Published July 1, 1981
Effects of 4-Aminopyridine on Potassium
Currents in a Molluscan Neuron
A. H E R M A N N and A. L. F. G O R M A N
From the Department of Physiology, Boston University School of Medicine, Boston, Massachusetts
02118. A. Hermann's present address is Department of Biology, University of Konstanz, D-7750
Konstanz, Federal Republic of Germany.
The aminopyridines block voltage-dependent potassium currents of nerve
a x o n ( P e l h a t e a n d Pichon, 1974; M e v e s a n d P i c h o n , 1975 a n d 1977; S c h a u f
et al., 1976; U l b r i c h t a n d W a g n e r , 1976; Y e h et al., 1976 a a n d 1976 b), s o m a
( T h o m p s o n , 1977), a n d a x o n - t e r m i n a l (Llin~is, et al., 1976) m e m b r a n e s a n d
o f m u s c l e m e m b r a n e (Gillespie a n d H u t t e r , 1975; F i n k a n d W e t t w e r , 1978;
M o l g o , 1978). M o r e o v e r , t h e y b l o c k the v o l t a g e - d e p e n d e n t K § c u r r e n t in
l o w e r c o n c e n t r a t i o n a n d in a different m a n n e r t h a n the b l o c k p r o d u c e d b y
t e t r a e t h y l a m m o n i u m . In m o l l u s c a n n e u r o n s the K + c u r r e n t o f the s o m a
m e m b r a n e c a n be d i v i d e d into a c o m p o n e n t a c t i v a t e d b y c h a n g e s in m e m b r a n e v o l t a g e a n d a c o m p o n e n t a c t i v a t e d b y the e n t r a n c e o f C a 2+ ions into
the c e l l - - t h e C a 2 + - a c t i v a t e d K + c u r r e n t ( M e e c h a n d S t a n d e n , 1975; H e y e r
J. GEN. PHYSIOL.9 The Rockefeller University Press 9 0022-1295/81/07/0063/24 $1.00
Volume 78 July 1981 63-86
63
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ABSTRACT The effects of4-aminopyridine (4-AP) on the delayed K + current
and on the CaZ+-activated K + current of the Aplysia pacemaker neurons R-15
and L-6 were studied. The delayed outward K + current was measured in Ca 2+free artificial seawater (ASW) containing tetrodotoxin (TTX), using brief
depolarizing clamp pulses. External (and internal) 4-AP blocks the delayed K +
current in a dose-dependent manner but does not block the leakage current.
O u r results show that one 4-AP molecule combines with a single receptor site
and that the block is voltage dependent with an apparent dissociation constant
(K4-Ap) of ~0.8 m M at 0 mV. K4-Ap increases e-fold for a 32-mV change in
potential, which is consistent with the block occurring - 0 . 8 of the distance
through the m e m b r a n e electrical field. The 4-AP block appears to depend uPon
stimulus frequency as well as upon voltage. The greater speed of onset of the
block produced by internal 4-AP relative to when it is used externally suggests
that 4-AP acts from inside the cell. The Ca2+-activated K + current was measured
in Ca2+-free ASW containing T T X , using internal Ca2+-ion injection to directly
activate the K § conductance. Low external 4-AP concentrations (<2 mM) have
no effect on the Ca2+-activated K + current, but concentrations of 5 m M or
greater increase the K + current. Internal 4-AP has the same effect. T h e opposing
effects of 4-AP on the two components of the K + current can be seen in
measurements of the total outward K § current at different m e m b r a n e potentials
in normal ASW and during the repolarizing phase of the action potential.
Published July 1, 1981
64
tHE
JOURNAL
OF GENERAL
PHYSIOLOGY
* VOLUME
78 9 1981
a n d L u x , 1976 b). T h e v o l t a g e - d e p e n d e n t K + c u r r e n t o f the m o l l u s c a n n e u r o n
s o m a m e m b r a n e has b e e n f u r t h e r d i v i d e d o p e r a t i o n a l l y into a fast o u t w a r d
K + c u r r e n t ( C o n n o r a n d Stevens, 1971; N e h e r , 1971) a n d the d e l a y e d o u t w a r d
K + c u r r e n t (or the " d e l a y e d o u t w a r d rectifier"), w h i c h was first described in
s q u i d a x o n b y H o d g k i n a n d H u x l e y (1952 a). It has b e e n r e p o r t e d t h a t
e x t e r n a l 4 - a m i n o p y r i d i n e (4-AP) selectively blocks the fast K + c u r r e n t o f the
m o l l u s c a n n e u r o n s o m a ( T h o m p s o n , 1977). T h e e x p e r i m e n t s r e p o r t e d in this
p a p e r w e r e c a r r i e d out to c o m p a r e the effects o f e x t r a c e l l u l a r a n d i n t r a c e l l u l a r
4 - A P on different c o m p o n e n t s o f the K § c u r r e n t o f a m o l l u s c a n p a c e m a k e r
n e u r o n . T h e y show t h a t 4-AP also blocks the d e l a y e d o u t w a r d K + c u r r e n t b u t
t h a t , at high c o n c e n t r a t i o n s , it increases the C a 2 + - a c t i v a t e d K + current. T h e
results suggest t h a t the a m i n o p y r i d i n e s c a n b e used to p r o v i d e a p h a r m a c o logical s e p a r a t i o n o f the voltage- a n d C a 2 + - d e p e n d e n t c o m p o n e n t s o f the K +
c u r r e n t . A p r e l i m i n a r y a c c o u n t o f s o m e aspects o f this w o r k has a p p e a r e d
elsewhere ( H e r m a n n a n d G o r m a n , 1978).
All experiments were performed on the pacemaker neurons R-15 and L-6 (Frazier et
al., 1967) in the abdominal ganglion of Aplysia californica. The ganglion was removed,
the overlying sheath was dissected away to expose the cells, and the ganglion was
pinned to the base of a chamber containing artificial seawater (ASW), where it was
maintained at a constant temperature (16~
The ASW contained (mmol/l) 478
NaC1, 10 KCI, 10 CaCl2, 50 MgCl2, and 15 Tris HC1, at p H 7.8. In Ca2+-free ASW,
Ca 2§ was replaced by Mg 2§ on an equimolar basis, so that the total divalent ion
concentration remained constant. The Ca2+-free ASW contained ~4 • l0 -6 M Ca 2§
(Gorman and Hermann, 1979). Unless otherwise specified, all ASW solutions contained 5 • 10-s M tetrodotoxin (TTX), which was added directly to the solution just
before its use. 4-Aminopyridine (4-AP) (Aldrich Chemical Co., Inc., Milwaukee, Wis.)
was added to the external solutions from freshly made stock solutions. The exchange
time for the chamber was determined with ASW solutions containing different K +
concentrations and a K+-sensitive microelectode (see Gorman and H e r m a n n [1979])
located near cell R-15 and was 95% complete in 5 s.
An identified neuron was impaled with up to four microelectrodes for the recording
of m e m b r a n e potential and passing current and for the injection of Ca 2+ ions and 4AP. Many of the procedural details have been given previously (see Gorman and
H e r m a n n [1979] and H e r m a n n and Gorman, [1979 a]); in brief, the microelectrodes
used for recording a n d stimulating were filled with 3 M KC1 and had resistances of
~5 and 2 M ~ in ASW, respectively. M e m b r a n e potential was measured differentially
with respect to a low-resistance extracellular electrode that was independent of bath
ground. The circuit used for measurements was similar to one described previously
(Thomas, 1977). The rest of the recording, voltage-clamp, and current-measuring
circuitry was conventional. Signals were displayed on an oscilloscope and on a threechannel rectilinear pen-recorder for analysis.
