Discrete B a 2+ Block as a Probe o f Ion
Occupancy and Pore Structure in the
High-Conductance CaZ+-activated K +
Channel
JACQUES NEYTON a n d CHRISTOPHER MILLER
From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254
ABSTRACT In this study, high-conductance Ca2+-activated K + channels from rat
skeletal muscle were i n c o r p o r a t e d into planar phospholipid bilayers, and discrete
blockade o f single channels by Ba ~+ was studied. With 150 mM K § held constant in
the internal solution, increasing external K + over the range 100-1,000 mM raises
the rate o f Ba 2+ dissociation. This " e n h a n c e m e n t effect," which operates at K §
concentrations 3 - 4 o r d e r s o f magnitude higher than those r e q u i r e d for the "lockin" effect described previously, d e p e n d s on applied voltage, saturates with K § concentration, and is not observed with Na § The voltage d e p e n d e n c e o f the Ba ~+
off-rate varies with external K + in a way suggesting that K § entering the channel
from the external side, forces Ba ~+ dissociation to the internal solution. With K §
held fixed in the external solution, the Ba 2+ off-rate decreases as internal K + is
raised over the range 0 - 5 0 mM. This "lock-in" effect is similar to that seen on the
external side (Neyton a n d Miller, 1988), except that the internal lock-in site is o f
lower affinity and shows only a fivefold preference for K § over Na § All the results
taken together argue strongly that this channel's conduction pathway contains
four sites o f very high affinity for K § all o f which may be simultaneously occupied
u n d e r normal conducting conditions. According to this view, the mutual destabilization resulting from this high ionic occupancy leads to the unusually high conductance o f this K+-specific channel.
INTRODUCTION
Like m a n y K+-specific ion channels, the h i g h - c o n d u c t a n c e Ca~+-activated K + (BK)
c h a n n e l o p e r a t e s by a m u l t i - i o n c o n d u c t i o n m e c h a n i s m , in which several K § ions
s i m u l t a n e o u s l y o c c u p y the p e r m e a t i o n p a t h w a y in t h e i r j o u r n e y across the m e m b r a n e . I n the c o m p a n i o n study ( N e y t o n a n d Miller, 1988), we s h o w e d that K + a n d
Ba ~+ s t r o n g l y i n t e r a c t within this c h a n n e l ' s c o n d u c t i o n p o r e . Specifically, a Ba 2+ ion
e n t e r i n g the c h a n n e l f r o m the i n t e r n a l s o l u t i o n dissociates to the e x t e r n a l side, as
l o n g as e x t e r n a l K + is absent; the a d d i t i o n o f K + to the e x t e r n a l s o l u t i o n p r e v e n t s
Address reprint requests to Dr. Christopher Miller, Graduate Department of Biochemistry,
Brandeis University, Waltham, MA 02254-9110.
j. GEN.PHYSIOL.9 The Rockefeller UniversityPress 90022-1295/88/11/0569/18 $2.00
Volume 92 November1988 569-586
570
THE JOURNAL OF GENERALPHYSIOLOGY.VOLUME 9 2 . 1988
Ba 2§ dissociation to that side, a n d effectively " l o c k s " the Ba ~§ ion inside the channel. T h e s u p r i s i n g a s p e c t o f this result is t h e very high affinity with which K § acts as
this e x t e r n a l " l o c k - i n " site; t h e c o n c e n t r a t i o n f o r half-inhibition is as low as 20
/~M.
W e a r g u e d that the existence o f a single K + - b i n d i n g site o f such high affinity
within the p e r m e a t i o n p a t h w a y is i n c o n s i s t e n t with the high c o n d u c t a n c e o f this
channel, that at least two a d d i t i o n a l K § sites m u s t b e o c c u p i e d d u r i n g c h a n n e l cond u c t i o n . I n this study, we f u r t h e r e x p l o i t the p h e n o m e n o n o f discrete b l o c k by the
Ba 2+ i o n to d e m o n s t r a t e the existence o f two a d d i t i o n a l K + - b i n d i n g sites within this
channel. O n e o f these sites, w h e n o c c u p i e d by K § accelerates the dissociation o f
Ba 2+ f r o m the c h a n n e l , a n d is l o c a t e d b e t w e e n the Ba ~+ b l o c k i n g site a n d the external lock-in site. T h e s e c o n d site is l o c a t e d t o w a r d s t h e i n t e r n a l face o f the channel,
a n d p r o d u c e s a Ba ~+ lock-in effect f r o m this side. T a k e n t o g e t h e r , these e x p e r i -
lOpA
FIGURE 1. Effects of external
K + on Ba ~+ block kinetics. Single BK channels were observed
in planar bilayers in the presence of 150 mM internal KCI
and 150 mM external NaCI
media, with additional KCI
added to the extemal medium.
Blocks were i n d u c e d with
internal Ba ~+, and the holding
voltage was 40 mV. Top trace:
no added K § in external solution; mean block time, 0.5 s.
Middle trace: 10 mM K §
added; mean block time, 4 s.
Bottom trace: 500 mM K §
added; mean block time, 1.7 s.
