we observed was attributable not to
an increase in burst duration but to a decrease in
closed,slow, which represents the interburst
interval and is consistent with an increase in the frequency of bursts.
Physiological roles of KATP channels
The tissue distribution of KATP channels is
widespread, and their physiological roles are equally diverse. The
coupling of cellular metabolism to membrane excitability produces a
variety of functional consequences in different tissues, e.g.,
regulation of local blood flow via effects on smooth muscle tone and
appetite control via effects on ventromedial hypothalamus neurons
(Ashcroft and Ashcroft 1990). In addition to being
regulated by the intracellular concentration of ATP,
KATP channels are modulated by a variety of
mechanisms including phosphorylation (Light 1996
) and
intracellular pH (Davies et al. 1992
). This is
particularly relevant in cardiac and skeletal muscle when even during
vigorous activity the concentration of intracellular ATP is maintained
at levels (5-10 mM) that are below the IC50 for
ATP, which is attributable to the buffering action of creatine
phosphate and creatine kinase (Carlson and Siger 1960
).
In light of their ubiquitous expression, ability to couple metabolism
to membrane excitability, and potential for modulation by a variety of
variables, it is not surprising that the therapeutic potential of
targeting KATP channels has been investigated in a variety of clinical conditions, e.g., reperfusion-induced cell death
and chronic metabolic stress-induced neurodegeneration implicated in
Parkinson's and Huntington's diseases (Lawson 2000).
KATP channels localized in pancreatic
cells
have been studied most and exist presently as an important therapeutic
target in the management of Type II diabetes. Glucose blocks the
activity of these KATP channels in a
concentration-dependent manner and results in membrane depolarization
and the activation of voltage-dependent Ca2+
channels (VDCCs), which produces a pattern of electrical activity consisting of slow oscillations in membrane potential between depolarized plateau phases with superimposed action potentials (bursts)
and repolarization phases (Ashcroft and Rorsman
1989
). While the voltage- and the
Ca2+-dependent inactivation kinetics of VDCCs
contribute to the interburst interval, it appears that the direct
modulation of KATP channel activity is the
primary mechanism for determining both the duration and the frequency
of bursts. Additionally, pancreatic
cells within an islet of
Langerhan are electrically coupled via gap junctions (Meda et
al. 1986
). Gap junctional communication permits the
transmission of electrical information rapidly throughout a local
syncytium of cells, which helps to synchronize the activity of the
coupled cells. Initiation of the electrical activity is attributable to
pacemaker cells, which possess characteristics that promote intrinsic
bursting, e.g., depolarized Vm, lower
spike threshold or higher glucose affinity.
KATP channel activity and seizures
The electrical behavior of cells is reminiscent of the
paroxysmal depolarizing shifts (PDSs) recorded intracellularly from principal cells in disinhibited brain slice preparations. The PDS is a
common feature of in vitro seizure models, and the clinical correlate,
the interictal discharge, is an electroencephalographic feature of an
epileptic brain (Hughes 1989
; Jensen and Yaari
1988
; Matsumoto and Marsan 1964
). Interestingly,
the same mechanisms, i.e., intrinsic membrane properties that
predispose cells to behave as pacemakers and electrical coupling via
gap junctions, promote synchronous activity in neurons and are believed
also to participate in various aspects of seizure activity
(McNamara 1994
; Perez Velazquez and Carlen
2000
; Prince and Connors 1986
); however, the
role of KATP channels in seizure activity has not
been investigated systematically. Because of the ubiquitous expression
of KATP channels in the brain and their role in
the maintenance of the RMP, in regions of the brain where there is a
high density of KATP channels in neurons coupled
by gap junction, modulation of these KATP
channels might influence seizure activity. Activation of the channels
would hyperpolarize the Vm, thus
taking the membrane further from the threshold for activation of VDCCs
and firing of action potentials. The former would decrease
Ca2+ influx, which might prevent the initiation
of Ca2+-dependent cell-death cascades
(Pelletier et al. 1999
). The latter might serve to
increase the seizure threshold of these cells or to limit the
propagation of the discharge. Another suggestive link between
KATP channel and seizure activity is the
influence of pH. Acidification of the intracellular pH has been
demonstrated to reduce the duration of epileptiform activity
(Xiong et al. 2000
), an effect that is thought to be
mediated in part by the closure of gap junctions (Perez
Velazquez et al. 1994
). In skeletal muscle
KATP channels, intracellular acidification
increased by an order of magnitude the
Ki for ATP (Davies et al.
1992
), an effect that would increase
Popen, which would hyperpolarize the Vm or produce a shunt current, or
both, consistent with an anticonvulsant influence. Conversely,
mechanisms that inhibit KATP channels might initiate an oscillation of the RMP, then following recruitment and
synchronization of a sufficient population of excitable cells, a local
epileptiform discharge would be initiated that may or may not be
propagated further.
The ability to influence membrane excitability also raises the
potential that modulation of KATP channels might
also influence the voltage-dependent mechanisms underlying nerve
conduction and the release of neurotransmitters (Parnas et al.
2000). Therefore it seems likely that
KATP channels localized in various regions of the
brain might exist as important therapeutic targets, and knowledge
concerning their molecular composition would promote the development of
drugs with selective actions on different tissues.
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ACKNOWLEDGMENTS |
---|
We thank F. Vidic for technical support.
This work was supported by grants from the Medical Research Council of Canada (P. A. Pahapill, P. S. Pennefather, and P. L. Carlen) and the Bloorview Epilepsy Program (M. R. Pelletier and P. L. Carlen).
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FOOTNOTES |
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Address for reprint requests: M. R. Pelletier, Bloorview Epilepsy Research Laboratory MCL 12-413, Div. of Cellular and Molecular Biology, Toronto Western Research Institute, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada (E-mail: pelch{at}uhnres.utoronto.ca).
Received 16 February 2000; accepted in final form 28 July 2000.
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REFERENCES |
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