Electrodes for intracellular injection were pulled with short shafts (<4 mm) and
tips with outside diameters of ~ 1.5/.tm. Ca2§
electrodes were filled with a
solution of 0.1 M CaC12 and had a resistance of ~3 Mfl when measured in ASW. 4AP-injection electrodes were filled with a solution of 0.1 M 4-AP, p H 7.0. At p H 7.0,
the 4-AP molecule is predominantly in the cationic form (pKa = 9.36 at 16~ Albert,
1963). 4-AP, therefore, can be ejected by making the microlectrode positive with
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METHODS
Published July 1, 1981
HZRMANN AND GORMAN
4-Aminopyridine Effects on K + Currents
65
Terminology
T h e o u t w a r d K + current of the molluscan neuron soma m e m b r a n e can be divided
into at least three components, which have been referred to by different names and
symbols by various groups. T w o of these K + currents are activated by changes in
voltage across the m e m b r a n e , whereas the third c o m p o n e n t is activated by changes
in the intracellular C a 2+ concentration. T h e voltage-dependent outward K + current
that activates quickly and undergoes appreciable inactivation during a voltage step
from hyperpolarized potentials is referred to as the fast outward I ~ current. It has also
been called the transient o u t w a r d K + current and is usually symbolized by IA. T h e
voltage-dependent outward K + current that activates with a delay and is slower to
inactivate during a voltage step is referred to as the delayed outward K + current. It has
also been called the voltage-dependent K + current, the delayed o u t w a r d rectifier, and
the voltage-activated late K + current and has been symbolized by IK and IK.v. T h e
third current depends on a change in the intracellular Ca 2 + concentration, which can
occur after Ca 2 + influx or after direct internal Ca 2+ ion injection, and referred to as
the Ca2+-activated K + current. It has also been called the Ca2+-dependent K + current,
the Ca2+-activated delayed o u t w a r d K + current, and the Ca"+-activated late K +
current and has been symbolized by IK,ca and Ic. Finally, the o u t w a r d current present
during positive potential steps under conditions where both the delayed o u t w a r d K +
and the Ca 2 + -activated K + currents are recorded simultaneously is referred to as the
total outward current but has also been referred to as the delayed current and as the
delayed K + current.
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respect to ground. Ca 2+ and 4-AP were electrophoretically injected into cells in the
voltage-clamp mode, so that there was no change in the net flow of current across the
m e m b r a n e during injection. T h e transport n u m b e r for the Ca 2+ electrodes has been
determined (Gorman a n d H e r m a n n , 1979) and on average is 0.29. T h e transport
n u m b e r for 4-AP electrodes is not known. Electrophoretic injections were m a d e with
an electrophoresis unit capable of maintaining a constant current of 10-6 A t h r o u g h
an electrode resistance of up to 10s ~ (model 160, W - P Instruments, Inc., H a m d e n ,
Conn.). T h e injection current was measured continuously and was constant for periods
of up to at least 90 s for Ca2+-containing electrodes and at least 250 s for 4-APcontaining electrodes. In some instances, 4-AP was pressure injected from similar
microelectrodes, with pressures between 15 and 25 lbs/sq in. There were no obvious
differences in the results obtained with the two methods for injecting 4-AP.
T h e resistance in series with the m e m b r a n e was determined by two methods.
Measurements of the series resistance (r~) were obtained under current-clamp conditions from A V / ~ I , where V is the amplitude of the initial j u m p in the voltage transient
in response to a brief current step, AI (see Moore and Cole [1963]), and under voltagec l a m p conditions from A V / L , where A V is the amplitude of a c o m m a n d potential
a n d /~ is the peak capacitive current (see N a t h a n and D e H a a n [1979]). T h e r e was
reasonable agreement between the two methods. Typically, the low-resistance extracellular recording electrode was positioned as close as possible to the cell to minimize
the series resistance, and u n d e r these conditions r~ was found to be 1-3 k~2. No
compensation was m a d e for this series resistance.
There is evidence that the interior of R-15 is isopotential under voltage-clamp
conditions (Adams and Gage, 1979). In most of the experiments, the axon of R-15
was undercut at a distance o f - 4 0 0 - 5 0 0 / ~ m from the soma in an attempt to minimize
the contribution of current from the axon, but it is most unlikely that the axonal
s t u m p was space clamped.
Published July 1, 1981
66
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
9 VOLUME
78
9
1981
RESULTS
Inhibition of the Delayed Outward K + Current by 4-AP
Fig. 1 illustrates the effect o f 5 m M e x t e r n a l 4 - A P on the K + o u t w a r d c u r r e n t s
m e a s u r e d w i t h d e p o l a r i z i n g c l a m p pulses o f short a n d long d u r a t i o n s a n d o f
various a m p l i t u d e s in Ca2+-free A S W c o n t a i n i n g 5 X 10 -5 M T T X . Ca2+-free
A S W was used to abolish the C a 2+ c u r r e n t a n d the C a 2 + - a c t i v a t e d K + c u r r e n t
c o m p o n e n t o f the total o u t w a r d c u r r e n t ( M e e c h a n d S t a n d e n , 1975; H e y e r
A
0 Co,4-AP
o~
'3
B
i (~)
2i F
+90
--
15
--
I
I
C
I
o
i
§
+55
+75
+55
I
i
i
l
15 30 45 60 75
I
0
i
I
*
|
15 30 45 60 75
I(pA)
§
+40
I
~
*2fi
+25
+5
I
I
I
I
I
o a2 0.4 0.60.B
t (s)
I
I
, r
I
I
I
I
0 (12 0 . 4 0 ~
t (s)
|
I
OB
I
FIGURE |. Effects of external 4-AP on R-15 membrane outward K + currents.
The left side shows outward currents measured in Ca2+-free ASW containing 5
• 10-5 M T T X , and the right side shows currents measured after 25 min in 5
m M 4-AP. The holding potential was - 4 5 inV. T h e membrane potential during
the pulse is indicated at the right of each current trace. In A and B the pulse
duration was 75 ms, and in C 1 s.
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o
Published July 1, 1981
HERMANN AND (~ORMAN 4-Aminopyridine Effects on K + Currents
67
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and Lux, 1976 b). T T X was used to eliminate the fast early inward current
that occurs in Ca2+-free A S W containing Na § (Geduldig and Gruener, 1970).
Finally, the m e m b r a n e was held at - 4 5 m V to inactivate the fast outward
current (Connor and Stevens, 1971; Neher, 1971). U n d e r these conditions the
residual o u t w a r d current is composed primarily of the delayed outward K +
current and the leakage current. In the absence of 4-AP, the outward current
rose to a peak with a delay and showed a slow decay during maintianed
depolarization. T h e delay can be attributed to the time necessary for the K §
channel to activate, and the slow decay to the inactivation of the same
channel, to K + ion accumulation in the extracellular space, or to both
processes. External 4-AP has several effects on outward K + currents. Fig. 1
shows that the m a x i m u m outward current, at any potential, was smaller than
the comparable current in the absence of 4-AP but that the reduction of the
outward current was less at very positive potentials and at long times. In
addition, the rise of the outward current was slowed by 4-AP and its decay
was less pronounced. The effects of 4-AP, particularly at high concentrations
(~ 1 m M or greater), were only partially reversible.
