Brain Research Phosphorylation

Brain Research 778 Ž1997. 107–119
Research report
Phosphorylation modulates the activity of the ATP-sensitive Kq channel in
the ventromedial hypothalamic nucleus
V.H. Routh
a
a, )
, J.J. McArdle a , B.E. Levin
b,c
Departments of Pharmacology and Physiology, UniÕersity of Medicine and Dentistry of New Jersey (UMDNJ), 185 S. Orange AÕenue, Newark, NJ
07104, USA
b
Department of Neurosciences, UniÕersity of Medicine and Dentistry of New Jersey (UMDNJ), Newark, NJ 07104, USA
c
Neurology SerÕice (127C), Departments of Veterans Affairs Medical Center, E. Orange, NJ 07018, USA
Accepted 12 August 1997
Abstract
Regulation of the ATP-sensitive Kq ŽK-ATP. channel was examined in cell-attached and inside-out membrane patches of freshly
isolated neurons from the ventromedial hypothalamic nucleus ŽVMN. of 7–14 day old male Sprague–Dawley rats. When inside-out
patches were exposed to symmetrical Kq, the reversal potential was y2.85 " 1.65 mV, the single channel conductance 46 pS, and the
total conductance varied as a multiple of this value. Glucose Ž10 mM. reversibly inhibited channel activity in cell-attached preparations by
81%. In the presence of 0.1 mM ADP, 10, 5, and 1 mM ATP reversibly inhibited VMN K-ATP channels in inside-out patches by 88, 83,
and 60%, respectively. This inhibition was not dependent on phosphorylation since 5 mM AMPPNP, the non-hydrolyzable analog of
ATP, reversibly inhibited channel activity by 67%. Relatively high concentrations of glibenclamide Ž100 mM. also reversibly inhibited
VMN K-ATP channel activity in cell attached and inside-out patches by 67 and 79%, respectively. Finally, the non-specific kinase
inhibitor H7 Ž200 mM. decreased channel activity by 53% while the non-specific phosphatase inhibitor microcystin Ž250 nM. increased
channel activity by 218%. These data suggest that while the inhibitory effect of ATP is not phosphorylation dependent, phosphorylation
state is an important regulator of the VMN K-ATP channel. q 1997 Elsevier Science B.V.
Keywords: ATP-sensitive Kq channel; Ventromedial hypothalamic nucleus; Phosphorylation; Glibenclamide; AMPPNP; Metabolism; Glucose
1. Introduction
The ventromedial hypothalamic nucleus ŽVMN. is a
key site for monitoring glucose status and initiating a
sympathoadrenal response. This control involves glucosensing neurons which increase their firing rate in the
presence of glucose w25x. Furthermore, glucose infusions
into the forebrain stimulate these neurons w7x and increase
sympathetic activity w16x. Although the mechanism by
which the VMN actually senses glucose is unknown, it is
likely to be similar to that of the pancreatic b-cell where
ATP inhibits a Kq channel ŽK-ATP.. When this channel
closes, the cell depolarizes and causes voltage sensitive
Ca2q channels to open. The resulting Ca2q influx ultimately causes insulin secretion w2x. This K-ATP channel is
the target for the anti-diabetic sulfonylurea drugs w1x.
)
Corresponding author. Fax: q1 Ž973. 972-4554; E-mail:
[email protected]
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 4 3 - 3
K-ATP channels are found in many tissues, including
brain w33x. Regulation of the activity of the K-ATP channel
in the periphery is very complex. For example, ATP is
known to exert a dual effect on the K-ATP channel; that is,
low levels of ATP are necessary to maintain channel
activity w34x, while higher levels are inhibitory w1x. Nucleoside diphosphates are also important regulators of K-ATP
channel activity w34x. Furthermore, protein kinases and
phosphatases have opposing effects on K-ATP channel
activity in a variety of peripheral tissues w5,15,21,22x.
Interestingly, the K-ATP channel described in the VMN
differs from that in the periphery. For example, it has a
reduced sensitivity to ATP w35x, a higher single channel
conductance w3x, and an insensitivity to sulfonylureas in
excised patches w4x. The present study was designed to
further investigate the regulation of this VMN K-ATP
channel. Our results are consistent with previous studies
indicating that the VMN K-ATP channel is less sensitive
to ATP than the peripheral K-ATP channel. However, we
found that single channel conductance may be similar to
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V.H. Routh et al.r Brain Research 778 (1997) 107–119
Punches were subjected to gentle trituration using firepolished Pasteur pipettes in order to isolate cells which
were then incubated in glucose free extracellular buffer for
30 min. This lack of glucose maximizes the possibility of
channel opening w3x.
2.2. Recording preparations
Fig. 1. Current–voltage relations for the K-ATP channel in an inside-out
patch of an isolated VMN neuron. The reversal potential for the VMN
K-ATP channel in symmetrical Kq was y2.85"1.65 mV Ž ns9. and
the single channel conductance was 46 pS Ž ns 7. at a holding potential
of 20 mV.
