Dynamics of Aminopyridine Block of Potassium Channels in Squid ...
Dynamics of Aminopyridine Block of Potassium Channels in Squid Axon Membrane J . z . Y E H , G. S. O X F O R D , C. H. W U , and T . N A R A H A S H I From the Department of Physiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710. Dr. Oxford's present address is the Department of Physiology, Universityof North Carolina, School of Medicine, Chapel Hill, North Carolina 27514.
A B S T R A C T Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a m a n n e r d e p e n d e n t u p o n the m e m b r a n e potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external m e m b r a n e surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. T h e K c u r r e n t in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a m a n n e r d e p e n d e n t u p o n the duration and interval of pulses. The recovery from block d u r i n g a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. T h e time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2AP interacts with the K channel more rapidly than 3-AP and 4-AP. T h e more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequencyd e p e n d e n t block which is different from that in 3-AP or 4-AP. T h e experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley ntype gating units. Rather, the data are consistent with a simple b i n d i n g scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules b i n d i n g to closed K channels and being released from open channels in a voltage-dependent m a n n e r . I N T R O D U C T I O N
I o n i c c h a n n e l s i n excitable m e m b r a n e s a r e s u b j e c t to v a r i o u s p h a r m a c o l o g i c a l , e n z y m a t i c , a n d c h e m i c a l m o d i f i c a t i o n s . T h e selective i n f l u e n c e o f s u c h m a n i p u l a t i o n s o n ionic c o n d u c t a n c e s is a s t r o n g a r g u m e n t f o r s e p a r a t e c h a n n e l s f o r t h e m o v e m e n t o f s o d i u m a n d p o t a s s i u m ions. B e c a u s e o f t h e specific a n d selective f e a t u r e s , c e r t a i n a g e n t s h a v e b e c o m e i n d i s p e n s a b l e tools f o r s t u d y i n g t h e b e h a v ior o f ionic c h a n n e l s . T e t r o d o t o x i n ( T T X ) w h i c h selectively blocks s o d i u m c h a n n e l s a n d t e t r a e t h y l a m m o n i u m i o n ( T E A ) w h i c h blocks p o t a s s i u m c h a n n e l s a r e two s u c h e x a m p l e s . THE JOURNAL
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4 - A m i n o p y r i d i n c has been r e p o r t e d to block selectively the potassium channel in axons o f the cockroach (Pelhate and Pichon, 1974), squid (Meves and Pichon, 1975; Yeh et al., 1976a), Myxicola (Schauf et al., 1976), and frog node o f Ranvier (Wagner and Ulbricht, 1975). In the squid axon and frog n o d e o f Ranvier the a m i n o p y r i d i n e block o f potassium channels is not a simple reduction o f conductance, but exhibits voltage-, time-, and f r e q u e n c y - d e p e n d e n t characteristics. T h e main objective o f this investigation is to characterize the dynamics o f interaction o f aminopyridines with the K channel. O n the basis o f the kinetic analysis o f the interaction to be p r e s e n t e d and two key assumptions, we f o r m u late a model which can account for m a n y o f the experimental observations. T h e first assumption is that the gating mechanism o f K channels is not significantly affected by aminopyridines, and the second is that a m i n o p y r i d i n e - b o u n d K channels c a n n o t pass K ions even when their activation gates are o p e n . A preliminary r e p o r t o f this work has been presented at the 20th annual meeting o f the Biophysical Society (Yeh et al., 1976b). METHODS
Experiments were performed on giant axons isolated from Loligo pealei obtained at the Marine Biological Laboratory, Woods Hole, Mass. Both intact axons and axons internally perfused by the roller technique of Baker et al. (1961) were used in these studies. Cleaned axons were mounted in a Plexiglas chamber designed for voltage clamping by conventional techniques described previously (Wu and Narahashi, 1973). Briefly, a "piggyback" double axial electrode assembly was inserted into the axon for measurement and control of membrane potential. Membrane current was measured by the virtual ground of an operational amplifier from a Pt-black electrode situated within a region of the chamber electrically guarded to maximize radial current flow. The response time of the clamp was --6 0ts (10-90% of step pulse command). Feedback compensation was used in all experiments to compensate for errors arising from approx, two-thirds of the measured 3-4 fl/ cm 2 of series resistance. Holding potentials were -80 mV for internal perfused axons and - 7 0 mV for intact axons. The axons were perfused externally with artificial seawater (ASW) containing ions in the following concentrations (mM): Na ÷, 450; K+, 10; Ca ÷÷, 50; HEPES buffer, 5; Ci-, 576. In several experiments an external bathing medium with elevated potassium concentration was used by equimolar replacement of 340 mM Na with K. External pH was adjusted to 8.0 in both cases. The standard internal solution (SIS) was composed as follows (mM): Na +, 50; K+, 350; glutamate-, 320; F-, 50; sucrose, 333; phosphate buffer, 15; and was adjusted to a pH of 7.3. All experiments were performed at a constant temperature of 8°-10°C. Most experiments were performed with 300 nM tetrodotoxin in the external bathing medium to eliminate current contributed by sodium channels. 2-, 3-, and 4-aminopyridine were obtained from Aldrich Chemical Company (Milwaukee, Wis.) and used without further purification.
Data Analysis, Terminology, and Computations Oscilloscope records of membrane current and voltage were captured on 35-mm film and analyzed by hand with the aid of a programmable calculator. Comparisons of data obtained with the Hodgkin-Huxley (H-H) model for potassium conductance (Hodgkin and Huxley, 1952) were performed by using the empirical formulae for an and/3, given by Palti (1971) and assuming a Q10 = 3. Actual simulations of voltage clamp experiments were performed on an HP9821 programmable calculator with 9864 digital plotter output
YEH, OXFORD, W u , AND NARAHASHI Dynamicsof Aminopyridine Block of K Channels
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(Hewlett-Packard Co., Cupertino, Calif.) by use of a kinetic equivalent of the H-H model (Armstrong, 1969) as described in the Discussion. The terminology of "gate" or "gating" as adopted in this paper implies no physical scheme of ionic channels but merely refers to the normal processes by which K channels allow ions to pass across the membrane whatever the mechanism may be. The three analogs of aminopyridine will be referred to collectively as n-AP in situations where differences in their effects are not readily distinguishable. RESULTS
A m i n o p y r i d i n e s (n-AP) selectively suppress potassium currents in the squid giant axon when applied to either internal or external m e m b r a n e surfaces (Fig. 1). Sodium currents remain u n a f f e c t e d by n - A P treatment. Potassium tail curControl
