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Pages 517-522
1990
AN ATP, CALCIUM AND VOLTAGE SENSITIVE POTASSIUM CHANNEL IN PORCINE CORONARY ARTERY SMOOTH MUSCLE CELLS
Shai D. Silberberg and Cornelis van Breemen Department of Molecular and Cellular Pharmacology University of Miami School of Medicine P.O. Box 016189 Miami, FL 33101 Received
August
24,
1990
There is increasing interest in the roles played by potassium channels of smooth muscle in protecting against ischemic and anoxic insults. Hence, potassium-selective channels were studied in freshly dispersed porcine coronary artery smooth muscle cells using the inside-out variant of the patch-clamp technique. The most abundant potassium channel had a conductance of 148 pS in a 5.4/140 mM K+ gradient, at 0 mV, and was regulated by cytoplasmic ATP (0.05-3.0 mM), cytoplasmic Ca2+ (0.1-10 PM) and voltage. ATP and AMPPNP (0.5 mM) reduced the probability of channel opening (P,) by 87 and 92%, respectively. This inhibition was partially reversed by the addition of 0.5 mM ADP. ADP on its own (2 mM) reduced POby 46%. It appears, therefore, that this channel shares properties with both the ATP-sensitive and the calcium-regulated potassium channels, raising the possibility that 2 1990Acddemlc press, Inc. it plays a central role in the regulation of coronary blood flow.
Potassium selective ion channels play an important role in the regulation of vascular tone (1). Factors released by the endothelium
have been shown to hyperpolarize
the
underlying smooth muscle cells by opening potassium channels, leading to vasorelaxation (2). In addition, drugs which activate potassium channels are being developed for the treatment of angina pectoris and hypertension (3). Of particular interest are a class of potassium channels modulated by intracellular ATP (K,,,
channels) (4,5) which have been proposed
to be the main site of action of the potassium-channel-activating the coronary vasorelaxation information
drugs (3) and to underlie
observed during ischemic conditions
(6). However,
little
is available on the properties and diversity of potassium channels in coronary
artery smooth muscle. KATP channels have been identified in a variety of tissues including cardiac (7), pancreatic (X,9), skeletal muscle (lo), neuronal (11) and vascular smooth muscle (12). All KATP channels described
to date show no apparent
intracellular
calcium
dependency and only slight voltage sensitivity (for review see 4 and 5). In this report we describe a KArP channel which shows ATP, Ca*+ and voltage sensitivity (K,,,,, The abundance and large conductance of this diversely modulated
channel).
potassium-selective
channel suggests that it plays a central role in the regulation of coronary artery tone,
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Sinale cell urepurution. Porcine (60-1.50 lb) hearts were collected from a local slaughterhouse within 30 min after death. Smooth muscle cells were enzymatically loosened from the proximal right coronary artery with papain, elastase and collagenase and dispersed by gentle tituration. The resulting suspension was placed on glass coverslips, where cells attached within 1 hr after dissociation. Experiments were performed within 16 hours of dissociation. EZectrical measurements. Single-channel events were recorded from membrane patches using the inside-out variant of the patch-clamp technique (13). The cytoplasmic side of the membrane was perfused with a solution which contained (in mM): 140 KCl, 10 Hepes, 10 glucose, 1 EGTA, (pH 7.4 with N-Methyl-D-Glucamine) and varying amounts of CaC& to attain desired levels of [Ca*+]i. The pipette solution contained (in mM): 137 NaCl, 5.4 KCI, 2 CaCl,, 5 Hepes and 10 glucose, (pH 7.4 with NaOH). Nucleotides were added directly to the experimental chamber after stopping perfusion. All experiments were performed at room temperature (20-24’C). The recording system included a List EPC-7 amplifier, a 4-pole Bessel filter (Ithaca) and a PCM4 (Medical Systems) data recorder. Current records stored on video-tape were low pass filtered at 2 kHz (-3dB) and digitized at 10 kHz (pclamp 5.5, Axon Instruments Inc.), unless otherwise stated. Analysis of single-channel records was performed on a 20 MHz 386 computer using custom made software. Unitary single channel currents were detected and idealized by an algorithm which uses both channel amplitude and slope information. Voltage protocols were applied with the pClamp software. Average values are the mean f S.E.M. Drugs. ATP and ADP were added as potassium salts. In view of proton concentration effects on the apparent association constant of EGTA for calcium (14) the pH of the stock solutions was adjusted to 7.4 with KOH and measurements were made which verified that the addition of K,ATP or K,ADP did not alter the pH of the solution. AMP-PNP was added as lithium salt. Stock solutions of AMP-PNP were made in 100 mM Hepes buffer preadjusted to pH 7.4 with KOH. Free Ca*+ concentrations were determined using a computer program based on published stability constants (15). RESULTS
A large-conductance, potassium-selective channel sensitive to voltage and cytoplasmic free calcium was present in 98 of 116 active inside-out patches. This channel had a chord conductance of 148 + 3 pS (n= 10) between -20 and 20 mV in [K+],/[K’]i Figure 1A demonstrates the voltage-dependency
of 5.4/140 mM.