Fig 2 A shows a plot of the peak K + outward current in CaZ+-free A S W
after the addition of 5 m M 4-AP. The K + outward current was fully suppressed
by 4-AP at m e m b r a n e depolarizations up to about +20 mV, but the block
was less effective at more positive potentials. The K + outward current,
however, includes both the delayed outward K + current and leakage current
component, which at positive m e m b r a n e potentials is carried primarily by K +
ions. T h e leakage current was estimated from the initial instantaneous j u m p
in current that occurs after the capacitative discharge of the membrane. It
was approximately linear between - 1 0 0 and about +50 mV, but increased
nonlinearly at more positive m e m b r a n e potentials. The leakage current was
unaffected (Fig. 2 B) or only slightly changed by 4-AP. In six cells, the leakage
current was unaffected (two cells), slightly increased (two cells), and slightly
decreased (two cells) at positive membrane potentials. Subtraction of the
leakage current from the total outward current in Ca2+-free A S W leaves the
delayed outward K + component, which was inhibited strongly by 5raM 4-AP
at all potentials (Fig. 2 C).
The fast outward K + current or A current (Connor and Stevens, 1971;
Neher, 1971) is seen in cell R-15 when depolarizing steps to positive potentials
are preceded by conditioning hyperpolarizations (Faber and Klee, 1972; Gola,
1976). In normal ASW, the fast outward K + current of cell R-15 is fully
inactivated at a holding potential of about - 5 0 mV, whereas the delayed
outward K + current is fully available for activation at this potential (Gola,
1976). Holding potentials between - 5 0 and - 4 5 mV, therefore, should
provide a convenient working range for studying the delayed K + current in
the absence of the fast K + current, without the inactivation that occurs at
more positive holding potentials. It is important, however, to investigate
whether the fast outward current is also fully inactivated at holding potentials
o f - 5 0 to - 4 5 m V in Ca2+-free ASW. This was investigated in two experiments
(performed in collaboration with Dr. D. Tillotson) in Na +- and Ca2+-free
Published July 1, 1981
68
THE
JOURNAL
OF
GENERAL
78 9 1981
PHYSIOLOGY 9 VOLUME
ASW. T h e fast o u t w a r d K + c u r r e n t was a c t i v a t e d b y d e p o l a r i z i n g pulses to
- 3 0 m V from c o n d i t i o n i n g steps (300-ms) b e t w e e n - 1 4 0 a n d - 3 0 mV. A
c o n d i t i o n i n g h y p e r p o l a r i z a t i o n to - 1 0 0 m V for 300 ms was long e n o u g h to
r e m o v e ~ 9 5 % o f the i n a c t i v a t i o n o f the fast K § c u r r e n t present at a h o l d i n g
p o t e n t i a l o f - 4 5 inV. T h e n o r m a l i z e d peak fast o u t w a r d K + c u r r e n t (the ratio
o f the p e a k K § c u r r e n t at a n y c o n d i t i o n i n g h y p e r p o l a r i z a t i o n to the m a x i m u m
p e a k K § current) was p l o t t e d vs. the c o n d i t i o n i n g h y p e r p o l a r i z i n g potential.
T h e results were similar to d a t a o b t a i n e d by C o n n o r a n d Stevens (1971) in
Anisodoris n e u r o n s in N a +- a n d Ca2+-free ASW. At - 4 5 m V , < 1 % a n d at - 5 0
A
B i0
6
/
2O
I(NA)
I
I
-20
I
I
I
I
+70
+I00
+130
I
/..../
Ir I
I
I
I
C ,or
i0 84
I
+I0
I
+40
I
§
V (mY)
I
I
+I00
+I~K)
o
I
I
-20
+I0
I
+40
V (mV}
FIGURE 2. Effect of external 4-AP on R-15 membrane outward current in
Ca2+-free ASW containing 5 • 10-5 M T T X . (A) Current-voltage relation for
outward current measured at the end of 75-ms voltage-clamp pulses from - 4 8
mV to the indicated potentials before (closed circles) and after 5 m M 4-AP
(closed triangles). (B) Current-voltage relation for the leakage current. (C)
Current-voltage relation for the delayed outward K + current for the same
neuron. Measurements were made 20 rain after the addition of 4-AP.
m V < 2 % o f the fast o u t w a r d K + c u r r e n t was available for activation. W i t h a
similar c o n d i t i o n i n g protocol the cell was also tested with d e p o l a r i z i n g pulses
to 0, +20, a n d + 7 0 m V to activate the d e l a y e d o u t w a r d K + current. C h a n g e s
in the rising phase o f the c u r r e n t wave form were m e a s u r a b l e only for
c o n d i t i o n i n g h y p e r p o l a r i z a t i o n s m o r e negative t h a n - 5 5 m V . T h i s is as
e x p e c t e d if the fast o u t w a r d K + c u r r e n t is almost fully i n a c t i v a t e d at h o l d i n g
potentials o f - 4 5 to - 5 0 m V a n d indicates that the effects shown in Figs. 1
a n d 2 represent a direct action o f 4-AP on the d e l a y e d o u t w a r d K + current.
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//
15
Published July 1, 1981
HERMANN AND GORMAN
4-ArainopyridineEffects on K + Currents
69
T h e effects o f c o n d i t i o n i n g h y p e r p o l a r i z a t i o n a n d d e p o l a r i z i n g steps bet w e e n - 1 2 0 a n d +30 m V on the d e l a y e d o u t w a r d K + c u r r e n t w e r e also s t u d i e d
in Ca2+-free A S W . A b o u t 18-22% o f the d e l a y e d o u t w a r d K § c u r r e n t was
i n a c t i v a t e d at h o l d i n g p o t e n t i a l s o f - 5 0
a n d - 4 5 m V , respectively. T h e
s t e a d y - s t a t e i n a c t i v a t i o n c u r v e (see H o d g k i n a n d H u x l e y [1952 b]) was not
a l t e r e d a p p r e c i a b l y b y the a d d i t i o n o f 5 m M 4 - A P to the Ca2+-free A S W .
Voltage Dependence of 4-AP Inhibition
~1KI4.~r I [ eztemol 4-AP
IK
i
" " " "
05I
0
"
* "
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I
i
I
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LK
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0
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50
tso
v (mv~
FIOURE 3. Effects of 4-AP on the K § current ratio of cell R-15. (A) Effects of
m e m b r a n e potential on the ratio of the delayed K + current in the presence of 2
m M external 4-AP to control K + current, IK.4-AP/IK (B) Effects of m e m b r a n e
potential on the ratio of the delayed K + current in the presence of internal 4-AP
to the control K + current from a different cell. The 4-AP was injected intracellularly for 1 rain at 250 nA. The dashed curves fitted to the experimental points
in B were calculated from the empirical relation IK,4-AP/IK = 1 -- exp (--V/b),
where b is a constant. For the cell shown in A, b = 40 mV, and for the cell shown
in B, b = 45 mV.