Single channel currents were studied in inside-out and
cell-attached membrane patches using patch clamp techniques w8x. Patch pipettes were drawn from 1.5 mm thick
walled borosilicate glass capillaries, coated with M-coat D
ŽMeasurements Group Inc., Raleigh, NC., and heat polished ŽMF-83, Narishige, Japan.. The patch pipettes had
resistances of 10–20 M V when filled with pipette solution. This solution contained ŽmM.: 140 KCl, 1.1 MgCl 2 ,
2.6 CaCl 2 , and 10 HEPES ŽpH 7.4.. The seals were
approximately 10 GV in resistance. The holding potential
was 0 and 20 mV for cell-attached and inside-out patches,
respectively. All experiments were carried out at room
temperature Ž21–258C..
2.3. Data collection and analysis
that found in the periphery and that high concentrations of
glibenclamide inhibit the activity of the VMN K-ATP
channel. Additionally, we present a model suggesting how
phosphorylation modulates the activity of the VMN K-ATP
channel as well as its sensitivity to ATP.
2. Materials and methods
2.1. Cell isolation
Brains from 8–18 day old Sprague–Dawley rats were
dissected and placed in ice-cold oxygenated Ž95% O 2r5%
CO 2 . isolation buffer. This buffer contained ŽmM.: 128
NaCl, 5 KCl, 1.2 NaH 2 PO4 , 2.4 CaCl 2 , 1.3 MgCl 2 , 26
NaHCO 3 , and 10 D-glucose ŽpH 7.4.. Brains were sliced
into 400 mm sections on a vibratome. Slices were then
enzymatically digested at room temperature for 20 min in
pronase Ž1 mgr6 ml oxygenated isolation buffer; Calbiochem, San Diego, CA. followed by 20 min in thermolysine Ž1 mgr6 ml oxygenated isolation buffer; Calbiochem.,
and then allowed to recover for 30 min in enzyme free
oxygenated isolation buffer. The VMN was punched with
a 500 mm blunt needle and placed in a small culture dish
in extracellular buffer. This buffer contained ŽmM.: 135
NaCl, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , and 10 HEPES ŽpH 7.4..
Recordings were made with an Axopatch 200A amplifier ŽAxon Instruments, Foster City, CA.. Single channel
data were stored on video tape using a pulse code modulator ŽMedical Systems Corp., Greenvale, NY. and subsequently analyzed via pClamp software ŽAxon Instruments..
Data were filtered at 2 kHz and digitized at 10 kHz. Due to
the fluctuations in channel open probability Ž Po ., channel
activity was quantified as the mean current Ž I . passed
through the K-ATP channel for 60 s segments of data;
I s NPori, where N is the number of channels and i is the
single channel conductance. Following a specified experimental protocol the data were expressed as the percent of
the peak response relative to the last recorded baseline
Žcontrol. value and analyzed using a 1 sample t-test ŽJMP
software, SAS Institute, Cary, NC.. Reversal potential for
inside-out preparations was determined from current–voltage plots derived from voltage ramps that ranged from
y50 to 50 mV. Both cell-attached and inside-out patches
were allowed to stabilize for at least 5 min prior to data
collection. Patches from cell-attached preparations were
exposed to glucose or glibenclamide for approximately
4–8 min before recording data and were allowed to recover during washout for up to 10 min. Inside-out patches
were exposed to the various treatments for 2–8 min and
allowed to recover for a similar time.
Fig. 2. The K-ATP channel in an inside-out preparation from an isolated VMN neuron has multiple subconductance states. This figure presents consecutive
records taken from the same inside-out patch. Conductance states are designated as integers from 1–5 at the right of each record. A: The VMN K-ATP
channel in control solution exhibits single channel conductances that are 2 to 5 times the magnitude of the 46 pS subconductance that is common to all
recordings of the VMN K-ATP channel. B: Although higher amplitude conductances appear occasionally when the K-ATP channel is exposed to 10 mM
ATPr0r1 mM ADP Župper record., it is the 46 pS subconductance that predominates under these conditions.
V.H. Routh et al.r Brain Research 778 (1997) 107–119
109
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V.H. Routh et al.r Brain Research 778 (1997) 107–119
2.4. Drugs
For inside-out patch preparations the control solution
contained 0.1 mM ATP and 0.1 mM ADP ŽKq salts,
vanadium free; Sigma Chemical Co., St. Louis, MO. in the
intracellular solution. This solution contained ŽmM.: 100
KCl, 40 KOH, 10 EDTA, and 10 HEPES ŽpH 7.2.. ATP,
ADP, and AMPPNP Žadenyl-5X-yl imidodiphosphate;
Sigma. were added to the intracellular solution within 1 h
of use. Glibenclamide Ž10 mM stock; Smith Klein
Beecham, King of Prussia, PA. was dissolved first in
DMSO followed by an equal volume of ethanol. The stock
solution was stored at 58C and used within 2 weeks.
Microcystin Ž25 mM stock; Bethesda Research Lab. ŽBRL.,
Bethesda, MD. was dissolved in 10% DMSO. H7 Ž20 mM
stock, BRL. was dissolved in water. Solutions were applied by gravity feed systems at a rate of approximately 1
ml miny1 .