4 -AP
Externol Application
n~/cnl 2
internal Applicotion
/z~=-----~=
FXGUR~ I. Effects of 4-aminopyridine on squid giant axons. Upper records are membrane action potentials, and middle records are clamp families before and after external application of 1 mM 4-AP. Lower records are clamp families from another axon before and after internal application of 1 mM 4-AP. Note only slight prolongation of action potential despite a dramatic reduction of K current. rents at the e n d o f 8-ms depolarizing steps are r e d u c e d due to both a suppression o f potassium c o n d u c t a n c e and a resultant reduction in K + ion accumulation in a periaxonal space. T h e block o f IK is dramatic but not complete for larger depolarizations. Despite the reduction in IK the action potential d u r a t i o n is only slightly p r o l o n g e d by n-AP. In the presence o f n-AP the potassium c u r r e n t rises more slowly than in control (Yeh et al., 1976a) reaching to a steady-state level only after tens o f milliseconds. T h e r e f o r e , the suppression o f K currents by nAP was evaluated at steady-state levels as well as at early times. Fig. 2 d e m o n strates that the inhibition o f IK at a given potential is less at the end o f a 70-ms pulse than after 8-ms (see also Table I). This d u r a t i o n - d e p e n d e n t block is shared by all three aminopyridines; however, there are some differences in potency a m o n g t h e m . Fig. 3 illustrates the m o r e rapid rise o f IK in 2-AP (b) than in 3-AP (a). T h e u p p e r trace in each case is a control IK in ASW alone. T h e curves in Fig. 3c and d are simulations o f the data with a kinetic m o d e l to be described later. Differences in potency a m o n g the
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a m i n o p y r i d i n e s for r e d u c i n g IK are s u m m a r i z e d in T a b l e I w h i c h clearly indicates that 2 - A P is the least p o t e n t in inhibiting K c u r r e n t s in squid a x o n s , w h e r e a s 3- a n d 4 - A P e x e r t a b o u t the s a m e e f f e c t . T h e c o n c e n t r a t i o n d e p e n d e n c e o f n - A P was difficult to e v a l u a t e b e c a u s e o f the e f f e c t b e i n g c o m p l i c a t e d by the m e m b r a n e p o t e n t i a l , a n d the d u r a t i o n and f r e q u e n c y o f p u l s e s . T a b l e I s h o w s that 30 m M 2 - A P is o n l y slightly m o r e O
18
o
o Control
15
o
V 3-AP, 8 ms
o o
3-AP ,70ms o
12 ©
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o
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&
o
&
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6 o
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Fbt~tk~l (my) FICURE 2. Time-dependent block of K current by 3-AP. Current-voltage relations of an axon were measured in the presence of 300 nM T T X externally. O, current measured at the end of 8-ms pulse before application of 3-AP; ~7 a n d / ~ , currents measured at the end of 8 ms and 70 ms, respectively, after external perfusion with 1 mM 3-AP. TABLE
I
EFFECT OF A M I N O P Y R I D I N E S ON POTASSIUM C U R R E N T MEASURED A T 0 AND 100 MV Percent inhibition* measured at 8 ms n-AP
70 ms
Concn
0
100
0
100
mM
mV
mV
mV
mV
80.4-+ 1.60 92.0
50.3---3,25 66.5
2-AP
1 30
74.8-+2.72 8 1 . 0 -+ 1.00
21.6±2.92 62.8-+2.85
3-AP
1 30
93.8-+2.20 99.5+0.05
73.3 + - 1.70 79.2 -+2.00
4-AP
0.03 0.10 0.30 1 10
70.0±3.79 88.3-+3,18 90.0 96.2-+ 1.92 94.0
55.3±4.06 59.0-+8.54 82.0 74.7-+2.29 88.0
* M e a n ± S E M (n = 3).
Yrri, OXFORD, Wu, AND NARAHASHI Dynamicsof ArainopyridineBlockof K Channels
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e f f e c t i v e t h a n 1 m M w h e n t h e e f f e c t is e v a l u a t e d at 0 m V a n d is t h r e e t i m e s m o r e e f f e c t i v e w h e n e v a l u a t e d at 100 m V . T h e e f f e c t o f 3 - A P is a l m o s t s a t u r a t e d at 1 mM.
Voltage-Dependent Block of IK A m i n o p y r i d i n e s s u p p r e s s t h e p o t a s s i u m c u r r e n t in a m a n n e r d e p e n d e n t u p o n m e m b r a n e p o t e n t i a l . I n Fig. 4 t h e r a t i o o f IK in 3 - A P to IK in A S W w i t h o u t 3 - A P is p l o t t e d as a f u n c t i o n o f m e m b r a n e p o t e n t i a l . T h e K c u r r e n t s w e r e m e a s u r e d in two ways. T h e t r i a n g l e s r e p r e s e n t m e a s u r e m e n t s o f s t e a d y - s t a t e IK at t h e e n d o f t h e first o f two 70-ms p u l s e s s e p a r a t e d by a 1-s i n t e r v a l . T h e circles a r e m e a s u r e m e n t s t a k e n a t 5 m s a f t e r t h e o n s e t o f t h e RXflN
0
b
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r~
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FIGURE 3. Patterns o f K c u r r e n t in 2- and 3-AP. M e m b r a n e currents d u r i n g a clamp pulse to + 100 mV before and after external application of 1 mM 3-AP (a) or 1 mM 2-AP (b). ASW contained 300 nM T T X in each case. Simulation o f c u r r e n t patterns in 3-AP (c) and 2-AP (d) with kinetic model described in Discussion. In controlk = 0,1 = 0, and rr = 0. In 3-APk = 0.06 ms -1,1 = 0.01 ms -~, and rrl = 3 s, and ~'r2 = 27 s. In 2-APk = 0.3 ms -~, l = 0.1 ms -~, and 7r = 0.8 s. At rest ( - 8 0 mV) U' = 0.95 in both cases. s e c o n d p u l s e o f f i x e d a m p l i t u d e ( + 100 m V ) . T h i s l a t t e r p r o c e d u r e is to m i n i m i z e e r r o r s d u e to K + a c c u m u l a t i o n in t h e p e r i a x o n a l s p a c e w h i c h d e c a y s with a t i m e c o n s t a n t o f - 3 0 m s ( F r a n k e n h a e u s e r a n d H o d g k i n , 1956), m u c h s h o r t e r t h a n t h a t f o r r e - e s t a b l i s h m e n t o f n - A P b l o c k a f t e r a l o n g p u l s e (see b e l o w ) . T h e v o l t a g e d e p e n d e n c e d e t e r m i n e d in this m a n n e r is a p p r o x i m a t e l y l i n e a r f o r t h e v o l t a g e r a n g e s t u d i e d a n d v e r y little a f f e c t e d b y t h e c h o i c e o f m e a s u r e m e n t procedure.
Frequency-Dependent Recovery from n-AP Block T h e i n h i b i t i o n o f p o t a s s i u m c u r r e n t s b y n - A P is m a r k e d l y d e p e n d e n t u p o n t h e f r e q u e n c y o f s t i m u l a t i o n ( Y e h et a l . , 1976a). A p r o g r e s s i v e r e c o v e r y o f IK o c c u r s d u r i n g r e p e t i t i v e a p p l i c a t i o n s o f d e p o l a r i z i n g s t e p s w h i c h is d e p e n d e n t u p o n t h e p u l s e f r e q u e n c y . As s e e n in Fig. 5 t h e r e m o v a l o f b l o c k in 3 - A P by successive
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p u l s e s is g r e a t e r f o r i n c r e a s i n g p u l s e f r e q u e n c i e s , s a t u r a t i n g at a p p r o x i m a t e l y 2 H z . Fig. 6 i l l u s t r a t e s t h e c u r r e n t p a t t e r n s o b t a i n e d in 3- a n d 2 - A P d u r i n g twin d e p o l a r i z i n g p u l s e s to + 100 m V s e p a r a t e d by a 1-s i n t e r v a l . T h e rise o f IK is m a r k e d l y a c c e l e r a t e d d u r i n g t h e s e c o n d p u l s e in 3 - A P (a) b u t a p p r o a c h e s t h e
/
cr
ms~t~
f.
