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A Ca2+-activated K+ channel from rabbit aorta: modulation by cromakalim
European Journal of Pharmacology, 167 (1989) 201-210 Elsevier
201
EJP 50924
A C a 2 +-activated K + channel from rabbit aorta: modulation by cromakalim Craig H. Gelband *, Nicholas J. Lodge and Cornclis Van Brcemen Department of Pharmacology (R-189), Universityof Miami School of Medicine, Miami, EL 33101, U.S.A.
Received 10 November 1988, revised MS received 3 March 1989, accepted 30 May 1989
A large conductance Ca2+-activated K + channel from rabbit aorta was incorporated into planar lipid bilayers. This channel had a conductance of 337 + 7 pS in symmetrical 250 mM KC1 solutions and had a N a + / K + permeability ratio of < 0.04. In asymmetrical solutions containing 300 mM KC1 cis (intracellular), 100 mM KC1 trans (extracellular) or 100 mM KCI cis 500 mM KC1 trans, the reversal potentials for the channel were - 3 0 and +46 mV, respectively. This channel possessed voltage-dependent activation and cis (intracellular) Ca 2+ sensitivity. Cromakalim (50 nM) added to the trans side of the bilayer significantly increased the Popen by 56% from 0.09 + 0.01 to 0.14 _ 0.01 (P < 0.01) at - 4 0 mV without altering the single channel conductance. This effect was dose-dependent, increasing at higher cromakalim concentrations. The primary effect of cromakalim was to decrease the %lowof the channel closed state from 266 + 32 to 1 4 7 _ 17 ms which is sufficient to account for the increase in Popen of the channel in the presence of cromakalim. Aorta; Planar lipid bilayer; Ca2+-activated K + channels; [Ca2+]i sensitivity; Cromakalim; (Rabbit)
1. Introduction L a r g e c o n d u c t a n c e Ca2+-activated K + channels have been i d e n t i f i e d in a n u m b e r of vascular a n d n o n - v a s c u l a r s m o o t h muscles (Bolton et al., 1985; I n o u e et al., 1985; M c C a n n a n d Welsh, 1986; Singer a n d Walsh, 1987). This K + c h a n n e l is a c t i v a t e d b y voltage a n d intracellular [Ca2+]. Its c o n d u c t a n c e is greater t h a n 200 pS, d e p e n d i n g on the e x p e r i m e n t a l c o n d i t i o n s used. A l t h o u g h this c o n d u c t a n c e is large, the channel is highly selective for K + ions over o t h e r cations a n d anions. Ca2+-activated K + channels have b e e n p r o p o s e d to p l a y a role in r e p o l a r i z a t i o n of action p o t e n t i a l s o r o t h e r d e p o l a r i z i n g stimuli of vascular s m o o t h m u s c l e a n d in slow wave p r o p a g a t i o n o f visceral s m o o t h muscle. M a n y c o m p o u n d s have been
* To whom all correspondence should be addressed: Department of Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101, U.S.A.
shown to b l o c k the large c o n d u c t a n c e C a 2+a c t i v a t e d K + channel of vascular s m o o t h muscle i n c l u d i n g T E A in r a b b i t p o r t a l vein ( I n o u e et al., 1985), Ba 2+ in guinea pig mesenteric artery (Benh a m et al., 1985), 4 - a m i n o p y r i d i n e in r a b b i t p u l m o n a r y a r t e r y ( O k a b e et al., 1987) a n d c h a r y b d o t o x i n in b o v i n e a o r t a ( T a l v e n h e i m o et al., 1988). C r o m a k a l i m ( B R L 34915), a novel a n t i - h y p e r tensive agent, lowers b l o o d pressure ( B u c k i n g h a m et al., 1986), h y p e r p o l a r i s e s s m o o t h muscle m e m b r a n e s ( H a m i l t o n et al., 1986), a n d relaxes prec o n t r a c t e d vascular s m o o t h muscle ( C l a p h a m a n d Wilson, 1984) b y a m e c h a n i s m which is h y p o t h e sized to involve the o p e n i n g of a class of K ÷ channels. T h e actions of B R L 34915 to relax contracted s m o o t h muscle a n d increase the 86Rb+ efflux f r o m s m o o t h muscle are a n t a g o n i z e d b y such K ÷ c h a n n e l a n t a g o n i s t s as T E A , 4 - a m i n o p y r i d i n e a n d p r o c a i n e (Wilson, 1987). H o w e v e r , a p a m i n , an a n t a g o n i s t of the small c o n d u c t a n c e
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
202 Ca2+-activated K + channel, is ineffective in antagonizing the effects of BRL 34915 in smooth muscle (Weir and Weston, 1986). Kreye and Weston (1986) have reported that the 86Rb+ efflux stimulated by BRL 34915 in rabbit aorta is Ca 2+dependent suggesting that the K + channel opened by BRL 34915 is also Ca2+-dependent. In addition Quast and Cook (1987) have reported that a crude venom of Leiurus quinquestriatus, a scorpion toxin which contains charybdotoxin (a putative Ca2+-activated K + channel antagonist), could inhibit BRL 34915-induced increases in 86Rb+ efflux from portal vein. In contrast, Beech and Bolton (1987) have reported that in portal vein BRL 34915 increases a K + conductance which is different from the large conductance Ca 2+activated K + channel. The aim of the present study was to incorporate the large conductance Ca 2 +-activated K + channel of rabbit aortic smooth muscle into planar lipid bilayers, characterize its activity, and investigate the actions of BRL 34915 on the isolated channel. Preliminary results have been presented (Gelband et al., 1988; Gelband and Van Breemen, 1989).
2. M a t e r i a l s
and methods
2.1. Microsomal isolation Microsomes were prepared according to the method of Kwan et'al. (1979) with some modifications. Adult rabbits (New Zealand white, 2-3 kg body weight) were anesthetized in a CO 2 chamber and the thoracic aorta was rapidly excised and placed in oxygenated PSS at 37 o C. Rabbit aortas were cleaned of fat and adventitia and stored at - 70 ° C until their use. When required, the tissues were thawed, minced and homogenized using a Polytron PT 10 homogenizer in 250 m M sucrose, 10 m M M O P S - K O H , p H 7.2. A Teflon pestle and a glass homogenizer was used to further disrupt the tissues. The homogenate was centrifuged at 10000 × g for 20 rain, and the resulting supernatant was recentrifuged at 27 000 X g for 20 rain. The 27000 x g pellet was discarded and the 27 000 x g supernatant was centrifuged at 100 000 x g for 1 h. The final pellet was resuspended in
250 m M sucrose, 10 m M M O P S - K O H , p H 7.2, aliquoted and frozen at - 7 0 ° C until its use. The final protein concentration was 1.5-3.0 m g / m l . All procedures were performed at 4 ° C. 2.2. Incorporation of Ca 2 +-activated K + channels into planar lipid bilayers A solution containing 27 m g / m l phosphatidylethanolamine, 23 m g / m l phosphatidyl serine (Avanti Polar Lipids, Birmingham, AL) dissolved in decane was used to form planar lipid bilayers. Planar lipid bilayers were formed across a 150-250 /~M diameter hole in a Lexan cup. The lipid bilayer separated two experimental chambers as described by Nelson et al. (1984). CaE+-activated K + channels from aortic microsomes were incorporated into the planar lipid bilayers by adding the sample to one side of the bilayer, designated the cis side. The side opposite to which the vesicles were added is designated trans. The cis solution was vigorously stirred to facilitate channel incorporation. The channel's sidedness was established on the basis of its voltage-dependent activation and its [Ca2+]i sensitivity. A consistent orientation was established by the observation that addition of Ca 2+ or E G T A to only the cis chamber increased the probability of channel opening or closing respectively. Depolarization of the membrane, Vcis-Vt..... also facilitated channel opening, while hyperpolarization of the m e m b r a n e decreased channel opening. Thus the solution in the cis chamber always represented the intracellular solution. [Ca 2+] was maintained low in the trans chamber of insure that if occasionally a channel was oriented in the opposite direction it would not be activated. The final protein concentration added to the cis side was 1-10/~g/ml. All experiments were performed at 22 o C. 2.3. Electrical recordings C o m m a n d potentials were applied to the bilayer under voltage clamp conditions using two A g / A g C l electrodes with the trans side held at virtual ground. Voltages are defined as Vcis-Vt.... . Current fluctuations across the bilayer were visualised on an oscilloscope, recorded on F M
203
tape and played back through an 8-pole Bessel filter at 315 Hz. The inherent filtering of the voltage clamp was 1 kHz. Data analysis was performed on these recordings, digitized at 4 kHz, using an IBM PC A T computer and the software p C l a m p 4.1. The total time of the records used in the analysis of probability of opening, Pope,, and open and closed time constants was 10 min in the absence and 10 min in the presence of the drug or vehicle. Six out of ten bilayers formed contained more than one channel; however, only those records from bilayers containing a single channel were used for analysis and are displayed in the figures.