of this channel and the time-dependency
of the macroscopic current obtained from single-channel ensembles. Repetitive voltage steps (illustrated in the top trace) were generated from a holding potential of -60 mV to 0 mV, in a bath solution containing 1 PM Ca*’ (pCa 6). Shown are five consecutive current traces and the average of 150 consecutive current traces (bottom trace). Channel activity was low at -60 mV, increasing gradually upon depolarization
to 0 mV. To asses the selectivity of the
channel for Kf over Na+ ions, current-voltage curves (I-V curves) were generated by either measuring single-channel current amplitudes at different holding potentials or by applying voltage ramps. Figure 1B illustrates I-V curves obtained for a single-channel using both techniques. Also shown is a fit to the data using the Goldman-Hodgkin-Katz
(GHK)
constant field equation (solid line) (16,17). The PNJPK permeability ratio, estimated from the fit, was less then 0.01, demonstrating the high selectivity for K’ over Nai. 518
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B -I
24 ”
nn
19
, ‘I’llm
‘li k
14 PA
r-L-J5
pA
-12pA
j -80
250 mS
-40
0 mv
40
a0
18
pea 5
14 PA lo
6
PA IO con.
+ wash M ATP
2 -21 -80
-40
0 mV
40
6 2 -21
a0
-80
pea 7 -40
0 mV
40
80
Figure 1. Properties of the KATP,oachannel. A. Current traces in response to voltage steps from a holding potential of -60 mV (inside negative) to 0 mV for 800 ms. The top trace illustrates the voltage step. Leak current was eliminated by digitally subtracting a current trace with no channel openings. Five consecutive individual current traces are shown along with the average of 150 consecutive traces (bottom trace). Filtered at 0.6 kHz (-3dB) and digitized at 2 kHz. B. Single-channel I-V curve constructed by either applying a voltage ramp between -80 and + 80 mV at 40 mV/s filtered at 0.3 kHz, or by averaging charmel amplitude over 30 seconds of continuous recording at different holding potentials, (filled circles). Solid line is a fit to the GHK constant field equation. C. Average of 25 consecutive single-channel current traces induced by voltage ramps prior to (con.), in the presence of 0.5 and 1.0 mM ATP, and following washout of ATP (wash). Same ramp protocol as in B. D. Average of 25 consecutive sin le-channel current traces induced by voltage ramps in 0.1 (pCa 7) and 10 (pCa 5) PM Cas,+ bath solutions. Same ramp protocol as in B.
Bath application of ATP (0.05-3.0 mM) reversibly reduced the probability of channel opening (P,) in a dose-dependent
manner, while having no apparent effect on single channel
conductance. The half-maximal effect of ATP was in the range of 0.3-0.5 mM ATP, at 0 mV, in pCa 6 solutions. The action of ATP, observed in 54 of 57 experiments, was studied over a wide range of voltages by applying voltage ramps to a single-channel patch (Figure 1C). At 0 mV, ATP (0.5 mM) reduced the current obtained by averaging 25 consecutive current
traces by approximately 50% while having little effect on P, above + 30 mV. 1 mM ATP had a more pronounced
effect over a wider range of voltages. The calcium sensitivity of the
channel is shown in Figure 1D. Figure 2A demonstrates calcium sensitivity and compares the ability of ATP, AMPPNP (a non hydrolyzable
analog of ATP) and ADP to inhibit PO of a single-channel.