Ba ~§ concentration was 1 #M
in top trace, and 50 tiM in two
other traces.
m e n t s strongly suggest that the BK c h a n n e l c o n t a i n s at least f o u r K + - b i n d i n g sites
within the c o n d u c t i o n pathway, at least t h r e e o f which a r e n e a r l y always o c c u p i e d
u n d e r n o r m a l c o n d u c t i n g conditions. T h e results r e c o n c i l e this c h a n n e l ' s p u z z l i n g
c o m b i n a t i o n o f high ionic selectivity a n d high c o n d u c t a n c e .
M A T E R I A L S AND M E T H O D S
Details of the experimental measurements were identical to those described in the companion
paper (Neyton and Miller, 1988) except for the following point. In this study, because many
experiments were carried out at negative potentials, we kept the channel highly activated by a
combination of high Ca 2+ and Mg~+ in the internal solution. Mg~+ is known to act aUosterically to raise the affinity of this channel for activator Ca ~+ (Golowasch et al., 1986; Oberhauser et al., 1988). Therefore, many experiments reported here were performed with 10
mM Mg2+ along with 1-10 mM Ca ~§ in the internal solution.
NEYTONAND MILLER K + Block of Ba 2+ Permecaion in C,a2+-activated K + Channels
571
RESULTS
E x t e r n a l K § at H i g h Concentration Speeds Dissociation o f B a z +
This study was i n i t i a t e d by t h e o b s e r v a t i o n illustrated in Fig. 1. I n this e x p e r i m e n t ,
the c h a n n e l was h e l d at + 4 0 m V a n d Ba g+ b l o c k kinetics w e r e a n a l y z e d as t h e e x t e r nal K + c o n c e n t r a t i o n was varied. A n i n c r e a s e in e x t e r n a l K + f r o m 0 to 10 m M p r o d u c e d the " l o c k - i n " effect d o c u m e n t e d in detail in the p r e c e d i n g p a p e r ( N e y t o n
a n d Miller, 1988): a p r o f o u n d decrease in the r a t e o f Ba g+ dissociation. As e x t e r n a l
K + was raised a b o v e 100 mM, h o w e v e r , the o p p o s i t e effect was o b s e r v e d : an increase
in Ba g+ off-rate. A n a c c e l e r a t i o n o f a b l o c k e r ' s dissociation r a t e b y raising the conc e n t r a t i o n o f p e r m e a n t ions is n o t in the least surprising. This sort o f effect has
b e e n o b s e r v e d in t h e case o f N a + b l o c k o f the d e l a y e d rectifier K + c h a n n e l o f squid
a x o n (Bezanilla a n d A r m s t r o n g , 1972) a n d o f t h e BK c h a n n e l in b o v i n e c h r o m a f f i n
cells (Yellen, 1984). I n d e e d , this is a n e x p e c t e d p r o p e r t y o f m u l t i - i o n channel. T h e
2
:o
I
I
400
t
800
[K+lout ,mM
1200
FIGURE 2. K + enhancement
of Ba 2+ dissociation. The Ba ~+
dissociation rate (ko~) was measured at different concentrations of external K +. Conditions were as described in Fig.
1, except that the holding voltage was - 2 0 mV, and internal
Ba 2§ was varied from 0.2 to 10
mM to obtain a high frequency
o f blocking events. Experimental points were fitted with Eq.
1, where K D = 490 raM.
b i n d i n g in a b l o c k e d c h a n n e l o f a n a d d i t i o n a l i o n destabilizes t h e b l o c k e r o n its
b i n d i n g site d u e to e l e c t r o s t a t i c r e p u l s i o n b e t w e e n t h e two ions (Hille a n d Schwarz,
1978). N o t i c e that f o r the B K c h a n n e l , a n i n c r e a s e in Ba 2+ off-rate is o b s e r v e d
a b o v e 100 m M e x t e r n a l K § i.e., u n d e r c o n d i t i o n s o f a l m o s t c o m p l e t e s a t u r a t i o n o f
t h e e x t e r n a l lock-in site (Kt~ ffi 0.3 mM). This s t r o n g l y suggests that it is the e n t r y o f a
s e c o n d K § ion into t h e e x t e r n a l p a r t o f t h e c o n d u c t i o n p a t h w a y that is r e s p o n s i b l e
f o r this " e n h a n c e m e n t effect," which is the e n h a n c e d r a t e o f Ba g§ dissociation
i n d u c e d by e x t e r n a l K §
I f this p i c t u r e is c o r r e c t , t h e n t h e r a t e c o n s t a n t o f Ba g+ dissociation s h o u l d
d e p e n d o n the o c c u p a n c y o f t h e e n h a n c e m e n t site, a n d h e n c e s h o u l d s a t u r a t e with
e x t e r n a l K + c o n c e n t r a t i o n . W e u s e d e x t e r n a l K + c o n c e n t r a t i o n s a b o v e 10 mM, so
t h a t t h e lock-in site c o u l d b e c o n s i d e r e d s a t u r a t e d , with Ba g+ dissociating to the
i n t e r n a l s o l u t i o n exclusively ( N e y t o n a n d Miller, 1988). W e a s s u m e that Ba g+ dissociates at a l o w e r r a t e w h e n t h e e n h a n c e m e n t site is e m p t y , hi, t h a n it d o e s w h e n it is
572
THE JOURNAL OF GENERAL PHYSIOLOGY. VOLUME 9 2 .