3. Results The activity of a single large conductance K + channel, in symmetrical 250 m M KC1 and 1 m M cis CaC12, is shown in fig. 1A. In symmetrical
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-20 PSS (in mM): NaC1 140, KCI 5, CaC12 1.5, MgC12 1, glucose 10, HEPES 5, p H 7.2. Experimental chambers contained the following (unless otherwise stated in the figure legends) KC1 250, M O P S - K O H 10, p H 6.8, E G T A 2. Ca 2÷ concentrations less than 50 ttM were calculated and prepared using an E G T A buffer assuming an apparent binding constant of the C a - E G T A complex to be 1 0 6 M - 1 at p H 6.8, 2 2 ° C (Saida and Nonomura, 1978). BRL 34915 was dissolved in dimethyl sulfoxide (Sigma).
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The open probability of a single channel, Popen, is defined as the amount of time the channel remained in the open state divided by the total time of the recording. Open and closed time histograms were described by exponentials using the method of least squares. The number of exponentials required to generate the best fit of the data was assessed using the statistical parameters provided within pClamp 4.1. N o corrections were made for rapid openings and closing not fully resolved by the voltage clamp system. All values shown are the means +__S.E.M. unless otherwise noted. Paired student t-tests were performed to evaluate the significance of the data. Data were considered significant with a P < 0.05.
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-2o_ ° -6o 4o- o o A 4b 6b 8b v (my} Fig. 1. Single channel records and current-voltage relationship of the large conductance Ca2+-activated K + channel. (A) Experimental chambers contained symmetrical (in mM): KCI 250, M O P S - K O H 10, p H 6.8. However 1 m M CaCI 2 was present on the cis side and 2 m M E G T A was present on thetrams side. Single channel records were filtered at 315 Hz. Solid line to the fight of the traces represents the closed state of t h e channel. (B) Current-voltage relationship (one experiment of ten).
204
solutions, the current flowing through the channel reversed at 0 mV. This channel possessed voltagedependent activation; as the membrane potential was hyperpolarized, not only did the current reverse, but the probability of the channel being open, Pope,, decreased. A representative single channel current-voltage relationship for this channel is shown in fig. lB. The slope conductance of the channel, estimated between +40 and - 4 0 mV, had an average value of 337 + 7 pS (n = 10). The selectivity of this large conductance K + channel from rabbit aorta was investigated and the results are shown in figs. 2 and 3. Figure 2 shows the effects of changing the K ÷ concentration on either side of the planar lipid bilayer. In the presence of 300 mM KC1 cis and 100 mM KC1 trans (closed squares), the Nernst equation for K ÷ predicts a reversal potential of - 2 8 mV. The
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Fig. 3. Effect of hi-ionic solutions on the single channel current-voltage relationship. The closed squares represent experiments (n = 3) under these conditions: trans (mM) NaC1 250, CaC1 z M O P S - N a O H 10, p H 6.8; cis (mM) KC1 250, MOPSK O H , p H 6.8. The closed circles, represent experiments (n = 3) performed under these conditions: trans (mM) KC1 250, CaC12 1, M O P S - K O H 10; p H 6.8; cis (mM) NaC1 250, M O P S - K O H 10, p H 6.8. The current-voltage relationship does not cross the zero current axis.
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Fig. 2. Effect of asymmetric K + solutions on the single channel current-voltage relationship. The closed squares represent experiments (n = 3) performed under these conditions: cis (mM) KCI 300, CaCI 2 1, M O P S - K O H 10, p H 6.8; trans (mM) KC1 100, M O P S - K O H 10, p H 6.8. The d o s e d circles represent (n = 3) experiments performed under these conditions, cis (mM) KCI 100, CaC12 1, M O P S - K O H 10, p H 6.8; trans (mM) KC1 500, M O P S - K O H 10 p H 6.8. Note that Ere v are - 3 0 and + 4 6 mV for the squares and circles, respectively.
20 40 60 80
(my)
single channel current reversed at - 3 0 mV. On the other hand, in the presence of 100 mM KC1 cis and 500 mM KC1 trans (closed circles), the single channel current reversed at +46 mV, in close agreement with + 41 mV for the Nernst potential for K + under these conditions. Thus the channel is highly selective for K + over C1-. Figure 3 examines the relative permeability of Na + through this K + channel. Na + ions have been reported to be impermeant through large Ca2+-activated K + channels (Blatz and Magleby, 1984). Under asymmetric NaC1/KC1 conditions, no Na + permeation can be measured if Na ÷ replaces K + on either side of the bilayer. It is apparent that the currents, inward or outward depending on the experimental conditions, became smaller appearing to approach the zero current level but did not reverse. Since the single channel currents did not reverse, an
205
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Fig. 4. The effect of voltage and [Ca 2+ ]i on Popenof the Ca2+-activated K + channel. Experimental chambers contained (in mM): symmetric KC1 250, MOPS-KOH 10 pH 6.8, and the Ca 2+ cxmcentration on the cis side is varied. EGTA, 2 raM, is present on the trans side of the bilayer.o(A) Records in the presence of 25 ~tM Ca 2+ cis. Records are filtered at 315 (n = 3). Solid line represents the closed state of the channel. (B) The effects of Ca 2 + on Po__.. Symbols: (e) 1/~M Ca 2 + (&) 25 ~M Ca 2+ ( 0 ) 50 ttM Ca 2+, (zx) 75 /~M Ca 2 + , (©) 100 #M Ca 2 + and (ll) 1 mM Ca 2 + (n = 3).
absolute Na+/K + permeability ratio cannot be calculated. However, an e s t i m a t e of the u p p e r limit of the N a + p e r m e a b i l i t y can b e m a d e b y e x t r a p o l a t i n g a reversal p o t e n t i a l f r o m the current-voltage relationship and would predict a N a + / K + p e r m e a b i l i t y r a t i o o f < 0.04, which is c o n s i s t e n t with o t h e r v a s c u l a r a n d visceral s m o o t h m u s c l e ( B e n h a m et al., 1986). T h e [Ca2+]i a n d voltage d e p e n d e n c y o f the large c o n d u c t a n c e Ca2+-activated K + channel was e x a m i n e d in fig. 4. F i g u r e 4 A shows single c h a n n e l r e c o r d s in the p r e s e n c e o f 25 v M CaC12 o n the cis (intracellular) side of the m e m b r a n e . D e p o l a r i z i n g the m e m b r a n e p o t e n t i a l f r o m - 6 0 to - 4 0 m V i n c r e a s e d the Pope, f r o m 0.07 to 0.27. A s the m e m b r a n e p o t e n t i a l is d e p o l a r i z e d further f r o m - 4 0 to + 20 mY, the Pop~, increased f r o m 0.27 to 0.97. F i g u r e 4B shows the effects o f v a r y i n g the cis
(intracellular) C a 2+ c o n c e n t r a t i o n on the Pope, of the channel. [Ca2+]i a c t i v a t i o n of the K + c h a n n e l was d e m o n s t r a t e d b y a shift to the left of the Popen voltage curve when the cis C a 2÷ was raised f r o m 1 /xM to 1 m M . T h e effects o f the novel a n t i h y p e r t e n s i v e a n d p u t a t i v e K + c h a n n e l a g o n i s t B R L 34915 a r e s h o w n in figs. 5, 6, 7 a n d in t a b l e 1. R e p r e s e n t a t i v e single c h a n n e l r e c o r d i n g s in the a b s e n c e a n d p r e s e n c e of 50 n M B R L 34915 on the trans (extracellular) side a r e seen in fig. 5. R e c o r d i n g s were m a d e in the presence o f s y m m a t r i c a l 250 m M K C I a n d 1 /xM CaC12 on the cis side of the m e m b r a n e at - 4 0 mV. T h e left h a n d p a n e l shows c o n t r o l records in the a b s e n c e of B R L 34915. O p e n i n g s were brief" a n d the t i m e the c h a n n e l r e m a i n e d in the closed state was l o n g c o m p a r e d to t h a t o f the o p e n state. T h e a c t i o n s o f 50 n M B R L 34915 a r e i l l u s t r a t e d in
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Fig. 5. Effects o f B R L 34915 o n single c h a n n e l r e c o r d i n g s . E x p e r i m e n t a l c h a m b e r s c o n t a i n e d the s a m e s o l u t i o n s as s t a t e d in M e t h o d s , except the cis C a 2+ c o n c e n t r a t i o n is 1 # M . T h e left h a n d p a n e l s h o w s c o n t r o l r e c o r d s a n d the r i g h t h a n d p a n e l s h o w s the effects o f 50 n M B R L 34915 a d d e d to the t r a n s side. M e m b r a n e p o t e n t i a l is - 4 0 mV. Pore, in t h e c o n t r o l is 0.10 a n d in t h e p r e s e n c e of B R L 34915 is 0.16. R e c o r d s are filtered a t 315 Hz. N o t e single c h a n n e l a m p l i t u d e is n o t a f f e c t e d b y the p r e s e n c e o f B R L 34915. Single c h a n n e l o p e n i n g s are d o w n w a r d . Scale b a r s r e p r e s e n t 20 p A (vertical) a n d 200 m s ( h o r i z o n t a l ) .