3. Results
3.1. VMN K-ATP channel characterization
The current–voltage relations of Fig. 1 demonstrate that
the reversal potential for the K-ATP channel in inside-out
patches from isolated VMN neurons in symmetrical Kq
was y2.85 mV. The channel did not rectify in the Mg 2q
free solution used in this study. Single channel conductance was 46 pS for single channel openings at a holding
potential of 20 mV. This 46 pS conductance may represent
a subconductance state since larger amplitude ATP sensitive conductances were observed in the same patches.
However, while these larger conductances were multiples
of 46 pS, they varied in total magnitude Žfrom 2–5 times
the single channel conductance. from patch to patch, making it difficult to determine the total conductance of a
single channel. This is illustrated in Fig. 2 which shows
records of the K-ATP channel in a single inside out patch
from an isolated VMN neuron that was exposed first to
control solution ŽFig. 2A. and then to 10 mM ATPr0.1
mM ADP ŽFig. 2B.. There is a small current of slightly
less than 1 pA shown in the lower record in Fig. 2B.
According to Ohms law, a conductance of 46 pS produces
a current of 0.92 pA at a holding potential of 20 mV. The
records in Fig. 2A show discrete single channel conductances that range from 2–5 times this amplitude. When the
channel is closed by ATP the lower amplitude conductances predominate ŽFig. 2B.. However, as seen in the
upper record in Fig. 2B, higher amplitude conductances
appear occasionally.
Glucose Ž10 mM. reversibly inhibited the mean current
passed through the VMN K-ATP channel by 81% in
cell-attached preparations ŽTable 1.. The baseline Žcontrol.
mean current and the time required for mean current to be
affected by 10 mM glucose varied from patch to patch.
Since this was also the case for all of the other treatments,
the data in Table 1 were quantified as the peak response
compared to the last recorded stable baseline Žcontrol.
value. A decrease in mean current in response to 10 mM
glucose was detectable within 3 to 5 min in most neurons
and achieved a new stable level within 5–8 min. Recovery
occurred between 5 and 10 min following washout of
glucose. The records of Fig. 3A–C depict this effect for a
single cell-attached preparation from an isolated VMN
neuron. This figure demonstrates that the single channel
current amplitude declined following exposure to glucose
for all cell-attached preparations. This has been attributed
to the decrease in the driving force for potassium between
the cell and the patch pipette which is caused by the
depolarisation resulting from the closure of a large number
of K-ATP channels w3x.
The VMN K-ATP channel was subject to rapid rundown following excision of an inside-out patch. Reducing
Table 1
Summary of the effects of metabolic and pharmacologic agents on the mean current passed by the K-ATP channel in isolated VMN neurons
Treatment
Cell attached
10 mM Glucose
100 mM Glibenclamide
Inside-out patch
10 mM ATPr0.1 mM ADP
5 mM ATPr0.1 mM ADP
1 mM ATPr0.1 mM ADP
5 mM AMPPNPr0.1 mM ADP
100 mM Glibenclamide
200 mM H7
250 nM Microcystin
n
Percent change from control
p value
Mean " S.E.M.
Range
6
6
x81 " 5
x67 " 12
64–98
37–90
20
7
5
4
3
7
6
x88 " 3
x83 " 4
x60 " 18
67 " 12
x79 " 4
x53 " 8
≠218 " 54
57–100
56–95
0–100
34–87
72–84
26–78
83–411
0.00003
0.0114
- 0.00001
0.00005
0.028
0.006
0.0021
0.0008
0.0099
The data were calculated as mean current for 60 s segments of data. Mean current was then expressed as the percent of the peak response relative to the
last stable baseline Žcontrol. value. This percent change from control was analyzed using a 1 sample test.
V.H. Routh et al.r Brain Research 778 (1997) 107–119
111
Fig. 3. The K-ATP channel in a cell-attached preparation from an isolated VMN neuron is closed by glucose and glibenclamide. This figure presents
consecutive records from the same cell-attached patch preparation. A: the channel fluctuates and is primarily in the open state in the absence of glucose
Žcontrol solution.; B: the channel is primarily in the closed state and the current being passed through the channel is very low following 5 min exposure to
10 mM glucose; C: channel activity and the mean current passed by the channel recover to control levels 7 min following washout of glucose; D: channel
activity and mean current are reduced 4 min after 100 mM glibenclamide was added to the control solution; E: this response is reversed 5 min after
washout of glibenclamide.
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V.H. Routh et al.r Brain Research 778 (1997) 107–119
Fig. 4. The K-ATP channel in an inside-out patch from an isolated VMN neuron is closed by ATP. This figure presents consecutive records from the same
inside-out patch preparation. A: the channel is primarily open in the control solution of 0.1 mM ATPr0.1 mM ADP; B: channel activity and mean current
are reduced by approximately 50% after 5 min in 1 mM ATPr0.1 mM ADP; C: the response to ATP is reversed after 3 min in control solution; D: channel
activity and the mean current passed by the channel are virtually abolished after 4 min in 10 mM ATPr0.1 mM ADP.
V.H. Routh et al.r Brain Research 778 (1997) 107–119
the Mg 2q concentration to very low levels w13x and inclusion of 0.1 mM ATP with 0.1 mM ADP in the control
internal solution ŽF.M. Ashcroft, personal communication.