1988
occupied, k~. Therefore, the observed dissociation rate, koer is the occupancyweighted average o f these two rates:
koer = kl + (k2 - k l ) ( [ K + ] / K D + [K+]),
(1)
where [K +] represents the external K + concentration, and K o is the dissociation constant for K § Fig. 2 confirms that the e n h a n c e m e n t effect does follow this expected
saturating behavior, and that it requires high K +, with K , in the range o f 3 0 0 - 6 0 0
mM. The m a x i m u m effect at very high K + is a 3 - 1 0 - f o l d increase in Ba ~+ dissociation rate.
Voltage Dependence
of B a 2 + Dissociation
We showed in the preceding p a p e r that Ba 2+ can dissociate f r o m the channel to
either the internal o r the external solution. With high internal K + and very low
B
A
[K+]ou, :
[K*]0ut
0
=
JSo ~,M
+ 5 0 mV
+ 20 m V
4-2 0 m Y
-IOmV
-lOmV
--40mV
SpA
............
- 30 m V
FIGURE3. Voltage dependence of Ba 2+ dissociation. Ba~+-blocking records were collected in
two separate single-channel experiments, using different external K + concentrations, and at
the indicated holding voltages. Ionic conditions were as described in Fig. 1. (A) No K + added
to external solution, 1 tzM Ba 2+. (B) 150 mM K § added to external medium, 3 mM Ba~+
Arrows mark the zero-current levels.
external K +, Ba ~+ nearly always dissociates to the external solution, and as e x t e m a l
K § is raised, dissociation to the internal solution is progressively favored. We therefore expected that most o f the Ba 2+ dissociation events observed at high external K §
would p r o c e e d to the internal solution. This expectation was confirmed by analysis
o f the voltage d e p e n d e n c e o f the Ba ~+ off-rate, shown in Fig. 3. I n K+-free external
solutions Ba ~+ block times b e c o m e shorter with depolarization (Fig. 3 A); in contrast, with 150 mM external K + present, the Ba ~+ blocks are lengthened by depolarization (Fig. 3 B).
Fig. 4 A shows these effects o f voltage in m o r e detail. T h e Ba 2+ dissociation rate
varies exponentially with applied voltage at b o t h high and low external K +, as
expected for a reaction in which an effective charge, z, moves t h r o u g h a fraction o f
NEYTONAND MILLER K + Block of Ba 2+ Permeation m Ca2+-activated K + Channels
573
the m e m b r a n e field, 6 (Woodhull, 1973):
kon(V) = kofr(0)ex p ( - z r F V / R T ) .
(2)
T h e figure shows that as e x t e r n a l K + is raised, the effective valence o f the dissociation reaction, zr, changes sign. I n o t h e r words, high e x t e r n a l K + i n d u c e s a polarity
reversal o f the voltage d e p e n d e n c e o f Ba 2+ dissociation, i n d i c a t i n g that Ba ~+ leaves
mainly to the external s o l u t i o n at low K § (6 > 0) a n d to the i n t e r n a l solution at high
K + (6 < 0). I n K+-free e x t e r n a l solutions, k ~ increased e-fold for 27 + 2 mV
d e p o l a r i z a t i o n (z6 = 0.9). I n 150 m M e x t e r n a l K +, kofr decreased e-fold for 35 + 2
m V d e p o l a r i z a t i o n (z6 = - 0.71).
A
10.0-
[
K
+
]
~
0
S
c...
1.0.
.2
0
0,1
-5[
+]out=150mM
I
I
I
I
-25
0
25
50
V, mV
1.0
0.00'5!~
OUTWARD
z6
INWARD
r
0
300
I
600
FIGURE 4. Influence of external K + on the voltage dependence of Ba2§ dissociation. (A)
Ba2+ off-rate vs. holding voltage was measured as described
in Fig. 3 at two different external K§ concentrations. Solid
lines are drawn according to
Eq. 2, with effective valence
measured from the slopes of
the lines. Open circles: no
added K +, zfi = 0.81. Filled circles: 150 mM external K +, z~$=
- 0 . 6 4 . (B) Variation of effective valence (z~) with external
K +. Ba 2+ off-rate as a function
of voltage was measured as in
A, at the indicated external K +
concentrations. For each set of
conditions, the effective valence was measured from three
to six voltage values. Each
point represents the mean _+
SE of three separate experiments.
[K+]out , mM
T h e reversal o f the voltage d e p e n d e n c e o f kofr by external K + is a progressive
effect. Fig. 4 B shows the effective valence for Ba ~+ dissociation as a f u n c t i o n o f
external K § This effective valence, initially positive at zero K +, rapidly a p p r o a c h e s
zero over the r a n g e 0 - 1 0 m M e x t e r n a l K § as the i n t e m a l lock-in site b e c o m e s
filled, a n d dissociation p r o c e e d s to b o t h sides at r o u g h l y equal rates (Neyton a n d
Miller, 1988). O n l y as K + is raised above 50 m M does the effective valence b e c o m e
increasingly negative, r e a c h i n g a s a t u r a t i o n value at very high K § It is precisely over
this r a n g e o f high K + c o n c e n t r a t i o n s that the e n h a n c e m e n t o f kofr by K § is observed.