Channel PO in bath solutions of pCa 5, 7 and 6 was 0.98, co.01 and 0.60, respectively (top
trace). ATP (0.5 mM) and AMP-PNP (0.5 mM) reduced PO to 0.095 and 0.013, respectively, 519
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A pCa
5
pCa
7
pCa
control
lpca
5)
ATP
control
(pea
5)
AMP-PNP(o.5
control
cpca
51
ADP
wash
(0.5 mM)
~pca 6)
wash (pea
mM1
(2
6
wash
mM1
6)
~pca 61
B control
ATP
ATP + ADP
Figure 2. Action of adenine nucleotides on single KATP,Ca channels. A. Continuous singlechannel record in different calcium solutions (top trace). Channel activity in pCa 5 solution preceding nucleotide addition is shown on the left of the bottom three traces (control). Channel activity in the presence of ATP (0.5 mM), AMP-PNP (0.5 mM) and ADP (2 mM) and during washout into a pCa 6 solution is shown to the right. Traces are from one experiment. B. Relief of ATP induced inhibition by ADP. Compared are channel activity in pCa 5 solution (control), in the presence of ATP (0.5 mM) and following the addition of ADP (0.5 mM) (ATP t ADP). Closed state is indicated by line at left of traces. Calibration bars: 5 pA and 10 seconds. Calibration bars apply to all traces.
while ADP
had weaker inhibitory effects; 2 mM ADP was required to attenuate P, to 0.36.
However, ADP (0.5 mM) could partially relieve the inhibition (Figure
2B). Figure
3 summarizes
2
the average
z
1.07
s a .’
0.8
1
2
0.6
‘_i j i
2 p .” 0 E i E
I
induced by 0.5 mM All’
effects of ATP, AMP-PNP
and ADP on P,,
-1_
0.4-1 0.2’ I 0.0
: ~
1,
pea6
ADP
rkil AMP-PNP
PIP
r-: 1 ATP + ADP
Figure 3. Effects of adenine nucleotides on KATP,ca channel activity. ADP (2 mM), AMPPNP (0.5 mM) and ATP (0.5 mM) attenuated P, to 0.54 + 0.16 (n=5), 0.08 + 0.04 (n=6) and 0.13 + 0.09 (n=6), respectively. P, in pCa 6 solution was reduced to 0.73 &- 0.06 (n=6). ADP (0.5 mM), added in presence of ATP (0.5 mM), increasing P, to 0.40 &- 0.19 (n=4). In each experiment, P, was estimated from 60 seconds of continuous recording at 0 mV in pCa 5 solution and normalized to a 60 second control period preceding nucleotide addition.
520
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at 0 mV, in pCa 5 solutions.
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Also shown is average P, in pCa 6 solution. These results
demonstrate that the action of ATP is predominantly
not due to Ca*+ chelation since 0.5
mM ATP was calculated to change [Ca*+] from 10 to 6.3 PM. DISCUSSION
Under the present experimental conditions, the most abundant potassium-selective channel identified
in excised inside-out patches from freshly dispersed porcine coronary
artery smooth muscle cells is sensitive to ATP, calcium and voltage. Other K,,,
channels
reported to be present in various cell types are not affected by varying the calcium concentration on the cytoplasmic side of the membrane (7,8,10,18). On the other hand, large conductance calcium-sensitive potassium channels (Bha
channels) have been reported to
be insensitive to cytoplasmic ATP (12,19). Hence, this channel has properties in common with K,,,
as well as BIQ, channels.
Similarities to KATp channels. The inhibitory
the K,,,
action of ATP on both the KATp channels and
oa channel does not involve ATP hydrolysis since magnesium ions are not a
prerequisite
for ATP action and non hydrolyzable
analogs of ATP mimic its action
(8,10,11,18,20). In pancreatic /3 cells, it is thought that the ratio of ATP to ADP governs K *rP channel activity since ADP partially overcomes the inhibition similar phenomenon
of ATP (21,22). A
applies to the KATP,oa channel in coronary smooth muscle (Figure 2B
and 3). Finally, the KATP,oa channel conductance (148 pS) resembles the conductance of the recently described smooth muscle K,rp channel (12) and Bc,
channels (23-25) measured
under similar experimental conditions but is much larger then that of cardiac, pancreatic p cell, skeletal muscle and neuronal KATP channels (7,9-11,20). Similadies to
BKC, channels. BK,, channels, described in several tissues and preparations
(for review see 26,27), share a number of properties with the K,,, foa channel. Smooth muscle B&, channels and the K,,, ,oa channel are sensitive to variations in [Ca*+J in the range of 0.01-10 PM (see 25 for references). Furthermore, potassium ions, BI& the K,,,
under physiological gradients of
channels have a similar conductance (loo-150 pS)(12,23-25). While
channels exhibit little or no membrane potential sensitivity (S-12,20), the KATP,Ca
and BQa channels are strongly activated by membrane depolarization It appears, therefore, that the K,,,
(26,27).