the right hand panel. Openings are again brief; however, the amount of time the channel remained closed was shortened. Pope, of the single channel increased by 60% from 0.10 to 0.16. In four experiments, an increase of 56% was evident from 0.09 + 0.01 to 0.14 + 0.01 (P < 0.01, table 1). The effect on Pope, was dose-dependent, increasing from 0.09 + 0.01 to 0.35 + 0.03 in the presence of 500 nM BRL 34915 at - 4 0 mV (see below, fig. 7). BRL 34915 did not have any effect on unitary current amplitude or single channel conductance. It was also evident that the channel gated between two distinct modes of opening. The first mode was characterized by its long closures between subsequent openings and the second contained groupings of rapid openings and closings. This activity is present in the absence and presence of BRL 34915. A long period of single channel activity was analyzed to take into account for the moding
behavior of the channel in calculating the effects of BRL 34915. Identical experiments were performed using the DMSO vehicle only. Pope, was found not to be significantly different during the 10 min exposure to DMSO when compared to the 10 min prior to the addition of DMSO (Pope, = 0.10 + 0.01 in the absence or presence of DMSO, P < 0.01). These data indicate that BRL 34915's effects can not be attributed to the vehicle or time dependent changes in the channel's probability of opening or states of inactivation. Kinetic analysis was performed on the records of fig. 5 and these results are depicted in fig. 6. In the upper panel, open time histograms are plotted for data in the absence and presence of 50 nM BRL 34915. Open time histograms were best fit by a single exponential. In the absence of BRL 34915, the open time constant (~) was 19 ms while in the
207 CONTROL
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Fig. 6. O p e n a n d closed time h i s t o g r a m s of the c h a n n e l in fig. 5 u n d e r control c o n d i t i o n s (left) a n d in the presence of 50 n M B R L 34915 trans (right). The o p e n time h i s t o g r a m s were best fit b y a single e x p o n e n t i a l a n d the closed t i m e h i s t o g r a m s were best fit b y the s u m of two exponentials. T i m e c o n s t a n t s for the e x p o n e n t i a l s are p r e s e n t e d in table 1.
presence of 50 nM BRL 34915, the open time constant was 17 ms. Table 1 shows the values for the open time constants for the experiment shown in figs. 5 and 6 and the values for the mean of the four experiments in the absence and presence of 50 nM BRL 34915. There was no significant
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-40
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;
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Voltage (mV) Fig. 7. Effects of B R L 34915 on Pop~. as a function o f voltage. E x p e r i m e n t a l c h a m b e r s c o n t a i n e d the s a m e s y m m e t r i c a l solutions as stated in Methods, except t h a t the cis [Ca 2+ ] w a s 1 # M . Symbols: control ( I ) , 50 n M B R L 34915 (Ill) a n d 500 n M B R L 34915 (&). B R L 34915 was a d d e d to the trans solution. Vo is - 5 , - 2 0 a n d - 3 0 m V for control, 50 a n d 500 n M B R L 34915, respectively. D a t a s h o w n are the m e a n s _+S.E.M. (n = 3). E r r o r bars are n o t present w h e n the s y m b o l is the s a m e size as the S.E.
change in the open time constant in the presence of 50 nM BRL 34915 (control 1 9 _ 1 ms, BRL 34915 18 _ 1 ms, n = 4). The major effect of BRL 34915 was resolved in the closed time analysis. Closed time histograms
TABLE 1 A n a l y s i s of single c h a n n e l records.
Open = Closed = Por~- a Open b Closed b Pop¢~ b
qq'fast %low ~ q-f,=t q-slow
Control
% of events
B R L 34915
% of events
19 m s 11 m s 233 m s 0.10 19 + 1 m s 31+13 ms 266 + 32 m s c 0.09+0.01 d
100 42 58
17 m s 13 m s 124 m s 0.16 18 + 1 m s 15+1 ms 147 _+ 17 m s c 0.14+0.01 d
100 57 43
100 32-I-8 68 + 8
100 36-4- 8 64 + 12
= Represents analysis of experiments in figs. 5 and 6 ([BRL 34915] = 50 nM). b M e a n + S . E . M . , n = 4. c Represents a significant decrease, P < 0.01. a Represents a significant increase, P < 0.01.
208 in the absence and presence of the drug were best fit by the sum of two exponentials with closed time constants of 11 and 233 ms under control conditions and 13 and 124 ms in the presence of 50 nM BRL 34915. There is no significant difference in the fast closed time constant in the presence of 50 nM BRL 34915; however, there is a significant decrease (P < 0.01) of 45% in the slow closed time constant (control 266 + 32 ms, BRL 34915 147 + 17 ms, table 1). A shift in the number of events exhibiting the faster closed time constant was also observed. This decrease in the slow time constant leads to the increase in Popenof 56% and the changes seen in the single channel records of fig. 5. BRL 34915 dose dependently shifted the Pope, voltage curve as shown in fig. 7. BRL 34915, 50 nM, shifted the midpoint of activation approximately 15 mV in the hyperpolarizing direction while 500 nM BRL 34915 shifted the curve approximately 30 mV. According to the Boltzman equation, Vo, the membrane potential were Pope, = 0.5, is - 5 mV in control, - 2 0 and - 3 5 mV in the presence of 50 and 500 nM BRL 34915, respectively. The slope factor of the curve did not change significantly in the presence of BRL 34915, suggesting little effect of BRL 34915 on the voltage-dependent activation of the channel.
4. Discussion
The Ca2+-activated K ÷ channels examined in this study have the same general properties as those first described in cultured bovine chromaffin cells (Marty, 1981) and in other vascular and non-vascular smooth muscle (Benham et al., 1985; Bolton et al., 1975; Inoue et al., 1985; McCann and Welsh, 1986; Singer and Walsh, 1987). These properties are: its large conductance, approximately 300 pS in symmetrical concentrations of KC1, high K + selectivity, dependence of the Pope~ on membrane potential and on [Ca2+]i and the impermeability to Na + ions. The use of planar lipid bilayers enables one to investigate various aspects of the channel under controlled conditions. Two new results are: first, the low [Ca2+]i sensitivity of the smooth muscle
K ÷ channel (fig. 4) and second, the effects of BRL 34915 on its open probability (figs. 5, 6, 7 and table 1). The [Ca2+]i sensitivity of this channel from rabbit aorta is less than that of some other vascular and non-vascular smooth muscles (Benham et al., 1986). There are several possible explanations for the observed low [Ca2+]i sensitivity of the Ca2+-activated K + channel from aortic smooth muscle cells. First, Mg 2÷ was left out of the experimental solutions in order to examine the actual [Ca2+]i sensitivity of the channel. It has been reported that [Mg2+]i (1-10 mM), in rat t-tubule membranes, increased the affinity of the channel for Ca 2+ and enhanced the sigmoidicity of the Ca 2÷ activation curve (Golowasch et al., 1986). The [Ca2+]i sensitivity of rat t-tubular channels is also low (Latorre et al., 1982) and though [Mg2÷]i (1-10 mM) did enhance the [Ca2+]i sensitivity of the channel, it was still less sensitive than that of the large Ca2+-activated K + channel in rabbit aorta (present data). In the presence of 10 mM [Mg2+]i and approximately 1 /xM [Ca2+]i , the Pown of the K ÷ channel in rabbit t-tubule was less than 0.1 at + 30 mV. In the rabbit aorta, under similar conditions (1 ~M [Ca2+]i and a holding potential of +30 mV), but in the absence of [Mg2+]i, the Pope, was 1.0. A second possible explanation for the low [Ca2+]~ sensitivity may be that a component essential for high [Ca2+]~ sensitivity is lost upon isolation of the smooth muscle cell membranes. A third possibility is that the [Ca2+]i sensitivity is indeed low but that the [Ca z+] just beneath the plasmalemma is greater than the average intracellular [Ca2+]. Variation in the [Ca 2÷] between different cytoplasmic regions of smooth muscle cell has been demonstrated (Williams et al., 1985). According to their fura-2 images, the [Ca2+]i just beneath the plasmalemma was higher than deep in the myoplasm. The postulate that [Ca2+]~ near the inner plasmalemmal surface is relatively high is further supported by the fact that Ca2+-activated K + channels of smooth muscle may be spontaneously activated without contractile activity being apparent (Benham et al., 1986; Ohya et al., 1987). The second result, which may be of pharmacological relevance, is that BRL 34915 increased the