Thus, the effects o f K § o n the effective valence o f the Ba 2+ off-rate are fully consistent with the p i c t u r e that external K + retards inward dissociation at low c o n c e n t r a -
574
THE JOURNAL OF GENERAL PHYSIOLOGY
9
VOLUME 9 2 9 1 9 8 8
tions ( 0 - 1 0 raM) a n d e n h a n c e s o u t w a r d dissociation at h i g h e r c o n c e n t r a t i o n s ( > 5 0
raM).
Voltage Dependence of the K+-enhancement Effect
T h e p r e c e d i n g section d e m o n s t r a t e d t h a t Ba 2+ dissociation is voltage d e p e n d e n t .
T h e efficacy o f e x t e r n a l K § in e n h a n c i n g Ba ~+ dissociation is also voltage d e p e n d e n t , as Fig. 5 d e m o n s t r a t e s . H e r e , we show K + - e n h a n c e m e n t curves at two values
o f a p p l i e d voltage. It is clear that b o t h the K D a n d t h e m a x i m u m effect a r e voltage
d e p e n d e n t . M o r e negative voltages, which t e n d to d r a w K + into the c h a n n e l f r o m
the e x t e r n a l solution, a n d Ba ~+ o u t o f it into the i n t e r n a l solution, t e n d to favor K +
b i n d i n g at the e n h a n c e m e n t site a n d to p r o d u c e a l a r g e r m a x i m u m increase in Ba 2+
off-rate. F r o m a series o f t h r e e e x p e r i m e n t s , KD was f o u n d to i n c r e a s e with d e p o l a r ization, with values o f 300 _+ 20 m M at - 30 mV, a n d 550 -+ 120 m M at 0 mV. T h e
fact that the K + dissociation c o n s t a n t at the e n h a n c e m e n t site is voltage d e p e n d e n t
a r g u e s that this site lies within the t r a n s m e m b r a n e voltage d r o p , as will b e discussed
later.
T=
,,=
Jro
i
i
i
400
800
1200
[ K + ] o u t ,raM
FIGURE 5. Voltage dependence o f K + - e n h a n c e m e n t
effect. Ba 2+ off-rate was measured, as in Fig. 2, as a function of external K § concentration, at two different holding
potentials. Both curves shown
were taken on the same channel. Triangles, - 3 0 mV. Solid
curve is drawn according to
Eq. 2, with k] = 0.1 s -j, k2 =
2.3 s -1, and K D = 580 mM.
Squares, zero voltage. Solid
curve is drawn with k] = 0.1
s -1, ks = 1.3 s -j, andKD = 580
mM.
Selectivity of the External Enhancement Site
Fig. 6 c o m p a r e s off-rate variations o b s e r v e d w h e n raising e x t e r n a l K +, R b +, a n d
N a +. As with K +, an e x t e r n a l Rb + c o n c e n t r a t i o n a b o v e 50 m M increases ko~. T h e
maximal a m p l i t u d e o f the R b + e n h a n c e m e n t effect, a n d also the value o f KD a r e
smaller t h a n f o r K § (for R b +, KD = 205 + 45 m M at z e r o voltage, less t h a n h a l f the
value f o r K+). Thus, R b + a p p e a r s to b i n d with a h i g h e r affinity to the e n h a n c e m e n t
site, b u t o n c e b o u n d it e x e r t s less o f a destabilizing f o r c e o v e r Ba 2+ t h a n d o e s K § I n
c o n t r a s t with the p e r m e a n t ions, e x t e r n a l N a + is u n a b l e to s p e e d Ba ~+ dissociation,
even at c o n c e n t r a t i o n s as high as 1.2 M. T h e d e c r e a s e in off-rate seen at high N a § in
Fig. 6 is the tail o f the Ba 2+ lock-in effect by e x t e r n a l N a + (Neyton a n d Miller,
1988). It is thus a p p a r e n t that only p e r m e a n t ions a r e able to e x e r t an e n h a n c e m e n t
effect.