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T h e 4 - A P i n h i b i t o r y effect o n the d e l a y e d o u t w a r d K § c u r r e n t d e p e n d s u p o n
v o l t a g e as well as u p o n c o n c e n t r a t i o n . T h e p o t e n t i a l d e p e n d e n c e o f the 4 - A P
i n h i b i t i o n c a n be seen in plots o f the r a t i o o f the d e l a y e d K + c u r r e n t in the
p r e s e n c e o f 4 - A P at a g i v e n c o n c e n t r a t i o n to the K + c u r r e n t u n d e r control
conditions. T h e r e was a progressive r e c o v e r y o f the d e l a y e d K + c u r r e n t d u r i n g
d e p o l a r i z i n g steps to positive m e m b r a n e p o t e n t i a l s at e a c h c o n c e n t r a t i o n (Fig.
3 A). T h i s r e c o v e r y d u r i n g d e p o l a r i z a t i o n also o c c u r r e d w h e n 4 - A P was
injected i n t r a c e l l u l a r l y (Fig. 3 B). T h e p o t e n t i a l d e p e n d e n c e c a n also b e seen
in dose-response plots o f the c u r r e n t ratio (IK,4-AP/IK) at different m e m b r a n e
Published July 1, 1981
70
THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME
78 9 1981
p o t e n t i a l s vs. the l o g a r i t h m o f the e x t e r n a l 4 - A P c o n c e n t r a t i o n (Fig. 4 A). I f
o n e 4 - A P m o l e c u l e b i n d s w i t h a single r e c e p t o r site, t h e n the l o g a r i t h m o f the
e x p e r i m e n t a l values for the c u r r e n t ratio (expressed as IR -- IR,4-AP/IR,4.Ap) at
a n y p o t e n t i a l s h o u l d fall a l o n g a s t r a i g h t line w i t h a slope o f 1 w h e n p l o t t e d
A
i
9 V:','20 mV
9 V:+~5 mV
'~ '~'~'II ~ ' 0
~,',.", ,~,,
"~ 9 \~',
\, xO
Xl;.
\ \ \A~X.\
0.5
O\
9 V:-.-5OmV
o V:+7OmV
%\
xx x
\
~,
0
!
I
(101
B
I
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[4-AP] (mM)
NAN
Downloaded from on June 18, 2017
x \ \\ iX\
A% \
\
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-,-,-.. -...
.
1.0
I
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/
K4-~o
/o "//~
/;,,.
/
o/
,o
//
9
///
.o~ M / "
///
/ / P9
/ //
/ / ,/
i
0.1
Ol
//
#,I
UZ[
/
.//z
/ e
5D
//
,~ / /=
9/
/
i i illll~
,0
i
I0
l i illlll
ID
K~
(rr )
05 F
,
0 + 20
,
40
,
80
,
80
I00
VCmV)
FIGURE 4. Effects of 4-AP concentration and membrane potential on R-15
delayed K + current. (A) Dose-response plot of the current ratio IK,4-AP/IK a t
four different test pulse potentials vs. the logarithm of the external 4-AP
concentration. The theoretical lines drawn through the experimental points
were calculated from Eq. 1. (B) Double logarithmic plot of the current ratio (IK
- IK,4-AP)/IK,4-AT" for the data in A vs. the external 4-AP concentration. The
parallel straight lines through the experimental points have a slope o f - 1. (C)
Semilogarithmic plot of the apparent dissociation constant (K4-Ap) vs. m e m b r a n e
potential. The data shown in A - C are from the same cell and were obtained in
Ca2+-free ASW containing 5 X 10-~ M T T X .
Published July 1, 1981
HERMANN AND GORMAN 4-Arninopyridine Effects on K + Currents
71
vs. the logarithm of the drug concentration. The experimental data in Fig. 4
A are replotted on a double logarithmic scale in Fig. 4 B and show that this
expectation is reasonably well met over a wide range of m e m b r a n e potentials.
T h e reaction of 4-AP with a receptor site, R, can be represented by
4-AP + R ~ 4-AP. R,
k- 1
where kx and k-1 are the forward and reverse rate constants and K4-AV = k l /
k-1 is the dissociation constant of the reaction. The experimental points in
Fig. 4 A were fitted with theoretical curves with the equation
IK,4-AP(V)
IK(V)
K4-AP(V),
~ K4-AP(V) + [4-AP]o'
(1)
K4-AP~v) ---- K4-AP~0)exp (zI~FV/RT),
(2)
where K~-AP~0)is the dissociation constant at zero membrane potential, z is the
charge of the 4-AP molecule, which is positive at the p H of the A S W (z --+1),/~ is a value between 0 and 1 and is the fraction of the potential difference
V across the m e m b r a n e experienced at the blocking site, and R T / F -~ 25 mV.
For the cell shown in Fig. 4 C, K4.Ap increased e-fold for a 32-mV change in
potential. For the straight line drawn through the points in Fig. 4 C, K4-AP~0)
---- 800 ~M and ~ - 0.78. Similar values were determined in an experiment on
a different cell (K4-AP~0) ----850/~M and/~ ---0.80).
During a clamping pulse, a current I will flow across the m e m b r a n e
resistance rm and across the resistance in series with the m e m b r a n e r~ (Hodgkin
et al., 1952; Taylor et al., 1960); therefore, the voltage clamp pulse Vc will
represent not only the voltage drop across the membrane (Vm -- Irm) but also
the voltage drop across the series resistance (V, -~ Ir,) or Vc -- Vm + Vs. The
error introduced into the current-voltage relation by rs can be significant when
large K § currents (~0.5/~A) flow across the membrane. The voltage dependence of the apparent dissociation constant K4-AP was altered but not abolished
when the current ratio (IK,4-AP/IK) was corrected for the estimated series
resistance error, using the relation Vm -- Vc - V~. For example, with the data
shown in Fig. 4 and an average value of r, of 1.5 k~, K4-AP increased e-fold for
a 29-mV change in potential.
Downloaded from on June 18, 2017
where IK~v) is the peak delayed K + current in Ca2+-free A S W at a given
potential V, IK,4-AP~V) is the peak K + current at the same potential in Ca2+-free
A S W containing 4-AP, [4-AP]o is the external 4-AP concentration, and K4.
APt V) is the apparent disassociation constant at V. The experimental points in
Fig. 4 A can be fitted with a series of parallel curves, each with a different
dissociation constant. The dissociation constant increases at positive membrane potentials and is plotted in Fig. 4 C as log K4-AP vs. membrane potential.
T h e points fall along a straight line over a wide range of membrane potentials.
The voltage dependence of the apparent dissociation constant can be described
by
Published July 1, 1981
72
T H E J O U R N A L OF GENERAL PHYSIOLOGY 9 V O L U M E 7 8 ~ 1 9 8 1
Effects of 4-AP on the Kinectics of the Delayed K + Current
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The delayed outward K + current in Ca2+-free ASW containing T T X rises to
a peak - 3 0 - 5 0 ms after the onset of a depolarizing clamp step and thereafter
declines slowly during maintained depolarization (see Fig. 1). The addition of
5 m M 4-AP to Ca2§
ASW slowed the rising phase and the decay of the
current wave form at all potentials. These results suggest that 4-AP may affect
both the activation and inactivation of the K + channel, but there is an
alternative explanation. In squid axon (Yeh et al, 1976 a and 1976 b; Meves
and Pichon, 1977) and in frog myelinated axon (Ulbricht and Wagner, 1976),
the removal of the 4-AP block is frequency as well as voltage dependent. The
frequency dependence has been satisfactorily explained by the hypothesis that
4-AP molecules are displaced from blocking sites during repetitive depolarizing
stimuli with a single time constant and rebound afterward, with a longer time
constant. In frog myelinated axon, full removal of the 4-AP block takes ~ 1 s
(Ulbright2+ and Wagner,. 1976). It is possible that the difference in kinetics"
" m"
Ca -free ASW, with and without 4-AP, reflects the time-course of the removal
of the 4-AP block during depolarization. This could account for the decrease
in the rising phase of the K + current and its slower decay in 4-AP.