113
prevented rundown. ATP in the presence of 0.1 mM ADP
reversibly inhibited the VMN K-ATP channel ŽTable 1.; 1
mM, 5 mM, and 10 mM ATP decreased mean current by
Fig. 5. The inhibitory effect of ATP on the K-ATP channel in an inside-out patch from an isolated VMN neuron is independent of phosphorylation. This
figure presents consecutive records from the same inside-out patch preparation. A: the channel is primarily open in the control solution of 0.1 mM
ATPr0.1 mM ADP; B: channel activity is reduced and mean current decreases to 40% of control values after 1 min in 5 mM AMPPNPr0.1 mM ADP; C:
Channel activity and mean current recover completely after 2 min in control solution; D: channel activity and mean current are reduced to 15% of control
values following 3 min exposure to 5 mM ATPr0.1 mM ADP; E: channel activity and mean current recover to 150% of control after a 1 min washout.
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V.H. Routh et al.r Brain Research 778 (1997) 107–119
60%, 83%, and 88%, respectively. In most cases, the
response to ATP appeared within the first minute and
became stable by approximately 3 min. Occasionally, mean
current continued to decrease until the ATP concentration
was lowered Ž5–6 min. to control levels. Similarly, mean
current increased within the first minute after return to
control solution and became stable 1–4 min later. The
records of Fig. 4 illustrate the effect of ATP on the K-ATP
channel in a single inside-out patch from an isolated VMN
neuron. In this case, 1 mM ATP reduced mean current by
approximately 50% ŽFig. 4B. and 10 mM ATP completely
abolished it ŽFig. 4D.. Finally, 5 mM AMPPNP, the
non-hydrolyzable analog of ATP, inhibited channel activity by 67% ŽTable 1.. This effect followed the same
time-course as ATP. Fig. 5 depicts this effect for a single
inside out patch.
Fig. 6. The K-ATP channel in an inside-out patch of an isolated VMN neuron is closed by glibenclamide. This figure presents consecutive records from the
same inside-out patch preparation. A: the channel is open in control solution; B: channel activity and mean current are virtually abolished 3 min after 100
mM glibenclamide is added to the control solution; C: channel activity and mean current are greater than control values after 2 min wash in the control
solution.
V.H. Routh et al.r Brain Research 778 (1997) 107–119
Although these data indicate that half maximal inhibition should occur below 1 mM ATP, it is important to
consider the variability in the response at that concentration ŽTable 1: range s 0–100%.. While the K-ATP channel in 1 out of the 5 inside-out patches was completely
shut by 1 mM ATP, it immediately reopened at a level
50% of control and remained at that level during continued
exposure to 1 mM ATP. Moreover, of the other 4 inside-out
patches containing K-ATP channels, 1 was unaffected, 2
almost completely shut but also immediately re-opened to
control values in the presence of 1 mM ATP, and mean
current decreased by 44% in the last. Thus, 1 mM ATP in
the presence of 0.1 mM ADP may actually inhibit the
mean current passed through the VMN K-ATP channel
slightly less than the 60% average indicated in Table 1.
3.2. Effects of sulfonylureas on cell-attached and inside-out
patch preparations
Within 1–5 min glibenclamide Ž100 mM. reversibly
decreased the mean current of the VMN K-ATP channel
by 67 and 79% in cell-attached and inside-out patches,
respectively ŽTable 1.. After wash in the control solution
for 1–5 min mean current recovered fully. This effect is
depicted for the K-ATP channel in a single cell-attached
115
patch in Fig. 3C–E and for a single inside-out patch in Fig.
6A–C. Control experiments revealed that vehicle had no
effect on channel activity.
3.3. Effects of phosphorylation on inside-out patch preparations
The non-specific phosphatase inhibitor microcystin Ž250
nM. increased mean current by 218% ŽTable 1.. In all
cases the response to microcystin appeared within the first
minute and caused a new stable level of activity by 2 min.
Recovery following washout was variable. The records in
Fig. 7 show that the mean current passed through the
K-ATP channel in a single inside-out patch was approximately doubled when phosphatase activity was inhibited
by microcystin. In contrast, the non-specific kinase inhibitor H7 Ž200 mM. decreased mean current by 53%
ŽTable 1.. In general, a response to H7 appeared within the
first minute and a new stable level of activity was achieved
within 2 min. Although mean current was increased within
the first minute after washout of H7, full recovery to
control levels generally required 4 to 10 min. However,
some K-ATP channels responded more slowly as depicted
in Fig. 8A–C for a single inside-out patch where 7 minutes
were required for the full effect of H7 Ž8B.. In this case,
recovery required 10 min of wash in control solution ŽFig.
Fig. 7. The K-ATP channel in an inside-out patch from an isolated VMN neuron is opened by persistent phosphorylation due to inhibiting
dephosphorylation. This figure presents consecutive records from the same inside-out patch preparation. A: channel activity and mean current are relatively
high in control solution; B: channel activity and mean current have more than doubled 5 min after the non-specific phosphatase inhibitor, microcystin Ž250
nM., was added to the control solution.