NEYTONAND MILLER K + Block of Ba 2§ Permeation in Ca2+-activated K + Channels
575
Internal K + Locks B a 2§ into the Channel
T h e e x p e r i m e n t s a b o v e a n d in t h e p r e c e d i n g s t u d y f o c u s e d o n t h e i n t e r a c t i o n s o f
Ba 9+ with e x t e r n a l K +. W e n o w r e v e r s e the system's p o l a r i t y a n d investigate interactions o f i n t e r n a l K + with Ba 2+ (which is still a d d e d to the i n t e r n a l s o l u t i o n in all
e x p e r i m e n t s ) . W e m a i n t a i n e d e x t e r n a l K + c o n s t a n t at 150 mM, a n d v a r i e d i n t e r n a l
K +. Fig. 7 shows t h a t the d u r a t i o n o f Ba 2+ blocks is strongly i n c r e a s e d by i n t e r n a l
K +. A t 300 m M i n t e r n a l K § t h e m e a n b l o c k time is o v e r 10 times l o n g e r t h a n K §
free c o n d i t i o n s . Raising K § also increases t h e d u r a t i o n o f t h e bursts, b u t the effect is
m u c h weaker: a t h r e e f o l d i n c r e a s e in t h e m e a n b u r s t time is o b s e r v e d w h e n the
i n t e r n a l c o n c e n t r a t i o n o f K § is c h a n g e d f r o m 0 to 300 mM. Thus, in analogy to the
effect o f e x t e r n a l K +, i n t e r n a l K + also i n d u c e s a Ba ~+ lock-in effect.
Fig. 8 illustrates quantitatively t h e effects o f i n t e r n a l K + o n the Ba~+-blocking
kinetics. I n the c o m p l e t e a b s e n c e o f i n t e r n a l K § the off-rate is r a p i d , a n d the addi-
,),
0
Rb+
.
I
I
400
800
[X§
t ,mM
FIGURE 6. Ion selectivity of
enhancement effect. Ba ~§ offrate was measured between
- 10 and 0 mV as a function of
the concentration of the indicated cation a d d e d to the
external medium. 150 mM
NMDG § was present in all
external solutions. Curves for
K + and Rb + are drawn according to Eq. 1, with a KD of 330
mM for K § and 160 mM for
Rb § Maximum values of offrates are 1.7 and 0.2 s-i for K +
and Rb § respectively. The
decrease in off-rate with added
Na + is due to the very lowaffinity "lock-in" effect seen
for this cation.
tion o f K + r e d u c e s this rate. I t is i m p o r t a n t to r e m e m b e r that these e x p e r i m e n t s
were c a r r i e d o u t in h i g h e x t e r n a l K +, w h e r e the e x t e r n a l lock-in site is always occupied; t h e r e f o r e , n e a r l y all the Ba 2+ dissociation events p r o c e e d to t h e i n t e r n a l solution, as i n d i c a t e d by the voltage d e p e n d e n c e above. As with the e x t e r n a l lock-in
effect, t h e i n h i b i t i o n o f Ba ~+ off-rate by i n t e r n a l K + follows a r e c t a n g u l a r h y p e r b o l i c
f u n c t i o n o f the i n t e r n a l c o n c e n t r a t i o n o f K § (Fig. 8 A). T h e K § c o n c e n t r a t i o n o f
half-inhibition (Ki) is in this case 8 mM. T h e Ba 2+ association r a t e also d e c r e a s e s
w h e n the i n t e r n a l c o n c e n t r a t i o n o f K + is r a i s e d (Fig. 8 B). H o w e v e r , m u c h h i g h e r
i n t e r n a l K § c o n c e n t r a t i o n s a r e r e q u i r e d to o b t a i n a significant r e d u c t i o n o f the onrate: K i was ~ 2 0 0 mM.
T h e similarity b e t w e e n the Ba ~+ lock-in effects i n d u c e d by e x t e r n a l a n d i n t e r n a l
K + a r g u e s f o r a similar u n d e r l y i n g m e c h a n i s m . I n strict a n a l o g y to t h e effects o f
e x t e r n a l K § ( N e y t o n a n d Miller, 1988), we p r o p o s e that the c o n d u c t i o n p a t h w a y
576
THE JOURNALOF GENERALPHYSIOLOGY.VOLUME 9 2 . 1988
contains a K§
site l o c a t e d o n the i n t e r n a l side o f the Ba~+-blocking site. This
internal lock-in site m u s t b e u n o c c u p i e d f o r a Ba 2+ ion to e n t e r the p o r e , o r f o r a
Ba ~+ ion inside the p o r e to leave it to t h e i n t e r n a l solution.
AjOlnity and Selectivity of the Internal Lock-In Site
T h e e x t e r n a l lock-in site was shown to b e highly selective f o r p e r m e a n t ions (Neyton
a n d Miller, 1988). This is n o t the case f o r the i n t e r n a l lock-in site. Fig. 9 shows that
b o t h K + a n d N a + in the i n t e r n a l s o l u t i o n a r e able to lock Ba 2+ in the channel. T h e
affinity o f the i n t e r n a l lock-in site is a b o u t fivefold l o w e r f o r N a + t h a n f o r K +. F r o m
a series o f eight e x p e r i m e n t s c a r r i e d o u t with 1 - 5 m M i n t e r n a l Ca + p r e s e n t , K i values were f o u n d to b e 7 + 1 m M f o r K + a n d 36 + 3 m M f o r N a +. I n t e r n a l R b §
p r o d u c e d a lock-in effect as well (Ki = 9 mM), as d i d Ca 2+ (Ki = 27 raM). Thus, the
i n t e r n a l lock-in site a p p e a r s p o o r l y selective a m o n g cations, b u t definitely favors
c o n d u c t i n g cations.
12# ,6
.L, ....