209 Popen of the CaZ+-activated K + channels in the p r e s e n c e of 1 # M [Ca2+]i (see figs. 5, 6, 7 a n d table 1); at - 40 m V the Porch of the large c o n d u c t a n c e CaZ+-activated K + c h a n n e l increased 56% a n d 200% in the presence of 50 a n d 500 n M B R L 34915, respectively. This is the first r e p o r t that a low c o n c e n t r a t i o n of B R L 34915 can m o d u l a t e the large c o n d u c t a n c e CaZ+-activated K + c h a n n e l of v a s c u l a r s m o o t h muscle at a p h y s i o l o g i c a l l y relev a n t m e m b r a n e p o t e n t i a l . D e t a i l e d analysis o f the results g e n e r a t e d with 50 n M B R L 34915 d e m o n s t r a t e d that the i n c r e a s e d Pop~ resulted f r o m the c h a n n e l r e m a i n i n g in its long closed state for a s h o r t e r p e r i o d o f time. T h e long closed state time c o n s t a n t decreased b y 45% f r o m 266 + 32 to 147 + 17 ms (table 1). T h e r e was also a s i m u l t a n e o u s increase in the n u m b e r of events exhibiting the s h o r t closed state of the channel in the presence of B R L 34915. This p h e n o m e n o n is also seen in the p r e s e n c e of 1 # M pinacidil, a n o t h e r p u t a t i v e K + c h a n n e l agonist in vascular s m o o t h muscle (Gelb a n d a n d V a n Breemen, 1989). B R L 34915's effect was m a n i f e s t as a d o s e - d e p e n d e n t shift in the Por~, voltage curve in the h y p e r p o l a r i z i n g direction. This shift was a t t a i n e d at relatively low c o n c e n t r a t i o n s o f B R L 34915 (50 a n d 500 n M ) . It is plausible; therefore, t h a t higher c o n c e n t r a t i o n s of B R L 34915 w o u l d further shift the a c t i v a t i o n curve a n d allow c h a n n e l o p e n i n g at the cell's resting m e m b r a n e potential. A l t h o u g h the o b s e r v e d i n c r e a s e d in Porch is n o t overwhelming, it m a y be sufficient to explain the r e p o r t e d effects of B R L 34915 on s m o o t h muscle c o n t r a c t i o n a n d 86Rb+ efflux. It has b e e n calcul a t e d that there are a p p r o x i m a t e l y 103-104 large c o n d u c t a n c e CaZ+-activated K + channels p e r s m o o t h muscle cell (Singer a n d Walsh, 1987). D u e to their large c o n d u c t a n c e , a c t i v a t i o n of o n l y a few o f these channels in the presence of B R L 34915 could increase the 86Rb+ efflux, a n d cause h y p e r p o l a r i z a t i o n of the cell m e m b r a n e a n d r e l a x a t i o n of the muscle. O t h e r m e c h a n i s m s ; however, c a n n o t b e e x c l u d e d at this time. Recently, it has been shown that g l y b u r i d e antagonizes the a c t i o n s of B R L 34915 in rat p o r t a l vein ( W i n q u i s t et al., 1988), suggesting that B R L 34915 m a y b e a c t i v a t i n g a n A T P - m o d u l a t e d K + c h a n n e l in s m o o t h muscle, in a d d i t i o n to activating the large
c o n d u c t a n c e C a 2 + - a c t i v a t e d K + channel. I n s u m m a r y , this s t u d y is the first to i n c o r p o r a t e large c o n d u c t a n c e C a 2 + - a c t i v a t e d K + c h a n n e l f r o m r a b b i t a o r t a i n t o p l a n a r lipid bilayers. T h e c h a n n e l shows the s a m e characteristics as o t h e r channels o f its type, n a m e l y its [Ca2+]i a n d volta g e - d e p e n d e n t activation, its i m p e r m e a b i l i t y to N a ÷, its K ÷ selectivity a n d its high c o n d u c t a n c e in s y m m e t r i c a l KC1. However, this a o r t i c s m o o t h muscle c h a n n e l has a r a t h e r low [Ca2+]i sensitivity in p l a n a r lipid bilayers. F i n a l l y , it has b e e n d e m o n s t r a t e d that B R L 34915 c a n i n c r e a s e the Por~n of the large c o n d u c t a n c e C a 2 + - a c t i v a t e d K ÷ c h a n n e l in v a s c u l a r s m o o t h muscle.
Acknowledgements This research was supported by the National Institute of Health Grants HL-40184 and HL-07188. The authors would like to thank Dr. J. McCullough (Squibb) for the synthesized BRL 34915, Drs. D. Adams and J. Talvenheimo for their helpful conversations, and G. Trebilcock for her secretarial assistance.
References Beech, D.J. and T.B. Bolton, 1987, Effects of BRL 34915 on membrane currents recorded from single smooth muscle cells from the rabbit portal vein, Br. J. Pharmacol. 92, 550P. Benham, C.D., T.B. Bolton, R.J. Lang and T. Takewaki, 1985, The mechanism of action of Ba 2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes, Pfliigers Arch. 403, 120. Benham, C.D., T.B. Bolton, R.J. Lang and T. Takewaki, 1986, Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery, J. Physiol. 371, 45. Blatz, A.L. and K.L. Magleby, 1984, Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle, J. Gen. Physiol. 84, 1. Bolton, T.B., R.J. Lang, T. Takewaki and C.D. Benham, 1985, Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells, Experientia 41, 887. Buckingham, R.E., J.C. Clapham, T.C. Hamilton, S.D. Longman, J. Norton and R.H. Poyser, 1986, BRL 34915, a novel antihypertensive agent: comparison of effects on blood pressure and other haemodynamic parameters with those of nifedipine in animal models, J. Cardiovasc. Pharmacol. 8, 798.
210 Clapham, J.C. and C. Wilson, 1984, Effects of the novel anti-hypertensive agent BRL 34915 in comparison with nifedipine on rabbit isolated mesenteric artery, Br. J. Pharmacol. 87, 77P. Gelband, C.H., N.J. Lodge, J.A. Talvenheimo and C. Van Breemen, 1988, BRL 34915 increases Po~n of the large conductance Ca 2+-activated K + channel isolated from rabbit aorta in planar lipid bilayers, Biophys. J. 53, 149a. Gelband, C.H. and C. Van Breemen, 1989, Reconstituted aortic Ca 2+-activated K + channels: selectivity and pharmacological modulation by cromakalim (BRL 34915) and pinacidil, Biophys. J. 55, 232a. Golowasch, J., A. Kirkwood and C. Miller, 1986, Allosteric effects of Mg 2+ on the gating of Ca2+-activated K + channels from mammalian skeletal muscle, J. Exp. Biol. 124, 5. Hamilton, T.C., S.W. Weir and A.H. Weston, 1986, Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein, Br. J. Pharmacol. 88, 103. Inoue, R., K. Kitamura and H. Kuriyama, 1985, Two Ca-dependent K-channels classified by the application of tetraethylamrnonium distribute to smooth muscle membranes of the rabbit portal vein, Pfliigers Arch. 405, 173. Latorre, R., C. Vergara and C. Hidalgo, 1982, Reconstitution in planar hpid bilayers of a Ca2+-dependent K + channel from transverse tubule membranes isolated from rabbit skeletal muscle, Proc. Natl. Acad. Sci. 79, 805. Kreye, V.A.W. and A.H. Weston, 1986, BRL 34915-induced stimulation of 86Rb + efflux in rabbit aorta and its dependence on calcium, J. Physiol. 374, 36P. Kwan, C.Y., R. Garfield and E.E. Daniel, 1979, An improved procedure for the isolation of plasma membranes from rat mesenteric arteries, J. Mol. Cell. Cardiol. 11,639. Marty, A., 1981, Ca-dependent K channels with large unitary conductance in chromaffin cell membranes, Nature 291, 797. McCarm, J.D. and M.J. Welsh, 1986, Calcium-activated potassium channels in canine airway smooth muscle, J. Physiol. 372, 113.
Nelson, M.T., R.J. French and B.K. Krueger, 1984, Voltage dependent calcium channels from brain incorporated into planar lipid bilayers, Nature (London) 308, 77. Ohya, K., K. Kitamura and H. Kuriyama, 1987, Cellular calcium regulates outward currents in rabbit intestinal smooth muscle cell, Am. J. Physiol. 252, C401. Okabe, K., K. Kitamura and H. Kuriyama, 1987, Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery, Pfliigers Arch. 409, 561. Quast, U. and N.S. Cook, 1988, Leirus quinquestriatus venom inhibits BRL 34915-induced S6Rb+ efflux from the rat portal vein, Life Sci. 42, 805. Saida, K. and Y. Nonomura, 1978, Characteristics of Ca 2+and Mg2+-induced tension development in chemically skinned smooth muscle fibers, J. Gen. Physiol. 72, 1. Singer, J.J. and J.V. Walsh, Jr., 1987, Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch clamp technique, Pfliigers Arch. 408, 98. Talvenheimo, J.A., G. Lam and C. Gelband, 1988, Charybdotoxin inhibitis the 250 pS Ca2+-activated K + channel in aorta and contracts aorta smooth muscle, Biophys. J. 53, 258a. Weir, S.W. and A.H. Weston, 1986, Effect of apamin on responses to BRL 34915, nicorandil and other relaxants in the guinea-pig taenia caeci, Br. J. Pharmacol. 85, 113. Williams, D.A., K.E. Fogarty, R.Y. Tsien and F.S. Fay, 1985, Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2, Nature 318, 558. Wilson, C., 1987, Antagonism of the vasorelaxant activity of BRL 34915 by K + channel blockers, Br. J. Pharmacol. 91, 401P. Winquist, R.J., L.A. Heaney and E.P. Baskin, 1988, Blockade of the relaxation to BRL 34915 and minoxidil by glyburide in isolated rat portal veins, FASEB J. 2, 784A.
201
EJP 50924
A C a 2 +-activated K + channel from rabbit aorta: modulation by cromakalim Craig H. Gelband *, Nicholas J. Lodge and Cornclis Van Brcemen Department of Pharmacology (R-189), Universityof Miami School of Medicine, Miami, EL 33101, U.S.A.