Ca channel described in this study is unique in
its sensitivity to both calcium and ATP. The ability of both intracellular to modulate
this channel suggests that it may play an important
conditions which are known to cause coronary artery vasodilation most probably
reduce intracellular
[Ca*+li, all of which hyperpolarization
would
ATP, cause membrane activate
521
role under ischemic (6). Anoxic conditions
depolarization
KATP ca channels,
and cell relaxation.
calcium and ATP
leading
and elevate to
membrane
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ACKNOWLEDGMENTS
We thank Drs. D.J. Adams and D.S. Weiss for helpful discussion, Dr. A.M.J. van Dongen and A. Mandveno
for the use of several analysis programs and M. Lam for
technical assistance. This work was supported by HL-35657. SDS is a Research Fellow of the American Heart Association, Florida Affiliate, Inc. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
Longmore, J., and Weston, A.H. (1990) In Potassium Channels, structure, classification, function and therapeutic potential (N.S. Cook, Ed.), pp. 259-278. Ellis Horwood Limited, Chichester, England. Tare, M., Parkington, H.C., Coleman, H.A., Neild, T.O., and Dusting, G.J. (1990) Nature 346, 69-71. Cook, N.S., and Quast, U. (1990) In Potassium Channels, structure, classification, function and therapeutic potential (N.S. Cook, Ed.), pp. 181-255. Ellis Horwood Limited, Chichester, England. Ashcroft, F.M. (1988) Ann. Rev. Neurosci. 11, 97-118. Rorsman, P., and Trube, G. (1990) In Potassium Channels, structure, classification, function and therapeutic potential (N.S. Cook, Ed.), pp. 96-116. Ellis Horwood Limited, Chichester, England. Daut, J., Maier-Rudolph, W., von Beckerath, N., Mehrke, G., Gunther, K., and Goedel-Meinen, L. (1990) Science 247, 1341-1344. Noma, A. (1983) Nature 305, 147-148. Cook, D.L., and Hales, C. N. (1984) Nature 311, 271-273. Ashcroft, F.M., Harrison, D.E., and Ashcroft, S.J.H. (1984) Nature 312, 446-448. Spruce, A.E., Standen, N.B., and Stanfield, P.R. (1985) Nature 316, 736-738. Ashford, M.L.J., Sturgess, N.C., Trout, N.J., Gardner, N.J., and Hales, C.N. (1988) Pfliigers Arch. 412, 297-304. Standen, N.B., Quayle, J.M., Davies, N.W., Brayden, J.E., Huang, Y., and Nelson, M.T. (1989) Science 245, 177-180. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and S&worth, F.J. (1981) Pfhigers Arch. 391, 85-100. Harrison, S.M., and Bers, D.M. (1987) Biochim. Biophys. Acta 925, 133-143. Martell, A.E., and Smith, R.M. (1974) Critical Stability Constants, Plenum Press, N.Y. Goldman, D.E. (1943) J. Gen. Physiol. 27, 37-60. Hodgkin, A.L., and Katz, B. (1949) J. Physiol. 108, 37-77. Spruce, A.E., Standen, N.B., and Stanfield, P.R. (1987) J. Physiol. 382, 213-236. Williams, Jr., D.L., Katz, G.M., Roy-Contancin, L., and Reuben, J.P. (1988) Proc. Natl. Acad. Sci. USA 85, 9360-9364. Kakei, M., Noma, A., and Shibasaki, T. (1985) J. Physiol. 363, 441-462. Kakei, M., Kelly, R.P., Ashcroft, S.J.H., and Ashcroft, F.M. (1986) FEBS Lett. 208, 63-66. Dunne, M.J., and Petersen, O.H. (1986) FEBS Lett. 208, 59-62. Benham, C.D., Bolton, T.B., Lang, R.J., and Takewaki, T. (1986) J. Physiol. 371,4567. Kume, H., Takai, A., Tokuno, H., and Tomita, T. (1989) Nature 341, 152-154. Hu, S.L., Yamamoto, Y., and Kao, C.Y. (1989) J. Gen. Physiol. 94, 833-847. Latorre, R., Oberhauser, A., Labarca, P., and Alvarez, O., (1989) Annu. Rev. Physiol. 51, 385-399. Haylett, D.G., and Jenkinson, D.H. (1990) In Potassium Channels, structure, classification, function and therapeutic potential (N.S. Cook, Ed.), pp. 70-95. Ellis Hok.lNood Limited, Chichester, England. 522