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V.H. Routh et al.r Brain Research 778 (1997) 107–119
Fig. 8. The K-ATP channel in an inside-out patch from an isolated VMN neuron is closed by inhibiting phosphorylation. This figure presents consecutive
records from the same inside-out patch preparation. A: the channel is open in control solution; B: channel activity and mean current are reduced 7 min after
the non-specific kinase inhibitor H7 Ž200 mM. was added to the control solution; C: channel activity and mean current have recovered 10 min after
washout to control solution, although not to the levels of the initial control.
8C.. Control experiments revealed that vehicle had no
effect on channel activity.
4. Discussion
The reversal potential of approximately 0 mV in symmetrical Kq and the inhibitory response to glucose as well
as ATP served to identify the K-ATP channel of VMN
neurons. The fact that the VMN K-ATP channel did not
inwardly rectify in our Mg 2q free control solution is not
surprising since inward rectification is considered to be
Mg 2q-dependent w34x. In agreement with earlier reports
w3,35x, we found that the neuronal VMN K-ATP channel
was much less sensitive to ATP ŽmM vs. mM. than the
V.H. Routh et al.r Brain Research 778 (1997) 107–119
peripheral K-ATP channel and that the non-hydrolyzable
ATP analog AMPPNP inhibited it.
The single channel conductance of the VMN K-ATP
channel which we studied was 46 pS. This is significantly
lower than the 160 pS conductance reported previously for
this tissue w3x, and in fact is much more similar to that
found in peripheral tissues w1x. While there may be different subtypes of K-ATP channel in the VMN it is more
likely that the 46 pS conductance is a minimal subconductance state, with much larger conductances arising as
multiples of this value. Since the data from earlier studies
w3x was filtered at a lower frequency Ž1 kHz. than ours Ž2
kHz., it would have been more difficult to observe this
smaller conductance. Thus, it may be that a subconductance existed in these studies, but filtering prevented its
detection. This would be compounded by any decrease in
the signal to noise ratio. Also, it is possible that the
recording conditions used by these investigators favored
one specific multiple of the subconductance. Krouse et al.
w14x described a similar phenomenon for a large anion
selective channel in mouse alveolar cells which has six
open states that are multiples of a smaller 60–70 pS
conductance. They suggested that this is one channel with
six ‘co-channels’ in parallel that share a common gating
mechanism capable of opening or closing one or all of
them simultaneously. As discussed below, co-channels
may also mediate the conductance of the K-ATP channel
of VMN neurons.
While it has been reported that the VMN K-ATP channel in cell-attached preparations is insensitive to low doses
Ž500 nM. of glibenclamide w4x, we found that high doses of
glibenclamide Ž100 mM. reversibly inhibited channel activity by 67%. This suggests that sulfonylureas act at a low
affinity receptor on VMN neurons. In fact, both high
ŽSUR1. and low ŽSUR2. affinity sulfonylurea receptors
have been cloned from rat brain w11,32x. Binding studies
show that 15–20% of sulfonylurea receptors in the VMN
117
are low affinity and these are completely abolished when
neuronal cell bodies are selectively destroyed with ibotenic
acid w6x. This lesion has no effect on the remaining high
affinity sites which are probably on GABA or glutamate
containing nerve terminals. Interestingly, SUR2 sites are
virtually non-existent in the VMN of a rodent model of
dietary obesity w20x, suggesting that the VMN K-ATP
channel may play a role in energy balance. Additionally,
we found that 100 mM glibenclamide reversibly inhibited
VMN K-ATP channel activity in inside-out patches, while
others did not w4x. Since the inside-out patch contains only
the excised membrane, the sulfonylurea binding site appears to be very closely associated with the channel protein, as suggested previously w32x. It is unclear why our
data differ from those of previous studies.
In order for the VMN K-ATP channel to serve as a
neuronal glucosensor two conditions must be met. First,
the channel must respond to a physiological ATPrADP
ratio. Second, the concentration of ATP monitored by the
K-ATP channel must vary with peripheral metabolism. In
this study, a 10:1 ATP to ADP ratio inhibited the channel
by approximately 50%. Although intraneuronal levels of
ATP and ADP are not known in the VMN, this ratio is
similar to that found in cortical neurons w26x. Thus, the
VMN K-ATP channel responds to alterations in the ATP
to ADP ratio found in the brain. The second condition is
more difficult to evaluate, since changes in intracellular
ATP are not easily measured. Moreover, cytosolic ATP
levels are believed to be very highly buffered. However, it
is conceivable that the local concentration of ATP around
the channel, rather than the bulk cytosolic concentration, is
the critical regulatory factor w10x. There is evidence to
suggest that ATP may be compartmentalized within the
cell w9x allowing intracellular ATP levels to regulate the
VMN K-ATP channel w37x. This makes the channel a
viable candidate as a neuronal glucosensing mechanism.