~ 0
Prepulse Potentiol (mv) FIGURE 4. Voltage-dependent block of K channels by aminopyridines. Two consecutive 70-ms voltage steps separated by a 1-s interval were applied to an axon bathed in ASW and then in 3-AP. T h e amplitude o f the first or conditioning pulse is varied while the second pulse is fixed at + 100 mV. K current measured in 3-AP relative to steady-state (8 ms) values in ASW is plotted as a function of the conditioning pulse amplitude. C u r r e n t was measured either at the end of the 70-ms prepulse (A) or after 5 ms of the test pulse (©). Solid line represents a least-squares regression fit to the data points obtained d u r i n g the test pulse.
5Hz ~
/5Hz
:3
~j~J
"2 Hz
%2
Io.Q~22Hz
1:12I - t z ~
2 mA/crn2
2 ms
Number of Pulses
FIGURE 5. F r e q u e n c y - d e p e n d e n t recovery from aminopyridine block. Potassium currents p r o d u c e d by six consecutive pulses to + 100 mV at the indicated frequencies after external application o f 1 mM 4-AP. A 3-mir/ rest period was allowed between each trial frequency. Increases in current d u r i n g consecutive pulses at several frequencies are shown to the right of the current records. Data points were measured after 2 ms of each pulse. Curves are drawn by eye through the points. s a m e s t e a d y - s t a t e level (b). T h e e f f e c t o f t h e c o n d i t i o n i n g p u l s e is less d r a m a t i c in 2 - A P (c), p r e s u m a b l y b e c a u s e t h e r e m o v a l o f b l o c k was e s s e n t i a l l y c o m p l e t e d d u r i n g t h e first p u l s e . Fig. 6d-f d e p i c t s s i m u l a t i o n s o f t h e t w i n p u l s e e x p e r i m e n t s with t h e k i n e t i c m o d e l d e s c r i b e d in t h e D i s c u s s i o n .
YEH, OXFORD, WU, AND NARAHASHI Dynamics of Aminopyridine Block of K Channels
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In order to examine f ur t her the frequency dependence of block, two series of e x p e r i m e n t s w e r e p e r f o r m e d . I n t h e f i r st series t h e t i m e c o u r s e f o r r e - e s t a b l i s h m e n t o f c h a n n e l b l o c k a f t e r r e m o v a l o f i n h i b i t i o n by a s i n g l e p u l s e was d e t e r m i n e d . T w i n d e p o l a r i z i n g p u l s e s to + 1 0 0 m V w e r e a p p l i e d a n d t h e i n t e r v a l b e t w e e n t h e first a n d s e c o n d p u l s e s v a r i e d . A 3 - m i n r e s t p e r i o d was a l l o w e d b e t w e e n e a c h p a i r o f p u l s e s to a s s u r e u n i f o r m c o n d i t i o n s f o r e a c h trial. Fig. 7
Rxo.
3-AP
2-AP
3-AP
a
b
c
e"
(
"
F-K K I NET I C M O D E L
r~ d
f
r~ e
[
~.1~
HS/DIV
I~i ~
HS/DIV 4
FIGURE 6. F r e q u e n c y - d e p e n d e n t block of K channels by aminopyridines. Current patterns obtained for two consecutive depolarizing steps to + 100 mV separated by a 1-s interval in 1 mM 3-AP (a, b) and 1 mM 2-AP (c). Simulations of each cu r r en t pattern by the kinetic model are shown (d-J) below the respective experimental pattern. T h e model parameters used in these simulations are identical to those used in Fig. 3 c o r r e s p o n d i n g to 2- and 3-AP. T i m e scales for a-c are the same as for d-f, respectively. T h e diminished frequency d e p e n d e n c e in 2-AP reflects the m o r e rapid interaction o f 2-AP molecules with the binding site (k = 0.3 ms -t, l = 0.1 ms -I , z = 0.8 s).
._xo.8~ .~0.6 -~0.4
~
~
_
s
Q2 I0 20 Pulse Interval
30
40
(s)
FU;URE 7. R e - e s t a b l i s h m e n t o f K c h a n n e l b l o c k by a m i n o p y r i d i n e s . T w o c o n s e c u tive pulses to + 100 mV separated by a variable time interval were applied to an axon
bathed in 1 mM 3-AP and 300 nM T T X . T h e ratio of the increase in IK d u r i n g the second pulse to the m a x i m u m observed increase is plotted as a function of pulse interval. Currents were measured at 0.5 ms of each pulse. Sample cu r r en t patterns for intervals o f 1, 10, and 60 s are shown in the insets. Block proceeds with two time constants of --3 s and - 2 7 s. Solid line is fit to the data points by the equation I/Imax = 0.55 exp (-t/3) + 0.45 exp (-t/27), where t = time in seconds.
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illustrates the results o f this type o f e x p e r i m e n t . T h e n o r m a l i z e d d i f f e r e n c e in IK between the first a n d second pulses was m e a s u r e d at 0.5 ms a f t e r each step a n d plotted as a function o f the interval between pulses r e p r e s e n t i n g the time course o f re-establishment o f inhibition. In the case o f 3-AP the time course exhibits two c o m p o n e n t s , a fast time constant (3 s) a n d a slower one (27 s). I n contrast, the reestablishment o f block in 2-AP h a d a m u c h faster time course. S u p e r i m p o s a b l e K c u r r e n t traces could be p r o d u c e d in 2-AP w h e n the two pulses were s e p a r a t e d by an interval o f only 2 s. Consequently, the time constant for re-establishment o f block in 2-AP is estimated to be less t h a n 1 s. Re-establishment o f block is m o r e r a p i d as the concentrations o f n-AP are increased. For e x a m p l e , in the p r e s e n c e o f 30 m M n-AP the potassium c u r r e n t s associated with the twin pulses could be r e p r o d u c e d for pulse intervals as short as 2 s in the case o f 3- or 4-AP a n d only 1 s in 2-AP.
Time Course of Removal of n-AP Block T h e second series o f e x p e r i m e n t s was designed to e x a m i n e the time course o f recovery f r o m block d u r i n g multiple pulses. T h e pulse schedule again used twin depolarizing pulses to + 100 m V in this case with a fixed 1-s pulse interval but with a variable first pulse d u r a t i o n . In Fig. 8 the recovery o f IK is plotted as the c u r r e n t m e a s u r e d at 3 ms o f the second pulse vs. the d u r a t i o n o f the first or conditioning pulse. T h e recovery f r o m 3-AP block usually followed a single e x p o n e n t i a l time course, with a time constant o f a p p r o x i m a t e l y 10-20 ms. This can p e r h a p s be visualized as r e p r e s e n t i n g the time constant g o v e r n i n g the interaction o f 3- or 4-AP with a site within the o p e n K channel. While the o p e n i n g o f the K channel gating m e c h a n i s m is p r o b a b l y r e q u i r e d for the pulsed e p e n d e n t recovery o f Ig, the rate-limiting step a p p e a r s to be the dissociation o f the n-AP molecule f r o m the o p e n channel. T h i s is a p p a r e n t , as the time constant for recovery f r o m block is a p p r o x i m a t e l y 10 times longer than that for n o r m a l activation o f K channels. T o the right o f the recovery plot in Fig. 8 are typical records used to construct the plot ( u p p e r traces) and the results o f simulating the same protocol on the kinetic m o d e l (lower curves).