FIGURE 7. Effect of internal
K + on Ba 2+ dissociation. Single-channel recordings were
collected with 150 mM external K + and 150 mM internal
NMDG + held fixed, while
internal K + was added to the
indicated concentrations. Ba 2+
was 100 #M and the voltage
was 20 inV. Mean block times
were 0.3, 1.2, and 3.0 s at 0,
10, and 300 mM internal K +,
respectively.
DISCUSSION
I n the p r e c e d i n g investigation ( N e y t o n a n d Miller, 1988) we s h o w e d that two ions,
Ba ~+ a n d K +, can s i m u l t a n e o u s l y b i n d with high affinity inside the p o r e o f the large
BK channel. By e x p l o i t i n g the i n t e r a c t i o n o f Ba ~+ with the c h a n n e l as a p r o b e , we
have now i d e n t i f i e d two a d d i t i o n a l effects o f K + o n Ba ~+ dissociation: an e x t e r n a l
e n h a n c e m e n t effect a n d an i n t e r n a l lock-in effect. W e t h e r e f o r e p r o p o s e that the
ion c o n d u c t i o n p a t h w a y o f the BK c h a n n e l c o n t a i n s f o u r i o n - b i n d i n g sites lined u p
in single file: two lock-in sites, an e n h a n c e m e n t site, a n d the Ba~+-blocking site. Several questions i m m e d i a t e l y arise. First, d o the t h r e e distinct effects o f K + o n Ba 2§
dissociation m e a n that t h e r e a r e t h r e e physically distinct K + - b i n d i n g sites involved?
H o w m a n y o f these sites m a y b e s i m u l t a n e o u s l y o c c u p i e d ? H o w a r e the o b s e r v e d
affinities o f these sites r e l a t e d to ionic c o n d u c t i o n a n d selectivity o f this channel? By
e x a m i n i n g these questions in light o f o u r e x p e r i m e n t a l results, we will see that a
minimal m o d e l f o r the c o n d u c t i o n p a t h w a y o f this c h a n n e l m u s t c o n t a i n f o u r phys-
NEe-CONANO MtLLEI~ K § Block e l b a z§ Permeation in Ca2 *-aairmted K § Channels
577
ically distinct K§
sites, all o f which are simultaneously o c c u p i e d most o f the
time d u r i n g normal K + conduction.
Are There Separate Sites?
Previous work has shown that the BK channel's c o n d u c t i o n pathway contains a site
that binds a single Ba ~+ ion with high affinity (Vergara and Latorre, 1983; Miller,
1987; Miller et al., 1987). It is very likely, t h o u g h n o t rigorously proven, that this
site is designed for K § conduction, a n d that Ba ~+ binds as a "substrate analogue"
because o f its similarity in size to K § (Latorre and Miller, 1983; Miller et al., 1987).
We know that as long as Ba 2+ ion resides o n this blocking site, the channel c a n n o t
A
4
0
200
o
~
400
600
=170m ~
I
200
[Kr
!
400
FIGURE 8. Lock-in effect induced
by internal K +. Ba ~+ off-rate (A) and
on-rate (B) were measured as a function of internal K +, under conditions
similar to those found of Fig. 7. Solid
curves were fit by rectangular hyperbolas, with half-inhibition concentrations of 8 mM for the off-rate and
160 mM for the on-rate.
!
600
c o n d u c t K + ions, since, as with m a n y K + channels (Hille and Schwarz, 1978), the
p o r e is so n a r r o w that ions c a n n o t pass o n e another.
The evidence for three additional ion-binding sites comes f r o m the present studies on K + interactions in the Ba*+-blocked channel. We f o u n d that low concentrations o f external K + greatly r e d u c e the rate o f Ba ~+ dissociation. The very high affinity o f K* in p r o d u c i n g this lock-in effect, and the high ion selectivity for c o n d u c t i n g
ions d e m o n s t r a t e that a specific site exists at which K + "blocks" the dissociation o f
Ba 2+ (Neyton a n d Miller, 1988). A n identical a r g u m e n t can be m a d e f o r the internal
lock-in site d o c u m e n t e d in this work; here, neither the affinity n o r the selectivity o f
the binding is as high as with the external site, but still they are high e n o u g h to rule
578
T H E J O U RN A L O F GENERAL PHYSIOLOGY 9 VOLUME
92
- 1988
out nonspecific effects, such as the screening o f local surface potentials. It is also
clear f r o m these results that the two lock-in sites are physically distinct, since they
are located o n different sides o f the blocking Ba 2+ ion.