Given the role of the VMN in autonomic activity and the
Fig. 9. Model for ATP regulation of the VMN K-ATP channel. K AT P indicates the channel in a non-phosphorylated and non-conducting state. K ATP P
results from the action of a cytoplasmic kinase ŽKNase-1.; the effect of KNase-1 vanishes when the patch is excised from the cell membrane. PHase-1 is a
membrane bound and Mg 2q-dependent phosphatase which converts K AT P P to K ATP . KNase-2 is a membrane bound kinase which phosphorylates K ATP P
to produce K AT P PP; PHase-2 is a phosphatase which reverses this process. Both K ATP P and K ATP PP can open to various subconductance states which are
determined by co-channels activation. These states are indicated by the letter suffix appended to OPEN. There is a corresponding OPEN state for K AT P P
and K AT P PP; i.e., OPENa – n and OPENaX – z . These OPEN states can be interconverted by either KNases 1 and 2 as well as PHases 1 and 2 or other Ž?.
similar enzymes. Because of the extra phosphate group, opening of co-channels is energetically favored for K AT P PP as compared to K ATP P. At the same
time, the affinity of open forms of K AT P PP for ATP is greater. Thus, our model predicts that while the mean current should be greater for K ATP PP, this
current will also be more sensitive to block by ATP. This form of feedback would provide for fine control of K-ATP channel activity.
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V.H. Routh et al.r Brain Research 778 (1997) 107–119
regulation of energy balance w7x, this channel could serve
as a link with glucose metabolism.
ATP exerts a dual effect on the K-ATP channel. In the
periphery, although ATP inhibits channel activity w12x, its
presence is also required for the maintenance of channel
activity w34x. Our data and that of others w3x showing that
the non-hydrolyzable analog of ATP, AMPPNP, mimics
the inhibitory effect of ATP on the VMN K-ATP channel
indicate that this inhibitory effect is not dependent on
phosphorylation. In contrast, the facilitating effect of ATP
is phosphorylation dependent w34x. In the periphery protein
kinases increase K-ATP channel activity w21,36x, while
protein phosphatases decrease channel activity w15,21x.
This is consistent with our data showing that inhibiting
phosphorylation decreased the activity of the VMN K-ATP
channel while inhibiting dephosphorylation increased activity. This may also explain the non-stationarity of the
VMN K-ATP channel w31x, as it does for other channels
w28x. Since our data were obtained using excised patches,
the regulatory kinases and phosphatases blocked in our
studies appear to be closely associated with the channel
protein andror the SUR2, both of which possess a number
of possible phosphorylation sites w11,22x.
Based on these phosphorylation data, we propose a
simple model for the regulation of the VMN K-ATP
channel ŽFig. 9. in which opposing actions of phosphatase
and kinase enzymes set a ‘steady state’ level of activity.
Phosphatase decreases phosphorylation and moves the
channel toward an inactive closed state. Kinase phosphorylates the channel, placing it in a more active state. When
one enzyme is blocked, activity of the opposing enzyme
predominates. While our data do not allow determination
of the type and number of kinases and phosphatases
involved, they do suggest that at least two sets of phosphatases and kinases are involved. When the channel is
excised into Mg 2q containing solution rundown occurs
w34x. This suggests the presence of a cytosolic kinase
ŽKNase-1. counteracting a membrane bound Mg 2q-dependent phosphatase ŽPHase-1. when the cell is intact. Excision of the patch strips the channel of the influence of the
kinase and the phosphatase dominates, producing rundown
ŽK ATP .. In contrast, when the channel is excised into
Mg 2q free solution, rundown does not occur. This is
presumably because the Mg 2q dependent PHase-1 is inactive. Thus, at excision, the channel exists in some phosphorylated state ŽK ATP P.. Since, at this point, blocking
phosphorylation decreases while blocking dephosphorylation increases channel activity, it seems likely that at least
one more set of Mg 2q-independent kinase and phosphatase
enzymes exist; i.e., KNase-2 and PHase-2 in Fig. 9. Conceptually, our model is not unprecedented. For example,
the activity of the K-ATP channel in kidney tubule cells is
inhibited by both okadaic acid-sensitive and Mg 2q-dependent phosphatases and stimulated by protein kinase A w15x.
In addition, K-ATP channels on ventricular myocytes are
similarly inhibited by a type 2A phosphatase and stimu-
lated by protein kinase C ŽPKC. w21x. Our model predicts
that the K-ATP channel can open a varying number of
co-channels from either of the phosphorylated states designated as K ATP P and K ATP PP. However, these two states
are envisioned to differ in two ways. First, the probability
that all co-channels are open increases with phosphorylation. Thus, the additional phosphate of K ATP PP provides
the energy allowing transition to the fully open state
ŽOPENz .. In contrast, the energy barrier to co-channel
opening is much greater for K ATP P so that a smaller
number of co-channels ŽOPENa – n . can be open simultaneously. We propose that even in their open states, K ATP P
and K ATP PP are substrates for kinases and phosphatases
which catalyze their interconversion; e.g., OPENa – n to
OPENaX – z and back. The phosphatase and kinase mediating these conversions may be KNase and PHase 1 and 2 or
others as indicated by the KNase-? and PHase-? in Fig. 9.
Secondly, we suggest that while any of the open states can
be reversibly blocked Ži.e., phosphorylation independent.
by ATP, the affinity of the blocking site for ligand is
conditioned by the level of phosphorylation.
It is important to note that our data do not permit the
determination of the exact number of phosphorylation
states and, therefore, the state of the channel which first
displays sensitivity to ATP inhibition. Rather, our model
attempts to depict how phosphorylation pathways required
for channel activity might interact with the blocking action
of ATP.