Steady-State Inhibition Is Less in High External K E x p e r i m e n t s in high external potassium have shown n-AP to suppress c u r r e n t s flowing in either direction t h r o u g h K channels (Yeh et al., 1976a). In 340 m M K seawater, the resting m e m b r a n e potential is a p p r o x i m a t e l y 0 m V a n d external application o f 1 m M 2-, 3-, or 4-AP p r o d u c e d no a p p r e c i a b l e change. T h e r e f o r e , the m e m b r a n e potential could be c l a m p e d at zero without polarizing the current-passing electrode. Step depolarizations to various potentials were applied a n d quasi-instantaneous c u r r e n t s were m e a s u r e d . T h e suppression o f K currents by 3-AP u n d e r these conditions is seen in Fig. 9. A linear I-V relation was f o u n d both in the control a n d n-AP axons (Yeh et al., 1976a). T h e decrease in slope of the I-V relation thus r e p r e s e n t s the steady-state inhibition o f K conductance at 0 m V by n-AP. T h e p e r c e n t a g e o f inhibition was not significantly d i f f e r e n t a m o n g these c o m p o u n d s , r a n g i n g f r o m 22% to 28%. I m p o r t a n t to note, however, is that the s u p p r e s s i o n is m u c h smaller t h a n o b s e r v e d at 0 m V (8 ms) for axons in n o r m a l K seawater (see T a b l e I). T h i s discrepancy m a y be
YEH, OXFORD, WU, AND NARAHASH] Dynamicsof Aminopyridine Block of K Channels
527
a s c r i b e d to d i f f e r e n c e s in t h e n u m b e r o f o p e n c h a n n e l s at t h e h o l d i n g p o t e n t i a l s u s e d in e a c h case, 0 m V in h i g h K a n d - 8 0 m V i n n o r m a l A S W . Also q u a n t i t a tive d i f f e r e n c e s i n t h e d e g r e e o f s t e a d y - s t a t e block a r e i n f l u e n c e d by the i n t e r a c t i o n s b e t w e e n K ions a n d n - A P m o l e c u l e s , i n a c t i v a t i o n ofgK, a n d K i o n a c c u m u -
RXON
=
.~OE "O.E
oo
Conditioning Pulse Duration (ms) FIGURE 8. Pulse-dependent recovery from aminopyridine block. Two consecutive pulses to + 100 mV with l-s interval were applied to an axon bathed in 1 mM 3AP and 300 nM T T X . T h e duration of the first pulse was varied and a 3-min rest period allowed between pulse pairs. K current was measured at 3 ms d u r i n g the second pulse. The ratio of IK to the m a x i m u m obtainable Is is plotted as a function of the first pulse duration. Solid line represents a single exponential fit to the data points with a time constant of 21 ms. Typical pattern used to extract such data is shown to the right with its simulation by the kinetic model. Conditioning pulse durations in the stimulation were 1, 2, 5, 10, 20, and 50 ms, consecutively. Model parameters are again identical to those used in Fig. 3 (k = 0.06 ms -1, l = 0.01 ms -a, Trl = 3 S, and Tr2 = 27 S).
Con~ol ~
~ . ~ _
3-AP )
' .....................
-
FIGURE 9. Nonrectifying block of IK by 3-AP. 3-AP (1 mM) was applied externally to an intact axon bathed in high-potassium seawater (340 mM K+). T h e axon was voltage clamped at the resting potential (0 mV) between depolarizing and hyperpolarizing voltage steps applied every 5 s. Note that both inward and outward IK were reduced, but by a smaller a m o u n t than in normal K seawater. l a t i o n . T h e s e factors will be d i s c u s s e d l a t e r . A t h i g h d e p o l a r i z a t i o n in e l e v a t e d e x t e r n a l [K+], c u r r e n t s u n d e r g o a t i m e - d e p e n d e n t r e c o v e r y r e a c h i n g c o n t r o l levels in 2- a n d 4 - A P a f t e r - 8 m s (Yeh et al., 1976a). S u c h a p h e n o m e n o n is n o t o b s e r v e d with 3 - A P (Fig. 9). T h i s m a y be r e l a t e d to t h e c h a r g e state o f t h e m o l e c u l e , as 3 - A P ( p K , = 6.03) exists a l m o s t exclusively i n its n e u t r a l f o r m at p H 8.
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T H E J O U R N A L OF GENERAL P H Y S I O L O G Y • VOLUME 6 8 ' 1 9 7 6
DISCUSSION
T h e interaction o f aminopyridines with potassium channels o f the squid axon m e m b r a n e has been shown to exhibit a complicated pattern o f voltage, time, and frequency d e p e n d e n c e . T h e steady-state reduction o f IK by n-AP is partially relieved at high depolarizations. In addition, the r e m a i n i n g IK exhibits m u c h slower a p p a r e n t t u r n - o n kinetics. While such p h e n o m e n a seem to suggest an alteration o f the conformational f r e e d o m o f K channel gating c o m p o n e n t s , f u r t h e r consideration o f the data reveals inconsistency with such an interpretation. Alternatively, it is suggested that, at the level o f a single channel, the effects o f aminopyridines are not intimately related to the molecules responsible for the o p e n i n g and closing o f the K ion pathway. Potassium currents obtained at the end o f 8-ms pulses in n-AP were c o n v e r t e d to c h o r d conductance values (GK) as a function o f m e m b r a n e potential. Amplitude scaling o f the GK-voltage relation in combination with shifts along the voltage axis results in inadequate fits o f the e x p e r i m e n t a l values in n-AP to steady-state GK in control. This implies that neither a reduction in the m a x i m u m conductance o f a single K channel n o r a shift o f the "set point" o f the gating voltage sensor can account for the observed effects. In addition, if one assumes the H o d g k i n - H u x l e y n p a r a m e t e r adequately to r e p r e s e n t the operation o f potassium channels, it is seen that n u m e r o u s alterations o f both the first o r d e r rate constants a , and /3, and the power function e x p o n e n t (Cole and Moore, 1960) fail to match adequately the kinetic pattern o f IK in n-AP (for example, see Fig. 10). This would seem to rule out a major change in the first o r d e r transition o f n-type gating units by n-AP, if one assumes that these or similarly behaving c o m p o n e n t s are involved in normal K channel gating. One might, however, conceive o f such alterations occurring in only a fraction o f the total channel population and being disguised in a functional summation with normally o p e r a t i n g channels. This possibility was e x a m i n e d by c o m p u t e r simulation using various combinations o f normal channels s u m m e d with channels having altered n parameters to yield a total GK. T h e progressive increase in the fractional population o f K channels having been either slowed by a factor o f 10 or delayed by increasing the power function e x p o n e n t results in a clearly distinguishable kinetic c o m p o n e n t (see Fig. 10b) which has no c o u n t e r p a r t in actual experimental data (for example, Fig. 3). Restricting the simulated change in o~, a n d / 3 , to five times results in a m o r e monotonic curve, but fails to describe adequately the experimentally observed slowing o f Ix. T h e lack o f success with this a p p r o a c h thus p r o m p t s us to suggest an alternative mechanism o f n-AP action which is i n d e p e n d e n t o f the gating properties o f K channels irrespective o f their origin. GK Kinetic Model T h e kinetics o f normal K channel gating can be described by a linear reaction sequence as follows: 4o~