M u c h of our data was obtained at relatively high concentrations of external
4-AP ( - 5 mM), because the primary purpose of our experiments was to
compare its effect on the delayed K + current to its effect on the Ca2+-activated
K + current (see below). High concentrations, however, tend to suppress the
frequency-dependent relief of the 4-AP block (Meves and Pichon, 1977). Fig.
5 shows the effect of a train of repetitive (1-Hz) depolarizing pulses to +20
m V from a holding potential o f - 4 5 m V on the peak delayed K + current in
Ca~+-free ASW and in the presence of 5 m M 4-AP. In cell R-15, the delayed
K + current undergoes a time- and frequency-dependent inactivation during
a repetitive train of pulses, and m" Ca2+-free ASW the peak outward current
was reduced by 45% at the end of 20 s of repetitive stimulation. This is a
characteristic feature of outward K + currents in m a n y molluscan neurons
(Gola, 1974; Lux and Eckert, 1974; Heyer and Lux, 1976 b; Aldrich et al.,
1979). The addition o f 4 - A P reduced the peak outward K + current measured
with the first depolarizing clamp pulse in a repetitive train of pulses to +20
m V by ~70% and also eliminated much of the subsequent frequency-dependent inactivation. This result is as expected if the frequency-dependent
inactivation is matched by a frequency-dependent relief of the 4-AP block
repetitive stimulation, but it is difficult to compare directly the outward
currents measured with identical voltage-clamp pulses under the two conditions because of the marked difference in their amplitudes. A different type of
comparison can be m a d e by increasing the size of the depolarizing pulse in 4AP to +50 mV, so that the outward K + current measured at the beginning of
the train is similar in magnitude to the current measured in Ca2+-free ASW.
Fig. 5 shows that the peak outward K § current in 4-AP was reduced by only
15% (to +50 mV) at the end of a 20-s train of repetitive depolarizing clamp
pulses. The results are consistent with a frequency-dependent relief of the 4-
Published July 1, 1981
HERM^r~N AND GORM^N
4-AminopyridineEffects on K* Currents
73
AP block but cannot be used to exclude the possibility that 4-AP inhibits
directly the frequency-dependent inactivation of the delayed K + channel.
Effect of 4-AP on the Ca2+-activated K + Current
Injection of Ca ~+ ions into molluscan neurons activates an outward current
carried by K + ions (Meech, 1974) whose amplitude depends on the magnitude
of charge passed through the electrode and the holding potential (Gorman
and H e r m a n n , 1979). Ca 2+ was injected electrophoretically (200 nA for 10 s)
into the cytoplasm at various membrane potentials between - 8 0 and - 3 0
A?_o
.o
9
B
i(pA)
~ Im I I I
1.5
II ~ I I I I
iii
i
iii
0i iCo,4-AP
ii
i ,~ i
9
§
B
~149149149149
§
0C~a
I0
oc4-AP
05
&&A&&A&AA&6,&
AA6
&6&&
C
20rm
&,
§
C) t
0
I
1
1
]
5
~3
~5
Z0
t(s)
~
.5~]
J
FIGURE 5. Effects of external 4-AP on R-15 delayed outward K + current
response to repetitive stimulation. (A) Plot of K + currents in Ca2+-free ASW
containing 5 X 10-5 M TTX before and after 20 rain in 5 mM 4-AP measured
with repetitive (1-Hz) 75-ms pulses to the indicated membrane potentials. (B)
Superimposed oscilloscope trace of K + currents measured with repetitive (1-Hz)
75-ms pulses to +20 mV before (top) and in 5 mM external 4-AP (bottom). (C)
Superimposed K + currents measured with repetitive (1-Hz) 75-ms pulses to +50
mV in 4-AP.
mV. T h e difference between the peak K + current and the holding current at
each potential in Ca2+-free ASW2is plotted in Fig. 6 A. At low external 4-AP
concentrations (<2 mM) the Ca -activated K current was unaffected, b u t
at higher concentrations ( - 5 raM) it was increased. T h e increase in the C a 2+activated K + current was proportionately greater when the current was
o u t w a r d but was also apparent when the current was inward. T h e reversal
potential (about - 7 2 mV), however, was unchanged by the addition of 4-AP.
The increase might be explained if 4-AP reduced the cell's ability to buffer
Ca 2+ intracellularly. In two experiments (carried out in collaboration with
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"~176
Published July 1, 1981
74
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY 9 VOLUME
78 9 1981
Dr. D. Tillotson), C a 2+ ions w e r e injected into cells c o n t a i n i n g the C a 2+sensitive d y e a r s e n a z o I I I , a n d the differential c h a n g e in d y e a b s o r b a n c e was
used to m o n i t o r the increase in free i n t r a c e l l u l a r C a 2+ d u r i n g injection. T h e
c h a n g e in i n t e r n a l C a 2+ was not g r e a t e r in the p r e s e n c e o f 5 m M e x t e r n a l 4AP.
A ,oo - .oc
B
V =+:35mY
I(nA)
OCo
C
F
OCO,4-AP
41-
0
-5
0 Co,4"AP
* Co- Inj.
' too,,.
-80
-40
V (mV)
']I+
-20
FIGURE 6. Effects of external 4-AP on R-15 Ca2+-activated K § currents (A)
Plot of K + current vs. m e m b r a n e holding potential before and in 5 m M external
4-AP. The current was measured as the difference between the holding current
at each potential and the peak current activated by an intracellular injection of
Ca 2+ for 10 s at an intensity of 200 nA. Current responses recorded at - 3 0 m V
in Ca2+-free ASW (bottom) and in Ca2+-free ASW with 5 m M 4-AP (top) are
also shown. The injection period is indicated below each record. (B) Outward
currents measured from a different cell before and at the end of a 10-s, 400-hA
intracellular Ca 2+ injection. (C) Outward currents measured from the same cell
in 5 m M 4-AP before and at the end of a 10-s, 400-hA Ca 2+ injection. The pulse
amplitude was +35 m V and the holding potential - 5 0 m V in B and C. All
currents were measured in Ca2+-free ASW containing 5 • 10-5 M T T X .
A different t y p e o f e x p e r i m e n t was used to d e t e r m i n e the effect o f 4 - A P on
the C a 2 + - a c t i v a t e d K + c u r r e n t at p o t e n t i a l s m o r e positive t h a n - 3 0 m V . It
has b e e n s h o w n ( G o r m a n a n d T h o m a s , 1980) t h a t the o u t w a r d c u r r e n t s
m e a s u r e d w i t h d e p o l a r i z i n g c l a m p pulses in Ca2+-free A S W are increased
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50
OCo
+ Co - inl.