This model allows for precise modulation of the ability
of the VMN to sense and respond to metabolic status. For
example, many monoamine neuromodulators control the
activity of protein kinases w24x. Thus, they could potentially regulate the activity of the K-ATP channel and its
sensitivity to ATP via phosphorylation. Our suggestion
that the VMN K-ATP channel may have a number of
co-channels might provide a site at which monoamine
induced phosphorylation could alter the number of open
co-channels and thus modulate the mean current in the
presence of the same level of ATP. Therefore, phosphorylation may modulate K-ATP channel activity by regulating
the mean channel conductance.
Our proposed interaction with monoamines could be
responsible for the malfunction of glucosensing systems in
obesity w16x and diabetes w27x. For example, multiple
monoamine and neuropeptide modulators are altered in the
VMN in rodent models of genetic w17,23,29,30x and dietary obesity w18x. These alterations in modulation may
affect the steady state activity of the K-ATP channel via
alterations in its phosphorylation state. This in turn could
alter the response to the same metabolic cue leading to the
altered ‘set-point’ seen in obesity.
Acknowledgements
The authors wish to thank Dr. F.M. Ascroft for advice
concerning methods of measuring channel activity and
V.H. Routh et al.r Brain Research 778 (1997) 107–119
preventing channel rundown, Dr. I.B. Levitan for advice
concerning phosphorylation, and Dr. J.-H. Ye for assistance with cell isolation. This work was supported by
grants from NIDDK ŽDK-3006. and the Research Service
of the Dept. of Veterans Affairs to BEL, an Individual
NRSA ŽNS10335. to VHR as well as a Grant-in-Aid from
the American Heart Association to JJM.
w17x
w18x
w20x
w21x
References
w1x F.M. Ashcroft, S.J.H. Ashcroft, D.E. Harrison, Properties of single
potassium channels modulated by glucose in rat pancreatic b-cells,
J. Physiol. 400 Ž1988. 501–527.
w2x F.M. Ashcroft, P. Proks, P.A. Smith, C. Ammala, K. Bokvist, P.
Rorsman, Stimulus-secretion coupling in pancreatic b-cells, J. Cell.
Biochem. 55s Ž1994. 54–65.
w3x M.L.J. Ashford, P.R. Boden, J.M. Treherne, Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive Kq
channels, Pflugers
Arch. 415 Ž1990. 479–483.
¨
w4x M.L.J. Ashford, P.R. Boden, J.M. Treherne, Tolbutamide excites rat
glucoreceptive ventromedial hypothalamic neurones by indirect inhibition of ATP-Kq channels, Br. J. Pharmacol. 101 Ž1990. 531–540.
w5x J.R. De Weille, H. Schmid-Automarchi, M. Fosset, M. Lazdunski,
Regulation of ATP sensitive Kq channels in insulinoma cells:
activation by somatostatin and protein kinase C and the role of
cAMP, Proc. Natl. Acad. Sci. USA 86 Ž1989. 2971–2975.
w6x A.A. Dunn-Meynell, V.H. Routh, J.J. McArdle, B.E. Levin, Low
affinity sulfonylurea binding sites reside on neuronal cell bodies in
the brain, Brain Res. 745 Ž1997. 1–9.
w7x A.A. Dunn-Meynell, E. Govek, B.E. Levin, Intracarotid glucose
infusions selectively increase FOS-like immunoreactivity in paraventricular and ventromedial nucleus neurons, Brain Res. 748 Ž1997.
100–106.
w8x O. Hamill, A. Marty, E. Neher, B. Sakmann, F.J. Sigworth, Improved patch-clamp techniques for high resolution recording from
cells and cell-free membrane patches, Pflugers.
Arch. 391 Ž1981.
¨
85–100.
w9x J.W. Han, R. Thielexczek, M. Varsanyi, L.M.G. Heilmeyer, Compartmentalized ATP synthesis in skeletal muscle triads, Biochemistry 31 Ž2. Ž1992. 377–384.
w10x C.D. Hardin, L. Raeymaekers, R.J. Paul, Comparison of endogenous
and exogenous sources of ATP in fueling Ca2q uptake in smooth
muscle plasma membrane vesicle, J. Gen. Physiol. 99 Ž1992. 21–40.
w11x N. Inagaki, T. Gonoi, J.P. Clement, C.Z. Wang, L. Aguilar-Bryan, J.
Bryan, S. Seino, A family of sulfonylurea receptors determines the
pharmacological properties of ATP-sensitive Kq channels, Neuron
16 Ž1996. 1011–1017.
w12x M. Kakei, A. Noma, T. Shibasaki, Properties of adenosine triphosphate regulated channels in guinea-pig ventricular cells, J. Physiol.
363 Ž1985. 441–462.
w13x R.Z. Kozlowski, M.L.J. Ashford, ATP sensitive Kq channel rundown is Mg 2q dependent, Proc. R. Soc. Lond. B 240 Ž1990.
397–410.
w14x M.E. Krouse, G.T. Schneider, P.W. Gage, A large anion-selective
channel has seven conductance levels, Nature 319 Ž1986. 58–60.
w15x M. Kubokawa, C.M. McNicholas, M.A. Higgins, W. Wang, G.