Received 10 November 1988, revised MS received 3 March 1989, accepted 30 May 1989
A large conductance Ca2+-activated K + channel from rabbit aorta was incorporated into planar lipid bilayers. This channel had a conductance of 337 + 7 pS in symmetrical 250 mM KC1 solutions and had a N a + / K + permeability ratio of < 0.04. In asymmetrical solutions containing 300 mM KC1 cis (intracellular), 100 mM KC1 trans (extracellular) or 100 mM KCI cis 500 mM KC1 trans, the reversal potentials for the channel were - 3 0 and +46 mV, respectively. This channel possessed voltage-dependent activation and cis (intracellular) Ca 2+ sensitivity. Cromakalim (50 nM) added to the trans side of the bilayer significantly increased the Popen by 56% from 0.09 + 0.01 to 0.14 _ 0.01 (P < 0.01) at - 4 0 mV without altering the single channel conductance. This effect was dose-dependent, increasing at higher cromakalim concentrations. The primary effect of cromakalim was to decrease the %lowof the channel closed state from 266 + 32 to 1 4 7 _ 17 ms which is sufficient to account for the increase in Popen of the channel in the presence of cromakalim. Aorta; Planar lipid bilayer; Ca2+-activated K + channels; [Ca2+]i sensitivity; Cromakalim; (Rabbit)
1. Introduction L a r g e c o n d u c t a n c e Ca2+-activated K + channels have been i d e n t i f i e d in a n u m b e r of vascular a n d n o n - v a s c u l a r s m o o t h muscles (Bolton et al., 1985; I n o u e et al., 1985; M c C a n n a n d Welsh, 1986; Singer a n d Walsh, 1987). This K + c h a n n e l is a c t i v a t e d b y voltage a n d intracellular [Ca2+]. Its c o n d u c t a n c e is greater t h a n 200 pS, d e p e n d i n g on the e x p e r i m e n t a l c o n d i t i o n s used. A l t h o u g h this c o n d u c t a n c e is large, the channel is highly selective for K + ions over o t h e r cations a n d anions. Ca2+-activated K + channels have b e e n p r o p o s e d to p l a y a role in r e p o l a r i z a t i o n of action p o t e n t i a l s o r o t h e r d e p o l a r i z i n g stimuli of vascular s m o o t h m u s c l e a n d in slow wave p r o p a g a t i o n o f visceral s m o o t h muscle. M a n y c o m p o u n d s have been
* To whom all correspondence should be addressed: Department of Pharmacology (R-189), University of Miami School of Medicine, P.O. Box 016189, Miami, FL 33101, U.S.A.
shown to b l o c k the large c o n d u c t a n c e C a 2+a c t i v a t e d K + channel of vascular s m o o t h muscle i n c l u d i n g T E A in r a b b i t p o r t a l vein ( I n o u e et al., 1985), Ba 2+ in guinea pig mesenteric artery (Benh a m et al., 1985), 4 - a m i n o p y r i d i n e in r a b b i t p u l m o n a r y a r t e r y ( O k a b e et al., 1987) a n d c h a r y b d o t o x i n in b o v i n e a o r t a ( T a l v e n h e i m o et al., 1988). C r o m a k a l i m ( B R L 34915), a novel a n t i - h y p e r tensive agent, lowers b l o o d pressure ( B u c k i n g h a m et al., 1986), h y p e r p o l a r i s e s s m o o t h muscle m e m b r a n e s ( H a m i l t o n et al., 1986), a n d relaxes prec o n t r a c t e d vascular s m o o t h muscle ( C l a p h a m a n d Wilson, 1984) b y a m e c h a n i s m which is h y p o t h e sized to involve the o p e n i n g of a class of K ÷ channels. T h e actions of B R L 34915 to relax contracted s m o o t h muscle a n d increase the 86Rb+ efflux f r o m s m o o t h muscle are a n t a g o n i z e d b y such K ÷ c h a n n e l a n t a g o n i s t s as T E A , 4 - a m i n o p y r i d i n e a n d p r o c a i n e (Wilson, 1987). H o w e v e r , a p a m i n , an a n t a g o n i s t of the small c o n d u c t a n c e
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
202 Ca2+-activated K + channel, is ineffective in antagonizing the effects of BRL 34915 in smooth muscle (Weir and Weston, 1986). Kreye and Weston (1986) have reported that the 86Rb+ efflux stimulated by BRL 34915 in rabbit aorta is Ca 2+dependent suggesting that the K + channel opened by BRL 34915 is also Ca2+-dependent. In addition Quast and Cook (1987) have reported that a crude venom of Leiurus quinquestriatus, a scorpion toxin which contains charybdotoxin (a putative Ca2+-activated K + channel antagonist), could inhibit BRL 34915-induced increases in 86Rb+ efflux from portal vein. In contrast, Beech and Bolton (1987) have reported that in portal vein BRL 34915 increases a K + conductance which is different from the large conductance Ca 2+activated K + channel. The aim of the present study was to incorporate the large conductance Ca 2 +-activated K + channel of rabbit aortic smooth muscle into planar lipid bilayers, characterize its activity, and investigate the actions of BRL 34915 on the isolated channel. Preliminary results have been presented (Gelband et al., 1988; Gelband and Van Breemen, 1989).
2. M a t e r i a l s
and methods
2.1. Microsomal isolation Microsomes were prepared according to the method of Kwan et'al. (1979) with some modifications. Adult rabbits (New Zealand white, 2-3 kg body weight) were anesthetized in a CO 2 chamber and the thoracic aorta was rapidly excised and placed in oxygenated PSS at 37 o C. Rabbit aortas were cleaned of fat and adventitia and stored at - 70 ° C until their use. When required, the tissues were thawed, minced and homogenized using a Polytron PT 10 homogenizer in 250 m M sucrose, 10 m M M O P S - K O H , p H 7.2. A Teflon pestle and a glass homogenizer was used to further disrupt the tissues. The homogenate was centrifuged at 10000 × g for 20 rain, and the resulting supernatant was recentrifuged at 27 000 X g for 20 rain. The 27000 x g pellet was discarded and the 27 000 x g supernatant was centrifuged at 100 000 x g for 1 h. The final pellet was resuspended in
250 m M sucrose, 10 m M M O P S - K O H , p H 7.2, aliquoted and frozen at - 7 0 ° C until its use. The final protein concentration was 1.5-3.0 m g / m l . All procedures were performed at 4 ° C. 2.2. Incorporation of Ca 2 +-activated K + channels into planar lipid bilayers A solution containing 27 m g / m l phosphatidylethanolamine, 23 m g / m l phosphatidyl serine (Avanti Polar Lipids, Birmingham, AL) dissolved in decane was used to form planar lipid bilayers. Planar lipid bilayers were formed across a 150-250 /~M diameter hole in a Lexan cup. The lipid bilayer separated two experimental chambers as described by Nelson et al. (1984). CaE+-activated K + channels from aortic microsomes were incorporated into the planar lipid bilayers by adding the sample to one side of the bilayer, designated the cis side. The side opposite to which the vesicles were added is designated trans. The cis solution was vigorously stirred to facilitate channel incorporation. The channel's sidedness was established on the basis of its voltage-dependent activation and its [Ca2+]i sensitivity. A consistent orientation was established by the observation that addition of Ca 2+ or E G T A to only the cis chamber increased the probability of channel opening or closing respectively. Depolarization of the membrane, Vcis-Vt..... also facilitated channel opening, while hyperpolarization of the m e m b r a n e decreased channel opening. Thus the solution in the cis chamber always represented the intracellular solution. [Ca 2+] was maintained low in the trans chamber of insure that if occasionally a channel was oriented in the opposite direction it would not be activated. The final protein concentration added to the cis side was 1-10/~g/ml. All experiments were performed at 22 o C. 2.3. Electrical recordings C o m m a n d potentials were applied to the bilayer under voltage clamp conditions using two A g / A g C l electrodes with the trans side held at virtual ground. Voltages are defined as Vcis-Vt.... . Current fluctuations across the bilayer were visualised on an oscilloscope, recorded on F M
203
tape and played back through an 8-pole Bessel filter at 315 Hz. The inherent filtering of the voltage clamp was 1 kHz. Data analysis was performed on these recordings, digitized at 4 kHz, using an IBM PC A T computer and the software p C l a m p 4.1. The total time of the records used in the analysis of probability of opening, Pope,, and open and closed time constants was 10 min in the absence and 10 min in the presence of the drug or vehicle. Six out of ten bilayers formed contained more than one channel; however, only those records from bilayers containing a single channel were used for analysis and are displayed in the figures.
3. Results The activity of a single large conductance K + channel, in symmetrical 250 m M KC1 and 1 m M cis CaC12, is shown in fig. 1A. In symmetrical
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-20 PSS (in mM): NaC1 140, KCI 5, CaC12 1.5, MgC12 1, glucose 10, HEPES 5, p H 7.2. Experimental chambers contained the following (unless otherwise stated in the figure legends) KC1 250, M O P S - K O H 10, p H 6.8, E G T A 2. Ca 2÷ concentrations less than 50 ttM were calculated and prepared using an E G T A buffer assuming an apparent binding constant of the C a - E G T A complex to be 1 0 6 M - 1 at p H 6.8, 2 2 ° C (Saida and Nonomura, 1978). BRL 34915 was dissolved in dimethyl sulfoxide (Sigma).