Published July 1, 1981
[-IERMANN AND GORMAN 4-AminopyridineEffects on K* Currents
75
during internal Ca 2+ injection. Fig. 6 B a n d C shows the results from an
exp2eriment in which the m e m b r a n e was briefly depolarized to +35 m V in
Ca -free A S W at the end o f a 10-s intracellular Ca z+ injection. T h e o u t w a r d
current was increased by ~ 1.4/.tA during Ca 9-+ injection (Fig. 6 B). T h e same
protocol was repeated in 5 m M external 4-AP, which altered the time-course
a n d reduced the o u t w a r d current but increased the o u t w a r d current to ~ 1.6
#A d u r i n g C a 2+ injection (Fig. 6 C).
Time-Course of the 4-AP Block
ml
IK(4-AP)IK /
--
V:+~:~]mV
I
20n'~
0.5
9
0 I
0
I
5
I
I0
I
15
f (min)
l
20
I
25
. . . .
J
50
FIGURE
7.
Time-course of onset of external 4-AP inhibitory effect on delayed
K + current. Plot of K + current ratio measured from two cells with +20-mV
pulses from a holding potential o f - 4 5 mV before and at the indicated times
after the addition of 5 mM 4-AP to Ca2+-free ASW containing 5 X 10-5 M
TTX. The inset shows representative oscilloscope current traces from one of these
cells recorded at different times in 4-AP. Data from cells R-15 and L-6.
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T h e aminopyridines are small, lipid-soluble molecules (Christen et al., 1975)
a n d thus might be expected to diffuse rapidly to and into the neuronal
m e m b r a n e when a d d e d to the b a t h i n g media. T h e onset o f block by 5 m M
external 4-AP was studied with 75-ms test pulses to -t-20 m V applied once per
minute. T h e time-course of the inhibitory action o f the drug on the delayed
o u t w a r d K + current in Ca~+-free A S W measured at the end o f the test pulse
was slow (Fig. 7). A steady-state blockade o f the current occurred (~77% o f
the current without 4-AP) after ~ 1 5 - 2 0 min. In five cells, the average time
necessary to p r o d u c e a 50% block was 1.25 + 0.17 min (SEM). Reversibility,
particularly after prolonged treatment, was poor. T h e time needed to achieve
Published July 1, 1981
76
THE. J O U R N A L
OF
GENERAL
PHYSIOLOGY
78 - 1981
9 VOLUME
a full block is only slightly longer than thc time (~13 rain) required for
external 4-AP to have the samc effect in myclinated axon (Ulbricht and
Wagner, 1976).
Effects of lnternal 4-AP on the K + Current
T h e r e is evidence from muscle that intracellular 4-AP blocks the K + current
(Fink and Wettwer, 1978) and that the effects o f extraeellular 4-AP occur
after passage through the m e m b r a n e (Gillespie a n d H u t t e r , 1975). This might
explain the slow time-course o f the effects o f external 4-AP. T h e intracellular
effect o f 4-AP on the delayed o u t w a r d K + current and on the Ca2+-activated
K § current was investigated. Fig. 8 A shows a plot o f the delayed K + current
A
I (IJA}
B 60
7
9 0 Ca
6
9
* 4-AP injected
/ e/"
I (nA)
40
./
20
/"
~~i_~___~___i
+
.......
0
I
-20
+10
I
+40
I
I
I
+ ' t O +100 +130
V (mV)
IO
I
I
I
1
-80
-60
-40
-20
V (mY)
FIGURE 8. Effects of internal 4-AP on R-15 delayed and Ca2+-activated K §
currents. (A). Plot of delayed K § current measured at 50 ms before and after
intracellular injection of 4-AP vs. membrane potential. The experimental points
in 4-AP were measured immediately after a drug injection of 3 min at 250 nA.
The leakage current was subtracted from all records (B) plot of Ca2+-activated
K § current vs. membrane holding potential before and after a 3-min, 300-hA
intracellular injection of 4-AP. The current was measured as described in Fig.
6A. All currents were measured in Ca2+-free ASW containing 5 • 10-5 M TTX.
measured with 200-ms pulses o f variable a m p l i t u d e in Ca2+-free A S W a n d
after electrophoretic injection o f 4-AP (250 nA for 3 min). A similar reduction
o f the K + current occurred when 4-AP was pressure injected internally. T h e
K + current was decreased at all potentials by internal 4-AP. In most respects
the effects of internal and external 4-AP were similar. T h e m a g n i t u d e o f the
block of the current d e p e n d e d on the length o f time 4-AP was injected and,
thus, presumably on the intracellullar 4-AP concentration. T h e voltage dependence o f the block by internal 4-AP can be seen in the progressive recovery
o f the delayed K + current during depolarizing steps to positive m e m b r a n e
potentials a n d is shown in Fig. 3 B, where the ratio of the current in the
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/~
OCa
9 4-APinjected /
Published July 1, 1981
HERMANN AND GORMAN
4-AminopyridineEffects on K + Currents
77
presence of internal 4-AP to the K + current under control conditions is plotted
vs. m e m b r a n e potential. T h e one major difference between external and
internal application of the drug is that a steady-state block was established
immediately after the electrophoretic or pressure injection of 4-AP. This
finding suggests that the receptor site for 4-AP may be more accessible from
the inside surface of the membrane. Fig. 8 B shows a plot of the K + current
activated by intracellular Ca 2+ injection (200 nA for 10 s) at different holding
potentials in Ca2+-free A S W and after injection of 4-AP in the same cell. The
CaZ+-activated K + current was increased by internal 4-AP without any effect
on the K + current reversal potential. T h e results are similar to those shown
above (Fig. 6 A), where a high external concentration of 4-AP was used. The
effects of intracellular injection of 4-AP were not reversible, at least not up to
2 h after injection (the longest period studied).
Effects of 4-AP on the Current- Voltage Relation in Normal A S W
Effects of 4-AP on the Action Potential and on Pacemaker Activity
In cockroach (Pelhate et al., 1972) and squid axon (Llin~s et al. 1976; Yeh et
al., 1976 a) the aminopyridines cause a small depolarization of membrane
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The m e m b r a n e current measured at the end of a (50-ms or greater) depolarizing clamp step to potentials more positive than about - 2 0 m V in normal
A S W is carried predominantly by K + ions (Heyer and Lux, 1976a). The
current-voltage relation is N-shaped at positive m e m b r a n e potentials (Fig. 9).
T h e reduction of the outward current between about +80 and + 120 m V has
been related to the reduction of the Ca2+-activated component of the total
o u t w a r d K + current as the m e m b r a n e potential approaches the Ca ~+ equilibrium potential (Meeeh and Standen, 1975; Heyer and Lux, 1976 a and 1976
b). T h e net inward flux of Ca 2+ ions and, therefore, the Ca2+-activated K +
current is reduced in this region, whereas the delayed K + and the leakage
currents continue to increase (see Fig. 2). The effects of 4-AP on the currentvoltage relation in normal A S W are consistent with its action on the individual
components of the K + current. Fig. 9 shows that the outward current in
external 4-AP (5 mM) was increased between about - 2 0 and +80 mV, where
the Ca2+-activated K + current predominates. T h e outward current, however,
was reduced at m e m b r a n e potentials positive to about +80 mV, where the
delayed K + and leakage currents predominate. The effect of external 4-AP is
quite different from the effect of external tetraethylammonium, which blocks
both the Ca2+-activated and the delayed K + currents and, therefore, reduces
the outward current at all potentials (Klee, 1978; H e r m a n n and Gorman,
1979 b and 1981). The current measured by depolarizing voltage-clamp pulses
to +20 m V contains a small, early inward current component that is increased
by the addition of 4-AP (see inset records in Fig. 9). It is possible that 4-AP
affects the inward current directly, but an equally likely explanation is that,
by slowing the onset and reducing the amplitude of the outward K + current
(see Fig. 1), 4-AP causes an increase in an underlying inward current (Hermann and Gorman, 1979 b).