3a
2o~
u
A B S T R A C T Aminopyridines (2-AP, 3-AP, and 4-AP) selectively block K channels of squid axon membranes in a m a n n e r d e p e n d e n t u p o n the m e m b r a n e potential and the duration and frequency of voltage clamp pulses. They are effective when applied to either the internal or the external m e m b r a n e surface. The steady-state block of K channels by aminopyridines is more complete for low depolarizations, and is gradually relieved at higher depolarizations. T h e K c u r r e n t in the presence of aminopyridines rises more slowly than in control, the change being more conspicuous in 3-AP and 4-AP than in 2-AP. Repetitive pulsing relieves the block in a m a n n e r d e p e n d e n t u p o n the duration and interval of pulses. The recovery from block d u r i n g a given test pulse is enhanced by increasing the duration of a conditioning depolarizing prepulse. T h e time constant for this recovery is in the range of 10-20 ms in 3-AP and 4-AP, and shorter in 2-AP. Twin pulse experiments with variable pulse intervals have revealed that the time course for re-establishment of block is much slower in 3-AP and 4-AP than in 2-AP. These results suggest that 2AP interacts with the K channel more rapidly than 3-AP and 4-AP. T h e more rapid interaction of 2-AP with K channels is reflected in the kinetics of K current which is faster than that observed in 3-AP or 4-AP, and in the pattern of frequencyd e p e n d e n t block which is different from that in 3-AP or 4-AP. T h e experimental observations are not satisfactorily described by alterations of Hodgkin-Huxley ntype gating units. Rather, the data are consistent with a simple b i n d i n g scheme incorporating no changes in gating kinetics which conceives of aminopyridine molecules b i n d i n g to closed K channels and being released from open channels in a voltage-dependent m a n n e r . I N T R O D U C T I O N
I o n i c c h a n n e l s i n excitable m e m b r a n e s a r e s u b j e c t to v a r i o u s p h a r m a c o l o g i c a l , e n z y m a t i c , a n d c h e m i c a l m o d i f i c a t i o n s . T h e selective i n f l u e n c e o f s u c h m a n i p u l a t i o n s o n ionic c o n d u c t a n c e s is a s t r o n g a r g u m e n t f o r s e p a r a t e c h a n n e l s f o r t h e m o v e m e n t o f s o d i u m a n d p o t a s s i u m ions. B e c a u s e o f t h e specific a n d selective f e a t u r e s , c e r t a i n a g e n t s h a v e b e c o m e i n d i s p e n s a b l e tools f o r s t u d y i n g t h e b e h a v ior o f ionic c h a n n e l s . T e t r o d o t o x i n ( T T X ) w h i c h selectively blocks s o d i u m c h a n n e l s a n d t e t r a e t h y l a m m o n i u m i o n ( T E A ) w h i c h blocks p o t a s s i u m c h a n n e l s a r e two s u c h e x a m p l e s . THE JOURNAL
OF GENERAL
PHYSIOLOGY
• VOLUME
68,
1976
• pages
519-535
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THE JOURNAL
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PHYSIOLOGY
• VOLUME
68 " 1976
4 - A m i n o p y r i d i n c has been r e p o r t e d to block selectively the potassium channel in axons o f the cockroach (Pelhate and Pichon, 1974), squid (Meves and Pichon, 1975; Yeh et al., 1976a), Myxicola (Schauf et al., 1976), and frog node o f Ranvier (Wagner and Ulbricht, 1975). In the squid axon and frog n o d e o f Ranvier the a m i n o p y r i d i n e block o f potassium channels is not a simple reduction o f conductance, but exhibits voltage-, time-, and f r e q u e n c y - d e p e n d e n t characteristics. T h e main objective o f this investigation is to characterize the dynamics o f interaction o f aminopyridines with the K channel. O n the basis o f the kinetic analysis o f the interaction to be p r e s e n t e d and two key assumptions, we f o r m u late a model which can account for m a n y o f the experimental observations. T h e first assumption is that the gating mechanism o f K channels is not significantly affected by aminopyridines, and the second is that a m i n o p y r i d i n e - b o u n d K channels c a n n o t pass K ions even when their activation gates are o p e n . A preliminary r e p o r t o f this work has been presented at the 20th annual meeting o f the Biophysical Society (Yeh et al., 1976b). METHODS
Experiments were performed on giant axons isolated from Loligo pealei obtained at the Marine Biological Laboratory, Woods Hole, Mass. Both intact axons and axons internally perfused by the roller technique of Baker et al. (1961) were used in these studies. Cleaned axons were mounted in a Plexiglas chamber designed for voltage clamping by conventional techniques described previously (Wu and Narahashi, 1973). Briefly, a "piggyback" double axial electrode assembly was inserted into the axon for measurement and control of membrane potential. Membrane current was measured by the virtual ground of an operational amplifier from a Pt-black electrode situated within a region of the chamber electrically guarded to maximize radial current flow. The response time of the clamp was --6 0ts (10-90% of step pulse command). Feedback compensation was used in all experiments to compensate for errors arising from approx, two-thirds of the measured 3-4 fl/ cm 2 of series resistance. Holding potentials were -80 mV for internal perfused axons and - 7 0 mV for intact axons. The axons were perfused externally with artificial seawater (ASW) containing ions in the following concentrations (mM): Na ÷, 450; K+, 10; Ca ÷÷, 50; HEPES buffer, 5; Ci-, 576. In several experiments an external bathing medium with elevated potassium concentration was used by equimolar replacement of 340 mM Na with K. External pH was adjusted to 8.0 in both cases. The standard internal solution (SIS) was composed as follows (mM): Na +, 50; K+, 350; glutamate-, 320; F-, 50; sucrose, 333; phosphate buffer, 15; and was adjusted to a pH of 7.3. All experiments were performed at a constant temperature of 8°-10°C. Most experiments were performed with 300 nM tetrodotoxin in the external bathing medium to eliminate current contributed by sodium channels. 2-, 3-, and 4-aminopyridine were obtained from Aldrich Chemical Company (Milwaukee, Wis.) and used without further purification.