Giebisch, Regulation of ATP-sensitive Kq channel by membranebound protein phosphatases in rat principal tubule cell, Am. J.
Physiol. 269 Ž38. Ž1995. F355–F362.
w16x B.E. Levin, Intra carotid glucose-induced norepinephrine response
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x
w32x
w33x
w34x
w35x
w36x
w37x
119
and the development of diet-induced obesity, Int. J. Obes. 16 Ž1992.
451–457.
B.E. Levin, B. Planas, V.H. Routh, J.S. Hamilton, J.S. Stern, B.A.
Horwitz, Altered a 1 -adrenoreceptor binding in intact and adrenalectomized obese Zucker rats Ž far fa., Brain Res. 614 Ž1993. 146–154.
B.E. Levin, Reduced norepinephrine turnover in organs and brains
of obesity-prone rat, Int. J. Obes. 16 Ž1995. 451–457.
B.E. Levin, K.L. Brown, A.A. Dunn-Meynell, Differential effects of
diet and obesity on high and low affinity sulfonylurea binding sites
in the rat brain, Brain Res. 739 Ž1996. 293–300.
P.E. Light, B.G. Allen, M.P. Walsh, R.J. French, Regulation of
adenosine triphosphate-sensitive potassium channels from rabbit
ventricular myocytes by protein kinase C and type 2A protein
phosphatase, Biochemistry 34 Ž1995. 7252–7257.
P. Light, Regulation of ATP-sensitive potassium channels by phosphorylation, Biochim. Biophys. Acta 1286 Ž1996. 65–73.
P.E. McKibbin, S.J. Cotton, S. McMillan, B. Holloway, R. Mayers,
H.D. McCarthy, G. Williams, Altered neuropeptide Y concentrations
in specific hypothalamic regions of obese Ž far fa. Zucker rats,
Diabetes 40 Ž1991. 1423–1429.
E.J. Nestler, P. Greengard, Protein phosphorylation and the regulation of neuronal function, in: G.J. Siegal ŽEd.., Basic Neurochemistry: Molecular, Cellular and Medicinal Aspects, 4th ed., Raven
Press, NY, 1989.
Y. Oomura, Glucose as a regulator of neuronal activity, in: A.J.
Szabo ŽEd.., Advances in Metabolic Disorders, New York Academic
Press, NY, 1983, pp. 31–65.
H. Onodero, K. Iijima, K. Kogure, Mononucleotide metabolism in
the rat brain after transient ischemia, J. Neurochem. 46 Ž1986.
1704–1710.
C. Rattarsaran, S. Dagogo-Jack, J.J. Zachweija, P.E. Cryer, Hypoglycemia-induced autonomic failure in IDDM is specific for stimulus of hypoglycemia and is not attributable to prior autonomic
activation, Diabetes 439 Ž1994. 809–818.
P.H. Reinhart, I.B. Levitan, Kinase and phosphatase activities intimately associated with a reconstituted calcium-dependent potassium
channel, J. Neurosci. 15 Ž6. Ž1995. 4572–4579.
V.H. Routh, D.M. Murakami, J.S. Stern, C.A. Fuller, B.A. Horwitz,
Neuronal activity in hypothalamic nuclei of obese and lean Zucker
rats, Int. J. Obes. 14 Ž1990. 879–891.
V.H. Routh, B.A. Horwitz, D.W. Geitzen, J.S. Stern, Hypothalamic
monoaminergic activity in 11-week cold-exposed female lean
Ž Far Fa. and obese Ž far fa. Zucker rats, Obes. Res. 2 Ž1994.
28–37.
V.H. Routh, J.J. McArdle, B.E. Levin, Fluctuations in activity of the
ATP-sensitive Kq channel in neurons isolated from the ventromedial hypothalamic nucleus, FASEB J. 10 Ž3. Ž1996. 1246.
H. Sakura, C. Ammala, F.M. Gribble, F.M. Ashcroft, Cloning and
functional expression of the cDNA encoding a novel ATP-sensitive
potassium channel subunit expressed in pancreatic b-cells, brain,
heart and skeletal muscle, FEBS Lett. 377 Ž1995. 338–344.
M. Takano, A. Noma, The ATP-sensitive Kq channel, Prog. Neurobiol. 41 Ž1993. 21–30.
A. Terzic, I. Findlay, Y. Hosoya, Y. Kurachi, Dualistic behavior of
ATP-sensitive Kq channels toward intracellular nucleoside diphosphates, Neuron 12 Ž1994. 1049–1058.
J.M. Treherne, M.L.J. Ashford, Extracellular cations modulate the
ATP sensitivity of ATP-Kq channels in rat ventromedial hypothalamic neurons, Proc. R. Soc. Lond. B 247 Ž1319. Ž1993. 121–124.
W. Wang, G. Giebisch, Dual effects of adenosine triphosphate on
the apical small conductance Kq channel of rat cortical collecting
duct, J. Gen. Physiol. 98 Ž1991. 35–61.
J.N. Weiss, S.T. Lamp, Cardiac ATP-sensitive Kq channels, J. Gen.
Physiol. 94 Ž1989. 911–935.