Published July 1, 1981
78
THE
JOURNAL
OF
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PHYSIOLOGY 9 VOLUME
78
9
1981
potential and spontaneous discharge but prolong only slightly the duration of
the action potential (Schauf et al., 1976; Yet et al., 1976 b). Their effect on
spontaneous pacemaker discharge in cell R-15 was somewhat more complex
and depended on the external drug concentration (Fig. 10 A). At low external
concentrations (>1 mM), 4-AP reduced the n u m b e r of action potentials per
burst. Higher concentrations caused an irregular bursting pattern or a t r a n -
2O
9
9 ASW
,, ASW,4-AP
I(pA)
/
Jf
/
15
/
//
t.'-"o,
"r-0
l(/
I
-20
+ 20
I
0
- ASW . . . .
+ 120
I
*50
'
I
+100
V (rnV)
I
+IF:K)
FIGURE 9. Effects of external 4-AP on the R-15 outward current in normal
ASW. Plot of the outward current measured at the end of 200-ms pulses before
and in 5 mM external 4-AP vs. membrane potential. The holding potential was
-50 mV. The open symbols show the current corrected for leakage currents,
and the closed symbols show the uncorrected currents. The inset shows representative responses to 200-ms clamp pulses to the indicated membrane potential
before and in 4-AP.
sition to continuous repetitive discharge. In concentrations of 10 m M or
greater, spontaneous discharge of action potentials stopped, and the membrane potential was stable at about - 5 5 inV.
The duration of the R-15 action potential was prolonged by external 4-AP
(-2.2-fold) without a significant change in its overshoot, but the afterhyperpolarization was increased (Fig. 10 B). These changes are consistent with the
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I0
Published July 1, 1981
4-Aminopyridine Effects on K + Currents
HERMANN AND GORMAN
79
finding that the delayed K + current is reduced, whereas the Ca2+-activated
K + current is increased by 4-AP. For comparison, external 4-AP was tested on
a n e u r o n in the same ganglion (cell L-11), in which the contribution o f the
Ca2+-activated K + current to the total o u t w a r d K + current is m u c h smaller
t h a n in cell R-15 ( H e r m a n n , 1978). External 4-AP at the same concentration
also prolonged the duration of the L-11 action potential but reduced the
afterhyperpolarization (Fig. 10 B).
A
ASW
ImM 4-AP
2raM 4-AP
5O
5raM ~ A P
B
~
s
lOmM 4-AP
R-15
I0ms
ASW
-ASW,4-AP ........
FIGURE 10. Effects of external 4-AP on pacemaker activity and on action
potentials. (A) Representative records of bursting pacemaker activity from cell
R-15 in different external 4-AP concentrations in normal ASW. The broken
line indicates 0 mV. (B) Superimposed spontaneous action potentials from cells
R-15 and L-11 before and in 5 mM external 4-AP in normal ASW. The
membrane potential was hyperpolarized to reduce rhythmic discharge in both
cases. Each record shows four action potentials. A and B show data from
different cells.
DISCUSSION
O u r results show that 4-AP blocks the delayed K + current but is either without
effect at low external concentrations or, at high concentrations, increases
the Ca2+-activated K + current o f the molluscan neuron soma m e m b r a n e .
T h e y confirm T h o m p s o n ' s (1977) conclusion that the aminopyridines can be
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20mY]
Published July 1, 1981
80
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OF
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PHYSIOLOGY ~ VOLUME
78
9 1981
Downloaded from on June 18, 2017
used to pharmacologically separate the voltage-dependent K + currents from
the Ca2+-activated K + current.
The blockade of the delayed K + current in axon (Meves and Pichon, 1977;
Ulbricht and Wagner, 1976; Yeh et al., 1976a and 1976b) and muscle
(Gillespie, 1977; Molgo, 1978) m e m b r a n e also depends on voltage, and there
is some indication that the block of the fast K + current in molluscan neurons
is influenced by changes in m e m b r a n e potential (Thompson, 1977). O u r
findings suggest that voltage affects a binding site within the membrane. A
similar conclusion has been reached for myelinated axon (Ulbricht and
Wagner, 1976) and can be inferred from the voltage dependency of the block
in squid axon (Meves and Pichon, 1977). O u r findings suggest also that the
binding site is more accessible from inside the cell, but data are not detailed
enough to rule out the possibility that external and internal 4-AP affect
different receptor sites (see Kirsch and Narahashi [1978]). Some authors
(Gillespie and Hutter, 1975; Thompson, 1977) have suggested that 4-AP has
a direct effect on the gating mechanism of K + channels. Although this
possibility cannot be excluded completely, the time- and frequency-dependent
relief of the 4-AP block (Meves and Pichon, 1977; Ulbricht and Wagner,
1976; Yeh et al., 1976 a and 1976 b) makes it very difficult to draw any firm
conclusions about changes in the gating mechanism from the kinetics of
m e m b r a n e currents in the presence of 4-AP.
An alternate explanation is that 4-AP occludes a portion of the delayed K +
channel, thereby preventing the normal movement of K + ions when the
channel gate is in the open configuration. The K + channel is thought to be a
pore with a "selectivity filter" measuring -3.0-3.3 ,~ in diameter that allows
the passage of small cations (up to 3.0 ,~, in diameter) but precludes the
movement of larger cations (Armstrong, 1975; Hille, 1975). Large, positively
charged molecules, such as tetraethylammonium and other quaternary amm o n i u m ions (diameter, ~8.0 A), are estimated to penetrate ~20% of the way
into a wider region of the channel near the inner m e m b r a n e surface, whereas
smaller, positively charged molecules, e.g., m e t h y l a m m o n i u m (diameter, ~3.3
A), are driven ~70% of the way through the channel when the inside of the
cell is m a d e sufficiently positive relative to the outside (Hille, 1975). These
estimates suggest that the selectivity filter is closer to the outside than to the
inner m e m b r a n e surface. The 4-AP molecule is a nonplanar, oblong structure
composed of a pyridine ring with an attached amine group (NH2), which, on
the basis of the microwave spectra measurements given by Christen et al.
(1975), measures ~4.6 • 6.4 A. At the normal intracellular pH of ~7.2, 4-AP
is expected to be almost totally in the cationic form (Albert, 1963). It has been
pointed out (Ulbricht and Wagner, 1976) that it is most unlikely for a
positively charged molecule the size of 4-AP to be driven through an opening
3.0-3.3 A in diameter during m e m b r a n e depolarization. Moreover, the block
and its relief during depolarization are independent of the direction and
amplitude of the K + current through the channel (Ulbricht and Wagner,
1976; Meves and Piehon, 1977), which excludes the possibility that 4-AP is
"flushed out" of occupied channels by K § ion movement.