Data Analysis, Terminology, and Computations Oscilloscope records of membrane current and voltage were captured on 35-mm film and analyzed by hand with the aid of a programmable calculator. Comparisons of data obtained with the Hodgkin-Huxley (H-H) model for potassium conductance (Hodgkin and Huxley, 1952) were performed by using the empirical formulae for an and/3, given by Palti (1971) and assuming a Q10 = 3. Actual simulations of voltage clamp experiments were performed on an HP9821 programmable calculator with 9864 digital plotter output
YEH, OXFORD, W u , AND NARAHASHI Dynamicsof Aminopyridine Block of K Channels
521
(Hewlett-Packard Co., Cupertino, Calif.) by use of a kinetic equivalent of the H-H model (Armstrong, 1969) as described in the Discussion. The terminology of "gate" or "gating" as adopted in this paper implies no physical scheme of ionic channels but merely refers to the normal processes by which K channels allow ions to pass across the membrane whatever the mechanism may be. The three analogs of aminopyridine will be referred to collectively as n-AP in situations where differences in their effects are not readily distinguishable. RESULTS
A m i n o p y r i d i n e s (n-AP) selectively suppress potassium currents in the squid giant axon when applied to either internal or external m e m b r a n e surfaces (Fig. 1). Sodium currents remain u n a f f e c t e d by n - A P treatment. Potassium tail curControl
4 -AP
Externol Application
n~/cnl 2
internal Applicotion
/z~=-----~=
FXGUR~ I. Effects of 4-aminopyridine on squid giant axons. Upper records are membrane action potentials, and middle records are clamp families before and after external application of 1 mM 4-AP. Lower records are clamp families from another axon before and after internal application of 1 mM 4-AP. Note only slight prolongation of action potential despite a dramatic reduction of K current. rents at the e n d o f 8-ms depolarizing steps are r e d u c e d due to both a suppression o f potassium c o n d u c t a n c e and a resultant reduction in K + ion accumulation in a periaxonal space. T h e block o f IK is dramatic but not complete for larger depolarizations. Despite the reduction in IK the action potential d u r a t i o n is only slightly p r o l o n g e d by n-AP. In the presence o f n-AP the potassium c u r r e n t rises more slowly than in control (Yeh et al., 1976a) reaching to a steady-state level only after tens o f milliseconds. T h e r e f o r e , the suppression o f K currents by nAP was evaluated at steady-state levels as well as at early times. Fig. 2 d e m o n strates that the inhibition o f IK at a given potential is less at the end o f a 70-ms pulse than after 8-ms (see also Table I). This d u r a t i o n - d e p e n d e n t block is shared by all three aminopyridines; however, there are some differences in potency a m o n g t h e m . Fig. 3 illustrates the m o r e rapid rise o f IK in 2-AP (b) than in 3-AP (a). T h e u p p e r trace in each case is a control IK in ASW alone. T h e curves in Fig. 3c and d are simulations o f the data with a kinetic m o d e l to be described later. Differences in potency a m o n g the
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a m i n o p y r i d i n e s for r e d u c i n g IK are s u m m a r i z e d in T a b l e I w h i c h clearly indicates that 2 - A P is the least p o t e n t in inhibiting K c u r r e n t s in squid a x o n s , w h e r e a s 3- a n d 4 - A P e x e r t a b o u t the s a m e e f f e c t . T h e c o n c e n t r a t i o n d e p e n d e n c e o f n - A P was difficult to e v a l u a t e b e c a u s e o f the e f f e c t b e i n g c o m p l i c a t e d by the m e m b r a n e p o t e n t i a l , a n d the d u r a t i o n and f r e q u e n c y o f p u l s e s . T a b l e I s h o w s that 30 m M 2 - A P is o n l y slightly m o r e O
18
o
o Control
15
o
V 3-AP, 8 ms
o o
3-AP ,70ms o
12 ©
~E
o
.29 E
&
o
&
©
6 o
V
&
Fbt~tk~l (my) FICURE 2. Time-dependent block of K current by 3-AP. Current-voltage relations of an axon were measured in the presence of 300 nM T T X externally. O, current measured at the end of 8-ms pulse before application of 3-AP; ~7 a n d / ~ , currents measured at the end of 8 ms and 70 ms, respectively, after external perfusion with 1 mM 3-AP. TABLE
I
EFFECT OF A M I N O P Y R I D I N E S ON POTASSIUM C U R R E N T MEASURED A T 0 AND 100 MV Percent inhibition* measured at 8 ms n-AP
70 ms
Concn
0
100
0
100
mM
mV
mV
mV
mV
80.4-+ 1.60 92.0
50.3---3,25 66.5
2-AP
1 30
74.8-+2.72 8 1 . 0 -+ 1.00
21.6±2.92 62.8-+2.85
3-AP
1 30
93.8-+2.20 99.5+0.05
73.3 + - 1.70 79.2 -+2.00
4-AP
0.03 0.10 0.30 1 10
70.0±3.79 88.3-+3,18 90.0 96.2-+ 1.92 94.0
55.3±4.06 59.0-+8.54 82.0 74.7-+2.29 88.0
* M e a n ± S E M (n = 3).
Yrri, OXFORD, Wu, AND NARAHASHI Dynamicsof ArainopyridineBlockof K Channels
523
e f f e c t i v e t h a n 1 m M w h e n t h e e f f e c t is e v a l u a t e d at 0 m V a n d is t h r e e t i m e s m o r e e f f e c t i v e w h e n e v a l u a t e d at 100 m V . T h e e f f e c t o f 3 - A P is a l m o s t s a t u r a t e d at 1 mM.
Voltage-Dependent Block of IK A m i n o p y r i d i n e s s u p p r e s s t h e p o t a s s i u m c u r r e n t in a m a n n e r d e p e n d e n t u p o n m e m b r a n e p o t e n t i a l . I n Fig. 4 t h e r a t i o o f IK in 3 - A P to IK in A S W w i t h o u t 3 - A P is p l o t t e d as a f u n c t i o n o f m e m b r a n e p o t e n t i a l . T h e K c u r r e n t s w e r e m e a s u r e d in two ways. T h e t r i a n g l e s r e p r e s e n t m e a s u r e m e n t s o f s t e a d y - s t a t e IK at t h e e n d o f t h e first o f two 70-ms p u l s e s s e p a r a t e d by a 1-s i n t e r v a l . T h e circles a r e m e a s u r e m e n t s t a k e n a t 5 m s a f t e r t h e o n s e t o f t h e RXflN
0
b
Control
~
Control
/ ~IC ICINE:TIC MOOL~.
r~
C
r~
d
FIGURE 3. Patterns o f K c u r r e n t in 2- and 3-AP. M e m b r a n e currents d u r i n g a clamp pulse to + 100 mV before and after external application of 1 mM 3-AP (a) or 1 mM 2-AP (b). ASW contained 300 nM T T X in each case. Simulation o f c u r r e n t patterns in 3-AP (c) and 2-AP (d) with kinetic model described in Discussion. In controlk = 0,1 = 0, and rr = 0. In 3-APk = 0.06 ms -1,1 = 0.01 ms -~, and rrl = 3 s, and ~'r2 = 27 s. In 2-APk = 0.3 ms -~, l = 0.1 ms -~, and 7r = 0.8 s. At rest ( - 8 0 mV) U' = 0.95 in both cases. s e c o n d p u l s e o f f i x e d a m p l i t u d e ( + 100 m V ) . T h i s l a t t e r p r o c e d u r e is to m i n i m i z e e r r o r s d u e to K + a c c u m u l a t i o n in t h e p e r i a x o n a l s p a c e w h i c h d e c a y s with a t i m e c o n s t a n t o f - 3 0 m s ( F r a n k e n h a e u s e r a n d H o d g k i n , 1956), m u c h s h o r t e r t h a n t h a t f o r r e - e s t a b l i s h m e n t o f n - A P b l o c k a f t e r a l o n g p u l s e (see b e l o w ) . T h e v o l t a g e d e p e n d e n c e d e t e r m i n e d in this m a n n e r is a p p r o x i m a t e l y l i n e a r f o r t h e v o l t a g e r a n g e s t u d i e d a n d v e r y little a f f e c t e d b y t h e c h o i c e o f m e a s u r e m e n t procedure.
Frequency-Dependent Recovery from n-AP Block T h e i n h i b i t i o n o f p o t a s s i u m c u r r e n t s b y n - A P is m a r k e d l y d e p e n d e n t u p o n t h e f r e q u e n c y o f s t i m u l a t i o n ( Y e h et a l . , 1976a). A p r o g r e s s i v e r e c o v e r y o f IK o c c u r s d u r i n g r e p e t i t i v e a p p l i c a t i o n s o f d e p o l a r i z i n g s t e p s w h i c h is d e p e n d e n t u p o n t h e p u l s e f r e q u e n c y . As s e e n in Fig. 5 t h e r e m o v a l o f b l o c k in 3 - A P by successive
524
T H E JOURNAL OF GENERAL P H Y S I O L O G Y " VOLUME 6 8 .
1976
p u l s e s is g r e a t e r f o r i n c r e a s i n g p u l s e f r e q u e n c i e s , s a t u r a t i n g at a p p r o x i m a t e l y 2 H z . Fig. 6 i l l u s t r a t e s t h e c u r r e n t p a t t e r n s o b t a i n e d in 3- a n d 2 - A P d u r i n g twin d e p o l a r i z i n g p u l s e s to + 100 m V s e p a r a t e d by a 1-s i n t e r v a l . T h e rise o f IK is m a r k e d l y a c c e l e r a t e d d u r i n g t h e s e c o n d p u l s e in 3 - A P (a) b u t a p p r o a c h e s t h e
/
cr
ms~t~
f.