-40
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--
400
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2.5. Statistics
50/
The open probability of a single channel, Popen, is defined as the amount of time the channel remained in the open state divided by the total time of the recording. Open and closed time histograms were described by exponentials using the method of least squares. The number of exponentials required to generate the best fit of the data was assessed using the statistical parameters provided within pClamp 4.1. N o corrections were made for rapid openings and closing not fully resolved by the voltage clamp system. All values shown are the means +__S.E.M. unless otherwise noted. Paired student t-tests were performed to evaluate the significance of the data. Data were considered significant with a P < 0.05.
0
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-2o_ ° -6o 4o- o o A 4b 6b 8b v (my} Fig. 1. Single channel records and current-voltage relationship of the large conductance Ca2+-activated K + channel. (A) Experimental chambers contained symmetrical (in mM): KCI 250, M O P S - K O H 10, p H 6.8. However 1 m M CaCI 2 was present on the cis side and 2 m M E G T A was present on thetrams side. Single channel records were filtered at 315 Hz. Solid line to the fight of the traces represents the closed state of t h e channel. (B) Current-voltage relationship (one experiment of ten).
204
solutions, the current flowing through the channel reversed at 0 mV. This channel possessed voltagedependent activation; as the membrane potential was hyperpolarized, not only did the current reverse, but the probability of the channel being open, Pope,, decreased. A representative single channel current-voltage relationship for this channel is shown in fig. lB. The slope conductance of the channel, estimated between +40 and - 4 0 mV, had an average value of 337 + 7 pS (n = 10). The selectivity of this large conductance K + channel from rabbit aorta was investigated and the results are shown in figs. 2 and 3. Figure 2 shows the effects of changing the K ÷ concentration on either side of the planar lipid bilayer. In the presence of 300 mM KC1 cis and 100 mM KC1 trans (closed squares), the Nernst equation for K ÷ predicts a reversal potential of - 2 8 mV. The
I
I
Fig. 3. Effect of hi-ionic solutions on the single channel current-voltage relationship. The closed squares represent experiments (n = 3) under these conditions: trans (mM) NaC1 250, CaC1 z M O P S - N a O H 10, p H 6.8; cis (mM) KC1 250, MOPSK O H , p H 6.8. The closed circles, represent experiments (n = 3) performed under these conditions: trans (mM) KC1 250, CaC12 1, M O P S - K O H 10; p H 6.8; cis (mM) NaC1 250, M O P S - K O H 10, p H 6.8. The current-voltage relationship does not cross the zero current axis.
(pA) 20 -
15 10 -
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-
.
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Fig. 2. Effect of asymmetric K + solutions on the single channel current-voltage relationship. The closed squares represent experiments (n = 3) performed under these conditions: cis (mM) KCI 300, CaCI 2 1, M O P S - K O H 10, p H 6.8; trans (mM) KC1 100, M O P S - K O H 10, p H 6.8. The d o s e d circles represent (n = 3) experiments performed under these conditions, cis (mM) KCI 100, CaC12 1, M O P S - K O H 10, p H 6.8; trans (mM) KC1 500, M O P S - K O H 10 p H 6.8. Note that Ere v are - 3 0 and + 4 6 mV for the squares and circles, respectively.
20 40 60 80
(my)
single channel current reversed at - 3 0 mV. On the other hand, in the presence of 100 mM KC1 cis and 500 mM KC1 trans (closed circles), the single channel current reversed at +46 mV, in close agreement with + 41 mV for the Nernst potential for K + under these conditions. Thus the channel is highly selective for K + over C1-. Figure 3 examines the relative permeability of Na + through this K + channel. Na + ions have been reported to be impermeant through large Ca2+-activated K + channels (Blatz and Magleby, 1984). Under asymmetric NaC1/KC1 conditions, no Na + permeation can be measured if Na ÷ replaces K + on either side of the bilayer. It is apparent that the currents, inward or outward depending on the experimental conditions, became smaller appearing to approach the zero current level but did not reverse. Since the single channel currents did not reverse, an
205
-60 mV
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Fig. 4. The effect of voltage and [Ca 2+ ]i on Popenof the Ca2+-activated K + channel. Experimental chambers contained (in mM): symmetric KC1 250, MOPS-KOH 10 pH 6.8, and the Ca 2+ cxmcentration on the cis side is varied. EGTA, 2 raM, is present on the trans side of the bilayer.o(A) Records in the presence of 25 ~tM Ca 2+ cis. Records are filtered at 315 (n = 3). Solid line represents the closed state of the channel. (B) The effects of Ca 2 + on Po__.. Symbols: (e) 1/~M Ca 2 + (&) 25 ~M Ca 2+ ( 0 ) 50 ttM Ca 2+, (zx) 75 /~M Ca 2 + , (©) 100 #M Ca 2 + and (ll) 1 mM Ca 2 + (n = 3).
absolute Na+/K + permeability ratio cannot be calculated. However, an e s t i m a t e of the u p p e r limit of the N a + p e r m e a b i l i t y can b e m a d e b y e x t r a p o l a t i n g a reversal p o t e n t i a l f r o m the current-voltage relationship and would predict a N a + / K + p e r m e a b i l i t y r a t i o o f < 0.04, which is c o n s i s t e n t with o t h e r v a s c u l a r a n d visceral s m o o t h m u s c l e ( B e n h a m et al., 1986). T h e [Ca2+]i a n d voltage d e p e n d e n c y o f the large c o n d u c t a n c e Ca2+-activated K + channel was e x a m i n e d in fig. 4. F i g u r e 4 A shows single c h a n n e l r e c o r d s in the p r e s e n c e o f 25 v M CaC12 o n the cis (intracellular) side of the m e m b r a n e . D e p o l a r i z i n g the m e m b r a n e p o t e n t i a l f r o m - 6 0 to - 4 0 m V i n c r e a s e d the Pope, f r o m 0.07 to 0.27. A s the m e m b r a n e p o t e n t i a l is d e p o l a r i z e d further f r o m - 4 0 to + 20 mY, the Pop~, increased f r o m 0.27 to 0.97. F i g u r e 4B shows the effects o f v a r y i n g the cis
(intracellular) C a 2+ c o n c e n t r a t i o n on the Pope, of the channel. [Ca2+]i a c t i v a t i o n of the K + c h a n n e l was d e m o n s t r a t e d b y a shift to the left of the Popen voltage curve when the cis C a 2÷ was raised f r o m 1 /xM to 1 m M . T h e effects o f the novel a n t i h y p e r t e n s i v e a n d p u t a t i v e K + c h a n n e l a g o n i s t B R L 34915 a r e s h o w n in figs. 5, 6, 7 a n d in t a b l e 1. R e p r e s e n t a t i v e single c h a n n e l r e c o r d i n g s in the a b s e n c e a n d p r e s e n c e of 50 n M B R L 34915 on the trans (extracellular) side a r e seen in fig. 5. R e c o r d i n g s were m a d e in the presence o f s y m m a t r i c a l 250 m M K C I a n d 1 /xM CaC12 on the cis side of the m e m b r a n e at - 4 0 mV. T h e left h a n d p a n e l shows c o n t r o l records in the a b s e n c e of B R L 34915. O p e n i n g s were brief" a n d the t i m e the c h a n n e l r e m a i n e d in the closed state was l o n g c o m p a r e d to t h a t o f the o p e n state. T h e a c t i o n s o f 50 n M B R L 34915 a r e i l l u s t r a t e d in
206 CONTROL l ~
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Fig. 5. Effects o f B R L 34915 o n single c h a n n e l r e c o r d i n g s . E x p e r i m e n t a l c h a m b e r s c o n t a i n e d the s a m e s o l u t i o n s as s t a t e d in M e t h o d s , except the cis C a 2+ c o n c e n t r a t i o n is 1 # M . T h e left h a n d p a n e l s h o w s c o n t r o l r e c o r d s a n d the r i g h t h a n d p a n e l s h o w s the effects o f 50 n M B R L 34915 a d d e d to the t r a n s side. M e m b r a n e p o t e n t i a l is - 4 0 mV. Pore, in t h e c o n t r o l is 0.10 a n d in t h e p r e s e n c e of B R L 34915 is 0.16. R e c o r d s are filtered a t 315 Hz. N o t e single c h a n n e l a m p l i t u d e is n o t a f f e c t e d b y the p r e s e n c e o f B R L 34915. Single c h a n n e l o p e n i n g s are d o w n w a r d . Scale b a r s r e p r e s e n t 20 p A (vertical) a n d 200 m s ( h o r i z o n t a l ) .