Published July 1, 1981
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The blocking effect of 4-AP on the delayed K + conductance and the relief
of this block during depolarization might be explained if the pyridine ring
inserts itself into the lipid phase of the m e m b r a n e on the external side of the
selectivity filter such that the amine group partially or fully occludes the
channel. This suggestion is consistent with our finding that the block by 4-AP
occurs ~0.8 of the w a y through the m e m b r a n e electric field, which is 10%
further than the beginning of the selectivity filter estimated from the fraction
of the field through which m e t h y l a m m o n i u m must be moved (0.7) to block
the channel (Hille, 1975). Ulbricht and Wagner (1976) estimated that the 4AP block was 0.77 of the distance through the electric field across the nodal
m e m b r a n e of myelinated axons, which agrees well with our results. W e assume
that the pyridine ring of the 4-AP molecule binds to a hydrophobic moiety
associated with the blocking site. This would be analogous to the hydrophobic
binding of the benzene ring of tetraethylammonium ion derivatives near the
internal m o u t h of the K § channel in Armstrong's model (1975) model for the
block of K § channels by internal quaternary a m m o n i u m ions. The delayed
K + channel is permeable to a m m o n i u m ions (NH4), which are presumed to
make hydrogen bonds with oxygen groups lining the channel wall, so that the
molecule can move through the selectivity filter (see Hille [1975]). The 4-AP
amine group is also capable of making hydrogen bonds that could hold the
nitrogen atom in a blocking position on the external side of the filter. The
bond strength for a hydrogen bond between hydrogen and oxygen is no more
than 2-4 kcal/mol. At 0 mV, the dissociation constant (KD) for the reaction
of 4-AP with the binding site was estimated to be 0.8 mM. Since the free
energy AG ~ ffi R T In KD, AG ~ is ~ 4 kcal/mol, which suggests that sufficient
energy is available to rupture hydrogen bonds. The time constant for the
removal of 4-AP block during repetitive depolarization is ~10 ms in squid
axon (Yeh et al., 1976 a; Meves and Pichon, 1977) and much slower (200 ms)
in myelinated axon. The positively charged 4-AP molecule could act like a
slow activation gate that swings slowly out of its blocking position during
depolarization, or it could be driven off the site into a region of the channel
adjacent to the external m e m b r a n e that can accomodate large molecules, such
as tetraethylammonium or other quaternary a m m o n i u m ions (Hermann and
Gorman, 1981). The model is speculative and is likely to remain so until there
is clear evidence that 4-AP does not interfere with channel gating and that
the block occurs within the K + channel, but the model does take into account
the charge of the 4-AP molecule, the relief of the block during m e m b r a n e
depolarization, and the location of the block within the membrane.
The increase in the Ca2+-activated K + current in high concentrations was
unexpected. W e can exclude the possibility that 4-AP decreases the affinity of
intracellular sequestering systems for Ca 2+, thereby causing a larger increase
99
in free int r a c ellular Ca 2+ per rejected
load and, thus, an increase in the
magnitude of the K § current. W e can also exclude the possibility that 4-AP
produces changes in cell volume causing the internal Ca2§
electrode
to move closer to the inner m e m b r a n e surface, thereby increasing the amount
of free Ca 2+ ions available at the m e m b r a n e to activate the K + current (see
Published July 1, 1981
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G o r m a n and H e r m a n n [1979]), because a similar increase in the Ca2+-acti vated K + current occurs under conditions where the K + current is activated
. . . .It is. possible that 4-AP affects
by Ca 2+ influx rathe r than by Ca 2+ rejection.
directly the activation of the K + conductance by Ca `'+ ions. It has been shown
(Gorman and Thomas, 1980) that the Ca2+-activated K + conductance increases exponentially with depolarization. This potential dependence has been
explained satisfactorily by a model in which a single Ca 2+ ion binds to a
negatively charged site that is about halfway through the electrical field across
the m e m b r a n e and that gates the K + channel (Gorman and Thomas, 1980).
If 4-AP enters into the K + channel then it might alter the position of the
binding site in the electrical field or alter its affinity for Ca ~+ ions, but these
and other possibilities need to be examined experimentally.
In a previous study of the effects of 4-AP on the outward K + currents in
molluscan neurons (Thompson, 1977), the delayed outward K + current and
the CaZ+-activated K + currents were studied together in normal ASW rather
than separately in Ca2+-free ASW. Most of our findings, therefore, are not
directly comparable. The increase of the total outward K + current in normal
ASW containing 4-AP shown in Fig. 9, however, differs from the previous
result in Tritonia neurons (Thompson, 1977), where a high concentration of 4AP had no effect on the total outward K + current studied under similar
conditions. T h e two results are not necessarily in conflict. O u r findings show
that high concentrations of 4-AP have opposite effects on the Ca2+-activated
and voltage-dependent K + currents. In normal ASW, both the Ca2+-activated
and the voltage-dependent components of the K + current (as well as inward
currents) contribute to the total outward current of the molluscan neuron
membrane, but the ratio of the Ca2+-activated to the voltage-dependent
(delayed outward) K + current depends upon cell type as well as upon
m e m b r a n e potential (Meech and Standen, 1975; Heyer and Lux 1976 a and
1976 b; H e r m a n n , 1978; Aldrich et al., 1979). In cell R-15, a significant
fraction of the total outward current is carried by the Ca2+-activated K +
current; e.g., at +20 mV, ~85% of the current is carried by the Ca2+-activated
K + current and ~15% by the delayed K + current (Hermann, 1978). It is not
surprising that there was a net increase in the total outward current of cell R15, since the increase in the Ca2+-activated K + current caused by a high
external concentration of 4-AP would be expected to mask any decrease in
the delayed K + current. T h e proportion of the total outward current carried
by the Ca~+-activated K + current appears to be about equal to that carried by
the delayed K + current in the Tritonia neurons studied by Thompson (1977).
We do not rule out the possibility that neurons differ in their response to 4AP, but an alternative explanation for the lack of effect of 4-AP on the total
outward current of Tritonia neurons is that the decrease in the delayed K +
current was masked by a proportional increase in the Ca2+-activated K +
current.
The aminopyridines increase synaptic activity (Schauf et al., 1976; Galindo
and R u d o m i n , 1978; Jacobs and Burley, 1978), enhance muscular contraction
(Harvey and Marshall, 1977) and at high concentrations act as a convulsant
Published July 1, 1981
HERMANN AND GORMAN 4-Aminopyridine Effects on If + Currents
83
We are indebted to Dr. Douglas Tillotson for his help in the experiments and to Mr. Mark
Sharer and Ms. Mary Ballew for their programming and technical assistance.
This work was supported by U. S. Public Health Service grant NS11429.
Received for publication 2June 1980.
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(Nicholson et al., 1976) t h a t 4-AP increases the d u r a t i o n o f slow potentials
a n d the e x t r a c e l l u l a r K + c o n c e n t r a t i o n . In a x o n terminals an increase in
i n t r a c e l l u l a r C a 2§ should also facilitate the release o f synaptic t r a n s m i t t e r
( K a t z a n d Miledi, 1969; Llin~s et al., 1976). A similar e x p l a n a t i o n m a y a p p l y
to the e n h a n c e m e n t o f m u s c u l a r contractility. Both nonspecific facilitation o f
synaptic transmission a n d increases in extracellular K § are likely to cause
convulsive activity in the central nervous system.
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