~ 0
Prepulse Potentiol (mv) FIGURE 4. Voltage-dependent block of K channels by aminopyridines. Two consecutive 70-ms voltage steps separated by a 1-s interval were applied to an axon bathed in ASW and then in 3-AP. T h e amplitude o f the first or conditioning pulse is varied while the second pulse is fixed at + 100 mV. K current measured in 3-AP relative to steady-state (8 ms) values in ASW is plotted as a function of the conditioning pulse amplitude. C u r r e n t was measured either at the end of the 70-ms prepulse (A) or after 5 ms of the test pulse (©). Solid line represents a least-squares regression fit to the data points obtained d u r i n g the test pulse.
5Hz ~
/5Hz
:3
~j~J
"2 Hz
%2
Io.Q~22Hz
1:12I - t z ~
2 mA/crn2
2 ms
Number of Pulses
FIGURE 5. F r e q u e n c y - d e p e n d e n t recovery from aminopyridine block. Potassium currents p r o d u c e d by six consecutive pulses to + 100 mV at the indicated frequencies after external application o f 1 mM 4-AP. A 3-mir/ rest period was allowed between each trial frequency. Increases in current d u r i n g consecutive pulses at several frequencies are shown to the right of the current records. Data points were measured after 2 ms of each pulse. Curves are drawn by eye through the points. s a m e s t e a d y - s t a t e level (b). T h e e f f e c t o f t h e c o n d i t i o n i n g p u l s e is less d r a m a t i c in 2 - A P (c), p r e s u m a b l y b e c a u s e t h e r e m o v a l o f b l o c k was e s s e n t i a l l y c o m p l e t e d d u r i n g t h e first p u l s e . Fig. 6d-f d e p i c t s s i m u l a t i o n s o f t h e t w i n p u l s e e x p e r i m e n t s with t h e k i n e t i c m o d e l d e s c r i b e d in t h e D i s c u s s i o n .
YEH, OXFORD, WU, AND NARAHASHI Dynamics of Aminopyridine Block of K Channels
525
In order to examine f ur t her the frequency dependence of block, two series of e x p e r i m e n t s w e r e p e r f o r m e d . I n t h e f i r st series t h e t i m e c o u r s e f o r r e - e s t a b l i s h m e n t o f c h a n n e l b l o c k a f t e r r e m o v a l o f i n h i b i t i o n by a s i n g l e p u l s e was d e t e r m i n e d . T w i n d e p o l a r i z i n g p u l s e s to + 1 0 0 m V w e r e a p p l i e d a n d t h e i n t e r v a l b e t w e e n t h e first a n d s e c o n d p u l s e s v a r i e d . A 3 - m i n r e s t p e r i o d was a l l o w e d b e t w e e n e a c h p a i r o f p u l s e s to a s s u r e u n i f o r m c o n d i t i o n s f o r e a c h trial. Fig. 7
Rxo.
3-AP
2-AP
3-AP
a
b
c
e"
(
"
F-K K I NET I C M O D E L
r~ d
f
r~ e
[
~.1~
HS/DIV
I~i ~
HS/DIV 4
FIGURE 6. F r e q u e n c y - d e p e n d e n t block of K channels by aminopyridines. Current patterns obtained for two consecutive depolarizing steps to + 100 mV separated by a 1-s interval in 1 mM 3-AP (a, b) and 1 mM 2-AP (c). Simulations of each cu r r en t pattern by the kinetic model are shown (d-J) below the respective experimental pattern. T h e model parameters used in these simulations are identical to those used in Fig. 3 c o r r e s p o n d i n g to 2- and 3-AP. T i m e scales for a-c are the same as for d-f, respectively. T h e diminished frequency d e p e n d e n c e in 2-AP reflects the m o r e rapid interaction o f 2-AP molecules with the binding site (k = 0.3 ms -t, l = 0.1 ms -I , z = 0.8 s).
._xo.8~ .~0.6 -~0.4
~
~
_
s
Q2 I0 20 Pulse Interval
30
40
(s)
FU;URE 7. R e - e s t a b l i s h m e n t o f K c h a n n e l b l o c k by a m i n o p y r i d i n e s . T w o c o n s e c u tive pulses to + 100 mV separated by a variable time interval were applied to an axon
bathed in 1 mM 3-AP and 300 nM T T X . T h e ratio of the increase in IK d u r i n g the second pulse to the m a x i m u m observed increase is plotted as a function of pulse interval. Currents were measured at 0.5 ms of each pulse. Sample cu r r en t patterns for intervals o f 1, 10, and 60 s are shown in the insets. Block proceeds with two time constants of --3 s and - 2 7 s. Solid line is fit to the data points by the equation I/Imax = 0.55 exp (-t/3) + 0.45 exp (-t/27), where t = time in seconds.
526
THE .JOURNAL OF GENERAL
PHYSIOLOGY
" VOLUME
68 "
1976
illustrates the results o f this type o f e x p e r i m e n t . T h e n o r m a l i z e d d i f f e r e n c e in IK between the first a n d second pulses was m e a s u r e d at 0.5 ms a f t e r each step a n d plotted as a function o f the interval between pulses r e p r e s e n t i n g the time course o f re-establishment o f inhibition. In the case o f 3-AP the time course exhibits two c o m p o n e n t s , a fast time constant (3 s) a n d a slower one (27 s). I n contrast, the reestablishment o f block in 2-AP h a d a m u c h faster time course. S u p e r i m p o s a b l e K c u r r e n t traces could be p r o d u c e d in 2-AP w h e n the two pulses were s e p a r a t e d by an interval o f only 2 s. Consequently, the time constant for re-establishment o f block in 2-AP is estimated to be less t h a n 1 s. Re-establishment o f block is m o r e r a p i d as the concentrations o f n-AP are increased. For e x a m p l e , in the p r e s e n c e o f 30 m M n-AP the potassium c u r r e n t s associated with the twin pulses could be r e p r o d u c e d for pulse intervals as short as 2 s in the case o f 3- or 4-AP a n d only 1 s in 2-AP.
Time Course of Removal of n-AP Block T h e second series o f e x p e r i m e n t s was designed to e x a m i n e the time course o f recovery f r o m block d u r i n g multiple pulses. T h e pulse schedule again used twin depolarizing pulses to + 100 m V in this case with a fixed 1-s pulse interval but with a variable first pulse d u r a t i o n . In Fig. 8 the recovery o f IK is plotted as the c u r r e n t m e a s u r e d at 3 ms o f the second pulse vs. the d u r a t i o n o f the first or conditioning pulse. T h e recovery f r o m 3-AP block usually followed a single e x p o n e n t i a l time course, with a time constant o f a p p r o x i m a t e l y 10-20 ms. This can p e r h a p s be visualized as r e p r e s e n t i n g the time constant g o v e r n i n g the interaction o f 3- or 4-AP with a site within the o p e n K channel. While the o p e n i n g o f the K channel gating m e c h a n i s m is p r o b a b l y r e q u i r e d for the pulsed e p e n d e n t recovery o f Ig, the rate-limiting step a p p e a r s to be the dissociation o f the n-AP molecule f r o m the o p e n channel. T h i s is a p p a r e n t , as the time constant for recovery f r o m block is a p p r o x i m a t e l y 10 times longer than that for n o r m a l activation o f K channels. T o the right o f the recovery plot in Fig. 8 are typical records used to construct the plot ( u p p e r traces) and the results o f simulating the same protocol on the kinetic m o d e l (lower curves).