the right hand panel. Openings are again brief; however, the amount of time the channel remained closed was shortened. Pope, of the single channel increased by 60% from 0.10 to 0.16. In four experiments, an increase of 56% was evident from 0.09 + 0.01 to 0.14 + 0.01 (P < 0.01, table 1). The effect on Pope, was dose-dependent, increasing from 0.09 + 0.01 to 0.35 + 0.03 in the presence of 500 nM BRL 34915 at - 4 0 mV (see below, fig. 7). BRL 34915 did not have any effect on unitary current amplitude or single channel conductance. It was also evident that the channel gated between two distinct modes of opening. The first mode was characterized by its long closures between subsequent openings and the second contained groupings of rapid openings and closings. This activity is present in the absence and presence of BRL 34915. A long period of single channel activity was analyzed to take into account for the moding
behavior of the channel in calculating the effects of BRL 34915. Identical experiments were performed using the DMSO vehicle only. Pope, was found not to be significantly different during the 10 min exposure to DMSO when compared to the 10 min prior to the addition of DMSO (Pope, = 0.10 + 0.01 in the absence or presence of DMSO, P < 0.01). These data indicate that BRL 34915's effects can not be attributed to the vehicle or time dependent changes in the channel's probability of opening or states of inactivation. Kinetic analysis was performed on the records of fig. 5 and these results are depicted in fig. 6. In the upper panel, open time histograms are plotted for data in the absence and presence of 50 nM BRL 34915. Open time histograms were best fit by a single exponential. In the absence of BRL 34915, the open time constant (~) was 19 ms while in the
207 CONTROL
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OPEN TIME HISTOGRAM 90"
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Fig. 6. O p e n a n d closed time h i s t o g r a m s of the c h a n n e l in fig. 5 u n d e r control c o n d i t i o n s (left) a n d in the presence of 50 n M B R L 34915 trans (right). The o p e n time h i s t o g r a m s were best fit b y a single e x p o n e n t i a l a n d the closed t i m e h i s t o g r a m s were best fit b y the s u m of two exponentials. T i m e c o n s t a n t s for the e x p o n e n t i a l s are p r e s e n t e d in table 1.
presence of 50 nM BRL 34915, the open time constant was 17 ms. Table 1 shows the values for the open time constants for the experiment shown in figs. 5 and 6 and the values for the mean of the four experiments in the absence and presence of 50 nM BRL 34915. There was no significant
-o0
-40
40
;
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Voltage (mV) Fig. 7. Effects of B R L 34915 on Pop~. as a function o f voltage. E x p e r i m e n t a l c h a m b e r s c o n t a i n e d the s a m e s y m m e t r i c a l solutions as stated in Methods, except t h a t the cis [Ca 2+ ] w a s 1 # M . Symbols: control ( I ) , 50 n M B R L 34915 (Ill) a n d 500 n M B R L 34915 (&). B R L 34915 was a d d e d to the trans solution. Vo is - 5 , - 2 0 a n d - 3 0 m V for control, 50 a n d 500 n M B R L 34915, respectively. D a t a s h o w n are the m e a n s _+S.E.M. (n = 3). E r r o r bars are n o t present w h e n the s y m b o l is the s a m e size as the S.E.
change in the open time constant in the presence of 50 nM BRL 34915 (control 1 9 _ 1 ms, BRL 34915 18 _ 1 ms, n = 4). The major effect of BRL 34915 was resolved in the closed time analysis. Closed time histograms
TABLE 1 A n a l y s i s of single c h a n n e l records.
Open = Closed = Por~- a Open b Closed b Pop¢~ b
qq'fast %low ~ q-f,=t q-slow
Control
% of events
B R L 34915
% of events
19 m s 11 m s 233 m s 0.10 19 + 1 m s 31+13 ms 266 + 32 m s c 0.09+0.01 d
100 42 58
17 m s 13 m s 124 m s 0.16 18 + 1 m s 15+1 ms 147 _+ 17 m s c 0.14+0.01 d
100 57 43
100 32-I-8 68 + 8
100 36-4- 8 64 + 12
= Represents analysis of experiments in figs. 5 and 6 ([BRL 34915] = 50 nM). b M e a n + S . E . M . , n = 4. c Represents a significant decrease, P < 0.01. a Represents a significant increase, P < 0.01.
208 in the absence and presence of the drug were best fit by the sum of two exponentials with closed time constants of 11 and 233 ms under control conditions and 13 and 124 ms in the presence of 50 nM BRL 34915. There is no significant difference in the fast closed time constant in the presence of 50 nM BRL 34915; however, there is a significant decrease (P < 0.01) of 45% in the slow closed time constant (control 266 + 32 ms, BRL 34915 147 + 17 ms, table 1). A shift in the number of events exhibiting the faster closed time constant was also observed. This decrease in the slow time constant leads to the increase in Popenof 56% and the changes seen in the single channel records of fig. 5. BRL 34915 dose dependently shifted the Pope, voltage curve as shown in fig. 7. BRL 34915, 50 nM, shifted the midpoint of activation approximately 15 mV in the hyperpolarizing direction while 500 nM BRL 34915 shifted the curve approximately 30 mV. According to the Boltzman equation, Vo, the membrane potential were Pope, = 0.5, is - 5 mV in control, - 2 0 and - 3 5 mV in the presence of 50 and 500 nM BRL 34915, respectively. The slope factor of the curve did not change significantly in the presence of BRL 34915, suggesting little effect of BRL 34915 on the voltage-dependent activation of the channel.
4. Discussion
The Ca2+-activated K ÷ channels examined in this study have the same general properties as those first described in cultured bovine chromaffin cells (Marty, 1981) and in other vascular and non-vascular smooth muscle (Benham et al., 1985; Bolton et al., 1975; Inoue et al., 1985; McCann and Welsh, 1986; Singer and Walsh, 1987). These properties are: its large conductance, approximately 300 pS in symmetrical concentrations of KC1, high K + selectivity, dependence of the Pope~ on membrane potential and on [Ca2+]i and the impermeability to Na + ions. The use of planar lipid bilayers enables one to investigate various aspects of the channel under controlled conditions. Two new results are: first, the low [Ca2+]i sensitivity of the smooth muscle
K ÷ channel (fig. 4) and second, the effects of BRL 34915 on its open probability (figs. 5, 6, 7 and table 1). The [Ca2+]i sensitivity of this channel from rabbit aorta is less than that of some other vascular and non-vascular smooth muscles (Benham et al., 1986). There are several possible explanations for the observed low [Ca2+]i sensitivity of the Ca2+-activated K + channel from aortic smooth muscle cells. First, Mg 2÷ was left out of the experimental solutions in order to examine the actual [Ca2+]i sensitivity of the channel. It has been reported that [Mg2+]i (1-10 mM), in rat t-tubule membranes, increased the affinity of the channel for Ca 2+ and enhanced the sigmoidicity of the Ca 2÷ activation curve (Golowasch et al., 1986). The [Ca2+]i sensitivity of rat t-tubular channels is also low (Latorre et al., 1982) and though [Mg2÷]i (1-10 mM) did enhance the [Ca2+]i sensitivity of the channel, it was still less sensitive than that of the large Ca2+-activated K + channel in rabbit aorta (present data). In the presence of 10 mM [Mg2+]i and approximately 1 /xM [Ca2+]i , the Pown of the K ÷ channel in rabbit t-tubule was less than 0.1 at + 30 mV. In the rabbit aorta, under similar conditions (1 ~M [Ca2+]i and a holding potential of +30 mV), but in the absence of [Mg2+]i, the Pope, was 1.0. A second possible explanation for the low [Ca2+]~ sensitivity may be that a component essential for high [Ca2+]~ sensitivity is lost upon isolation of the smooth muscle cell membranes. A third possibility is that the [Ca2+]i sensitivity is indeed low but that the [Ca z+] just beneath the plasmalemma is greater than the average intracellular [Ca2+]. Variation in the [Ca 2÷] between different cytoplasmic regions of smooth muscle cell has been demonstrated (Williams et al., 1985). According to their fura-2 images, the [Ca2+]i just beneath the plasmalemma was higher than deep in the myoplasm. The postulate that [Ca2+]~ near the inner plasmalemmal surface is relatively high is further supported by the fact that Ca2+-activated K + channels of smooth muscle may be spontaneously activated without contractile activity being apparent (Benham et al., 1986; Ohya et al., 1987). The second result, which may be of pharmacological relevance, is that BRL 34915 increased the