O u r results f u r t h e r d e m o n s t r a t e the existence o f a third K+-binding site located
o n the external side o f the blocking Ba 2+ ion, the e n h a n c e m e n t site. This site, when
o c c u p i e d by a K + ion, destabilizes Ba 2+ o n its binding site, and thus speeds its dissociation to the internal solution; this is analogous to the conventional "knock-off''
effects o f p e r m e a n t ions o n blockers, long known in a variety o f K § channels
(Bezanilla and A r m s t r o n g , 1972; A d e l m a n and French, 1978; Yellen, 1984; Cecchi
et al., 1987). This e n h a n c e m e n t site, requiring K + at concentrations > 5 0 mM, is o f
very low affinity, but it maintains high selectivity; only c h a n n e l - p e r m e a n t ions produce the effect. This selectivity allows us to conclude that the e n h a n c e m e n t effect is
the result o f specific o c c u p a n c y o f a site, and, t o g e t h e r with the voltage d e p e n d e n c e
o f the e n h a n c e m e n t site affinity, that this site is most likely located within the chan-
z
2
~
~
I
I
I
tO0
200
300
FIGURE 9. Ion selectivity of
internal lock-in effect. The
Ba '2+ off-rate was measured
against internal concentrations
of either K § or Na +, under
conditions similar to those
found in Fig. 7. Half-inhibition
constants of solid curves under
8 mM for K + (triangles) and 41
mM for Na + (squares).
Ix+]in,raM
nel's c o n d u c t i o n pathway. Moreover, it is clear that the e n h a n c e m e n t site is distinct
f r o m either o f the lock-in sites. Concentrations o f K + p r o d u c i n g the e n h a n c e m e n t
effect are orders o f m a g n i t u d e higher than those required to saturate the lock-in
sites.
In studying ion channels, or any o t h e r types o f enzymes, it is usually i m p r o p e r to
identify a saturating process with o c c u p a n c y o f a binding site. T h e reason that we
can make this identification here without apology is that all o u r experiments are
designed so that the K + interactions u n d e r scrutiny are at true t h e r m o d y n a m i c equilibrium. T h e reason for this is that these experiments focus on the dissociation o f
Ba 2+ f r o m a blocked, n o n c o n d u c t i n g channel, i.e., a channel which K § c a n n o t permeate. Because the Ba ~+ ion prevents K + m o v e m e n t across the pore, any K + ion
inside the channel must leave it to the same solution f r o m which it had entered. This
means that as long as Ba 2+ remains b o u n d , K + o c c u p y i n g a site within the channel is
in equilibrium with the bulk solution o n the same side o f the Ba 2+. By focusing o n
the Ba 2+ dissociation process, we are necessarily studying the channel while it is
NEYrONANDMILLER K § Block of Ba2§ Permeation in CaZ§
K § Channels
579
blocked in this way. We assume, o f course, that K + enters and leaves the channel on
the conduction time-scale (nanoseconds to microseconds) many orders o f magnitude m o r e rapidly than Ba 2+ dissociates f r o m its blocking site. It is for these reasons
that we can validly identify the saturating effects of K + on Ba ~+ dissociation kinetics
with occupancy o f sites within the conduction pathway.
How Many Sites Can Be Simultaneously Occupied?
The arguments above identify the three K+-binding sites as distinct, but they do not
tell us whether these sites can be occupied simultaneously. The apparent dissociation constants of both the external and internal lock-in sites are low: ~20 #M and 8
raM, respectively, when measured in 150 mM N-methyl-~glucamine (NMDG+)-con taining solutions, Each o f these dissociation constants was measured in the presence
of 150 mM K + in the opposite compartment, i.e., with the opposite lock-in site
almost saturated. Thus, in 150 mM K + on both sides, both lock-in sites are nearly
always occupied simultaneously, with Ba ~+ on its blocking site between them.
Is the enhancement site occupied simultaneously with the lock-in sites? The
enhancement site, located on the external side o f the blocking Ba ~+, starts to be
significantly occupied only above 50 mM K +, many orders o f magnitude above the
dissociation constant o f the external lock-in site. The externally located lock-in and
enhancement sites can therefore be occupied at the same time. Moreover, enhancement of Ba ~+ dissociation by external K + was analyzed in 150 mM internal K +, i.e.,
with the internal lock-in site highly occupied. It might be argued that u n d e r the very
high external concentration o f K + required to fill the enhancement site, the affinity
o f the internal lock-in site for K + might be lower. This is not the case, however, since
identical lock-in effects are seen at all external K + concentrations tested; at 150,
300, and 500 mM external K +, KD values for the lock-in effect were 10, 11, and 8
raM, respectively. In conclusion, in a Ba2+-blocked channel, the external lock-in and
enhancement sites as well as the internal lock-in site can be occupied at the same
time. Moreover, the simultaneous occupancy o f these three K+-binding sites occurs
along with a Ba r+ ion residing inside the conduction pathway in addition. These
four cation-binding sites u n d e r study must therefore be separate entities.
A Physical Picture o f K+-Ba 2+ Interactions Inside the Pore
The lock-in effects observed at low K + concentrations are easily rationalized in
terms of the single-filing behavior of this K § channel (Neyton and Miller, 1988).
H o w can we understand the ability o f external K + at high concentrations to speed
Ba 2+ dissociation? O u r results show that this effect is due to the low-affinity binding,
in the external part o f the channel's p o r e already occupied by the lock-in K +, of a
second K + ion. Given that this is a single-filing channel, we would like to know which
o f these sites is located m o r e externally, and which is deeper inside, closer to the
Ba 2+ ion.