Steady-State Inhibition Is Less in High External K E x p e r i m e n t s in high external potassium have shown n-AP to suppress c u r r e n t s flowing in either direction t h r o u g h K channels (Yeh et al., 1976a). In 340 m M K seawater, the resting m e m b r a n e potential is a p p r o x i m a t e l y 0 m V a n d external application o f 1 m M 2-, 3-, or 4-AP p r o d u c e d no a p p r e c i a b l e change. T h e r e f o r e , the m e m b r a n e potential could be c l a m p e d at zero without polarizing the current-passing electrode. Step depolarizations to various potentials were applied a n d quasi-instantaneous c u r r e n t s were m e a s u r e d . T h e suppression o f K currents by 3-AP u n d e r these conditions is seen in Fig. 9. A linear I-V relation was f o u n d both in the control a n d n-AP axons (Yeh et al., 1976a). T h e decrease in slope of the I-V relation thus r e p r e s e n t s the steady-state inhibition o f K conductance at 0 m V by n-AP. T h e p e r c e n t a g e o f inhibition was not significantly d i f f e r e n t a m o n g these c o m p o u n d s , r a n g i n g f r o m 22% to 28%. I m p o r t a n t to note, however, is that the s u p p r e s s i o n is m u c h smaller t h a n o b s e r v e d at 0 m V (8 ms) for axons in n o r m a l K seawater (see T a b l e I). T h i s discrepancy m a y be
YEH, OXFORD, WU, AND NARAHASH] Dynamicsof Aminopyridine Block of K Channels
527
a s c r i b e d to d i f f e r e n c e s in t h e n u m b e r o f o p e n c h a n n e l s at t h e h o l d i n g p o t e n t i a l s u s e d in e a c h case, 0 m V in h i g h K a n d - 8 0 m V i n n o r m a l A S W . Also q u a n t i t a tive d i f f e r e n c e s i n t h e d e g r e e o f s t e a d y - s t a t e block a r e i n f l u e n c e d by the i n t e r a c t i o n s b e t w e e n K ions a n d n - A P m o l e c u l e s , i n a c t i v a t i o n ofgK, a n d K i o n a c c u m u -
RXON
=
.~OE "O.E
oo
Conditioning Pulse Duration (ms) FIGURE 8. Pulse-dependent recovery from aminopyridine block. Two consecutive pulses to + 100 mV with l-s interval were applied to an axon bathed in 1 mM 3AP and 300 nM T T X . T h e duration of the first pulse was varied and a 3-min rest period allowed between pulse pairs. K current was measured at 3 ms d u r i n g the second pulse. The ratio of IK to the m a x i m u m obtainable Is is plotted as a function of the first pulse duration. Solid line represents a single exponential fit to the data points with a time constant of 21 ms. Typical pattern used to extract such data is shown to the right with its simulation by the kinetic model. Conditioning pulse durations in the stimulation were 1, 2, 5, 10, 20, and 50 ms, consecutively. Model parameters are again identical to those used in Fig. 3 (k = 0.06 ms -1, l = 0.01 ms -a, Trl = 3 S, and Tr2 = 27 S).
Con~ol ~
~ . ~ _
3-AP )
' .....................
-
FIGURE 9. Nonrectifying block of IK by 3-AP. 3-AP (1 mM) was applied externally to an intact axon bathed in high-potassium seawater (340 mM K+). T h e axon was voltage clamped at the resting potential (0 mV) between depolarizing and hyperpolarizing voltage steps applied every 5 s. Note that both inward and outward IK were reduced, but by a smaller a m o u n t than in normal K seawater. l a t i o n . T h e s e factors will be d i s c u s s e d l a t e r . A t h i g h d e p o l a r i z a t i o n in e l e v a t e d e x t e r n a l [K+], c u r r e n t s u n d e r g o a t i m e - d e p e n d e n t r e c o v e r y r e a c h i n g c o n t r o l levels in 2- a n d 4 - A P a f t e r - 8 m s (Yeh et al., 1976a). S u c h a p h e n o m e n o n is n o t o b s e r v e d with 3 - A P (Fig. 9). T h i s m a y be r e l a t e d to t h e c h a r g e state o f t h e m o l e c u l e , as 3 - A P ( p K , = 6.03) exists a l m o s t exclusively i n its n e u t r a l f o r m at p H 8.
528
T H E J O U R N A L OF GENERAL P H Y S I O L O G Y • VOLUME 6 8 ' 1 9 7 6
DISCUSSION
T h e interaction o f aminopyridines with potassium channels o f the squid axon m e m b r a n e has been shown to exhibit a complicated pattern o f voltage, time, and frequency d e p e n d e n c e . T h e steady-state reduction o f IK by n-AP is partially relieved at high depolarizations. In addition, the r e m a i n i n g IK exhibits m u c h slower a p p a r e n t t u r n - o n kinetics. While such p h e n o m e n a seem to suggest an alteration o f the conformational f r e e d o m o f K channel gating c o m p o n e n t s , f u r t h e r consideration o f the data reveals inconsistency with such an interpretation. Alternatively, it is suggested that, at the level o f a single channel, the effects o f aminopyridines are not intimately related to the molecules responsible for the o p e n i n g and closing o f the K ion pathway. Potassium currents obtained at the end o f 8-ms pulses in n-AP were c o n v e r t e d to c h o r d conductance values (GK) as a function o f m e m b r a n e potential. Amplitude scaling o f the GK-voltage relation in combination with shifts along the voltage axis results in inadequate fits o f the e x p e r i m e n t a l values in n-AP to steady-state GK in control. This implies that neither a reduction in the m a x i m u m conductance o f a single K channel n o r a shift o f the "set point" o f the gating voltage sensor can account for the observed effects. In addition, if one assumes the H o d g k i n - H u x l e y n p a r a m e t e r adequately to r e p r e s e n t the operation o f potassium channels, it is seen that n u m e r o u s alterations o f both the first o r d e r rate constants a , and /3, and the power function e x p o n e n t (Cole and Moore, 1960) fail to match adequately the kinetic pattern o f IK in n-AP (for example, see Fig. 10). This would seem to rule out a major change in the first o r d e r transition o f n-type gating units by n-AP, if one assumes that these or similarly behaving c o m p o n e n t s are involved in normal K channel gating. One might, however, conceive o f such alterations occurring in only a fraction o f the total channel population and being disguised in a functional summation with normally o p e r a t i n g channels. This possibility was e x a m i n e d by c o m p u t e r simulation using various combinations o f normal channels s u m m e d with channels having altered n parameters to yield a total GK. T h e progressive increase in the fractional population o f K channels having been either slowed by a factor o f 10 or delayed by increasing the power function e x p o n e n t results in a clearly distinguishable kinetic c o m p o n e n t (see Fig. 10b) which has no c o u n t e r p a r t in actual experimental data (for example, Fig. 3). Restricting the simulated change in o~, a n d / 3 , to five times results in a m o r e monotonic curve, but fails to describe adequately the experimentally observed slowing o f Ix. T h e lack o f success with this a p p r o a c h thus p r o m p t s us to suggest an alternative mechanism o f n-AP action which is i n d e p e n d e n t o f the gating properties o f K channels irrespective o f their origin. GK Kinetic Model T h e kinetics o f normal K channel gating can be described by a linear reaction sequence as follows: 4o~
3a
2o~
u