209 Popen of the CaZ+-activated K + channels in the p r e s e n c e of 1 # M [Ca2+]i (see figs. 5, 6, 7 a n d table 1); at - 40 m V the Porch of the large c o n d u c t a n c e CaZ+-activated K + c h a n n e l increased 56% a n d 200% in the presence of 50 a n d 500 n M B R L 34915, respectively. This is the first r e p o r t that a low c o n c e n t r a t i o n of B R L 34915 can m o d u l a t e the large c o n d u c t a n c e CaZ+-activated K + c h a n n e l of v a s c u l a r s m o o t h muscle at a p h y s i o l o g i c a l l y relev a n t m e m b r a n e p o t e n t i a l . D e t a i l e d analysis o f the results g e n e r a t e d with 50 n M B R L 34915 d e m o n s t r a t e d that the i n c r e a s e d Pop~ resulted f r o m the c h a n n e l r e m a i n i n g in its long closed state for a s h o r t e r p e r i o d o f time. T h e long closed state time c o n s t a n t decreased b y 45% f r o m 266 + 32 to 147 + 17 ms (table 1). T h e r e was also a s i m u l t a n e o u s increase in the n u m b e r of events exhibiting the s h o r t closed state of the channel in the presence of B R L 34915. This p h e n o m e n o n is also seen in the p r e s e n c e of 1 # M pinacidil, a n o t h e r p u t a t i v e K + c h a n n e l agonist in vascular s m o o t h muscle (Gelb a n d a n d V a n Breemen, 1989). B R L 34915's effect was m a n i f e s t as a d o s e - d e p e n d e n t shift in the Por~, voltage curve in the h y p e r p o l a r i z i n g direction. This shift was a t t a i n e d at relatively low c o n c e n t r a t i o n s o f B R L 34915 (50 a n d 500 n M ) . It is plausible; therefore, t h a t higher c o n c e n t r a t i o n s of B R L 34915 w o u l d further shift the a c t i v a t i o n curve a n d allow c h a n n e l o p e n i n g at the cell's resting m e m b r a n e potential. A l t h o u g h the o b s e r v e d i n c r e a s e d in Porch is n o t overwhelming, it m a y be sufficient to explain the r e p o r t e d effects of B R L 34915 on s m o o t h muscle c o n t r a c t i o n a n d 86Rb+ efflux. It has b e e n calcul a t e d that there are a p p r o x i m a t e l y 103-104 large c o n d u c t a n c e CaZ+-activated K + channels p e r s m o o t h muscle cell (Singer a n d Walsh, 1987). D u e to their large c o n d u c t a n c e , a c t i v a t i o n of o n l y a few o f these channels in the presence of B R L 34915 could increase the 86Rb+ efflux, a n d cause h y p e r p o l a r i z a t i o n of the cell m e m b r a n e a n d r e l a x a t i o n of the muscle. O t h e r m e c h a n i s m s ; however, c a n n o t b e e x c l u d e d at this time. Recently, it has been shown that g l y b u r i d e antagonizes the a c t i o n s of B R L 34915 in rat p o r t a l vein ( W i n q u i s t et al., 1988), suggesting that B R L 34915 m a y b e a c t i v a t i n g a n A T P - m o d u l a t e d K + c h a n n e l in s m o o t h muscle, in a d d i t i o n to activating the large
c o n d u c t a n c e C a 2 + - a c t i v a t e d K + channel. I n s u m m a r y , this s t u d y is the first to i n c o r p o r a t e large c o n d u c t a n c e C a 2 + - a c t i v a t e d K + c h a n n e l f r o m r a b b i t a o r t a i n t o p l a n a r lipid bilayers. T h e c h a n n e l shows the s a m e characteristics as o t h e r channels o f its type, n a m e l y its [Ca2+]i a n d volta g e - d e p e n d e n t activation, its i m p e r m e a b i l i t y to N a ÷, its K ÷ selectivity a n d its high c o n d u c t a n c e in s y m m e t r i c a l KC1. However, this a o r t i c s m o o t h muscle c h a n n e l has a r a t h e r low [Ca2+]i sensitivity in p l a n a r lipid bilayers. F i n a l l y , it has b e e n d e m o n s t r a t e d that B R L 34915 c a n i n c r e a s e the Por~n of the large c o n d u c t a n c e C a 2 + - a c t i v a t e d K ÷ c h a n n e l in v a s c u l a r s m o o t h muscle.
Acknowledgements This research was supported by the National Institute of Health Grants HL-40184 and HL-07188. The authors would like to thank Dr. J. McCullough (Squibb) for the synthesized BRL 34915, Drs. D. Adams and J. Talvenheimo for their helpful conversations, and G. Trebilcock for her secretarial assistance.
References Beech, D.J. and T.B. Bolton, 1987, Effects of BRL 34915 on membrane currents recorded from single smooth muscle cells from the rabbit portal vein, Br. J. Pharmacol. 92, 550P. Benham, C.D., T.B. Bolton, R.J. Lang and T. Takewaki, 1985, The mechanism of action of Ba 2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes, Pfliigers Arch. 403, 120. Benham, C.D., T.B. Bolton, R.J. Lang and T. Takewaki, 1986, Calcium-activated potassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery, J. Physiol. 371, 45. Blatz, A.L. and K.L. Magleby, 1984, Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle, J. Gen. Physiol. 84, 1. Bolton, T.B., R.J. Lang, T. Takewaki and C.D. Benham, 1985, Patch and whole-cell voltage clamp of single mammalian visceral and vascular smooth muscle cells, Experientia 41, 887. Buckingham, R.E., J.C. Clapham, T.C. Hamilton, S.D. Longman, J. Norton and R.H. Poyser, 1986, BRL 34915, a novel antihypertensive agent: comparison of effects on blood pressure and other haemodynamic parameters with those of nifedipine in animal models, J. Cardiovasc. Pharmacol. 8, 798.
210 Clapham, J.C. and C. Wilson, 1984, Effects of the novel anti-hypertensive agent BRL 34915 in comparison with nifedipine on rabbit isolated mesenteric artery, Br. J. Pharmacol. 87, 77P. Gelband, C.H., N.J. Lodge, J.A. Talvenheimo and C. Van Breemen, 1988, BRL 34915 increases Po~n of the large conductance Ca 2+-activated K + channel isolated from rabbit aorta in planar lipid bilayers, Biophys. J. 53, 149a. Gelband, C.H. and C. Van Breemen, 1989, Reconstituted aortic Ca 2+-activated K + channels: selectivity and pharmacological modulation by cromakalim (BRL 34915) and pinacidil, Biophys. J. 55, 232a. Golowasch, J., A. Kirkwood and C. Miller, 1986, Allosteric effects of Mg 2+ on the gating of Ca2+-activated K + channels from mammalian skeletal muscle, J. Exp. Biol. 124, 5. Hamilton, T.C., S.W. Weir and A.H. Weston, 1986, Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein, Br. J. Pharmacol. 88, 103. Inoue, R., K. Kitamura and H. Kuriyama, 1985, Two Ca-dependent K-channels classified by the application of tetraethylamrnonium distribute to smooth muscle membranes of the rabbit portal vein, Pfliigers Arch. 405, 173. Latorre, R., C. Vergara and C. Hidalgo, 1982, Reconstitution in planar hpid bilayers of a Ca2+-dependent K + channel from transverse tubule membranes isolated from rabbit skeletal muscle, Proc. Natl. Acad. Sci. 79, 805. Kreye, V.A.W. and A.H. Weston, 1986, BRL 34915-induced stimulation of 86Rb + efflux in rabbit aorta and its dependence on calcium, J. Physiol. 374, 36P. Kwan, C.Y., R. Garfield and E.E. Daniel, 1979, An improved procedure for the isolation of plasma membranes from rat mesenteric arteries, J. Mol. Cell. Cardiol. 11,639. Marty, A., 1981, Ca-dependent K channels with large unitary conductance in chromaffin cell membranes, Nature 291, 797. McCarm, J.D. and M.J. Welsh, 1986, Calcium-activated potassium channels in canine airway smooth muscle, J. Physiol. 372, 113.
Nelson, M.T., R.J. French and B.K. Krueger, 1984, Voltage dependent calcium channels from brain incorporated into planar lipid bilayers, Nature (London) 308, 77. Ohya, K., K. Kitamura and H. Kuriyama, 1987, Cellular calcium regulates outward currents in rabbit intestinal smooth muscle cell, Am. J. Physiol. 252, C401. Okabe, K., K. Kitamura and H. Kuriyama, 1987, Features of 4-aminopyridine sensitive outward current observed in single smooth muscle cells from the rabbit pulmonary artery, Pfliigers Arch. 409, 561. Quast, U. and N.S. Cook, 1988, Leirus quinquestriatus venom inhibits BRL 34915-induced S6Rb+ efflux from the rat portal vein, Life Sci. 42, 805. Saida, K. and Y. Nonomura, 1978, Characteristics of Ca 2+and Mg2+-induced tension development in chemically skinned smooth muscle fibers, J. Gen. Physiol. 72, 1. Singer, J.J. and J.V. Walsh, Jr., 1987, Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch clamp technique, Pfliigers Arch. 408, 98. Talvenheimo, J.A., G. Lam and C. Gelband, 1988, Charybdotoxin inhibitis the 250 pS Ca2+-activated K + channel in aorta and contracts aorta smooth muscle, Biophys. J. 53, 258a. Weir, S.W. and A.H. Weston, 1986, Effect of apamin on responses to BRL 34915, nicorandil and other relaxants in the guinea-pig taenia caeci, Br. J. Pharmacol. 85, 113. Williams, D.A., K.E. Fogarty, R.Y. Tsien and F.S. Fay, 1985, Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2, Nature 318, 558. Wilson, C., 1987, Antagonism of the vasorelaxant activity of BRL 34915 by K + channel blockers, Br. J. Pharmacol. 91, 401P. Winquist, R.J., L.A. Heaney and E.P. Baskin, 1988, Blockade of the relaxation to BRL 34915 and minoxidil by glyburide in isolated rat portal veins, FASEB J. 2, 784A.