We can distinguish the two possible arrangements o f sites by taking advantage o f
the fact that K + binds to these sites at true thermodynamic equilibrium, as discussed
above. Because o f this, the observed "half-saturation concentration" for each effect
rigorously represents the dissociation constant of the K + ion on the relevant site. In
such a case, the voltage dependence of the K D provides an unequivocal indicator o f
580
THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME
92 9 1988
the position of the site in the applied voltage drop within the channel, fi (Eq. 2). We
therefore measured the voltage dependence o f KD for all three K+-binding sites (Fig.
10). The binding o f external K § to the external lock-in and enhancement sites weakens with depolarization, while binding of internal K + to the corresponding lock-in
site strengthens. From a series of five experiments, the values of fi for the externally
facing site were 0.18 _ 0.02 for the external lock-in site, and 0.57 _+ 0.13 for the
enhancement site. The external lock-in site is therefore located in the most external
part of the channel's conduction pathway. The enhancement of Ba l+ dissociation by
external K + arises f r o m the occupancy o f a site located more deeply in the pore,
closer to the Ba~+-blocking site. The most natural explanation o f this effect is simple
electrostatic destabilization of Ba 2+ on its site by the K § ion nearby. Fig. 10 further
shows that the internal lock-in site experiences about one-third of the voltage drop,
as measured f r o m the inside (~, - 0 . 3 6 _+ 0.04).
An immediate objection may arise to the picture of the enhancement effect just
offered. The very high affinity of the lock-in site ensures that this externally located
site is virtually always occupied at the high K § concentrations required to load the
Ext lock
0
v
Y
0.5
0.2
'
,
-40
I
0
~
'
~
~
V, m V
Int lock
I
40
,
f
80
FIGURE 10. Voltage dependence of the three K § binding
effects. Variation of the apparent dissociation constants for
the three effects of K + on Ba2+
off-rate, measured in three
separate experiments. Open
circles: external lock-in site, zfi
= 0.18. Filled circles: enhancement site, z~ = 0.42. Open
triangles: internal lock-in site,
z~ = -0.45.
enhancement site. How, then, can K + ions ever gain access to the more internally
located site if the single-filing rule applies? There are two answers to this objection.
First, we may remind ourselves that in a true binding equilibrium, the kinetic path
for going from an initial state to a final state need not be specified. The enhancement effect simply represents a change f r o m the initial state with only the externally
facing lock-in site occupied, to a final state with both K+-binding sites occupied. The
voltage dependence of the K D tells us that regardless o f how this reaction occurs, a
net charge moves about half-way down the voltage gradient.
Despite the thermodynamic purity of this argument, it might be worthwhile to
suggest a concrete way for the enhancement site to become occupied even though
the m o r e externally located lock-in site is of much higher apparent affinity. When
only one of these K+-binding sites is occupied, there will be a dynamic equilibrium
of the single K § ion between the two sites. Since the external site is of much higher
affinity, the K § ion resides on this site nearly all the time. Very rarely but inevitably,
however, the K + ion will occupy the lower affinity enhancement site, and during
such moments the externally located site will become available for attack by a K § ion
NEYTONANDMILLER K § Block of Ba z+ Pen,neation in Ca2 +-activated K + Channels
581
in bulk solution. The net result o f this reaction will be to fill the e n h a n c e m e n t site by
a conventional vacancy mechanism. T h e r e is really n o theoretical p r o b l e m at all, as
long as the rates o f ion m o v e m e n t a m o n g the K+-binding sites are fast c o m p a r e d
with the Ba ~+ dissociation rate.
Ironically, the site a b o u t which we have the least information is the Ba~+-blocking
site itself. We know neither its affinity for K + n o r its ion selectivity. Since we rely
u p o n the Ba~+-blocked channel as the object o f all o u r m e a s u r m e n t s here, we cannot manipulate the blocking site. We should ask if this even is a single, well-defined
site, o r w h e t h e r the Ba ~+ ion can exchange a m o n g the o t h e r K+-binding sites, as
apparently occurs in the squid a x o n delayed rectifier (Armstrong et al., 1982). The
present model asserts that when either lock-in site is empty, the Ba ~+ moves into this
site just before dissociation; the high Ba 2+ off-rate at zero K + o n either side (>4 s -1)
would represent the rate o f m o v e m e n t f r o m the high-affinity blocking site into the
lock-in site f r o m which Ba ~+ rapidly exits. This would be possible with b o t h lock-in
sites filled (K + > l m M external and 100 mM internal), but when the e n h a n c e m e n t
site is empty, Ba ~+ would be able to exchange between the blocking site and the
e n h a n c e m e n t site. Thus while Ba ~+ may not be physically confined to a single site, it
must remain in the region between the two lock-in sites for times on the o r d e r o f
h u n d r e d s o f milliseconds.
O n e puzzle f o r which we d o n o t have a satisfactory explanation is the fact that as
K + o n b o t h sides raised, the Ba ~+ off-rate a p p r o a c h e s a n o n z e r o value o f ~0.2 s -1. A
model with only a lock-in site o n the internal side would predict that at fixed high
external K +, as internal K + is increased without limit, the Ba 2+ off-rate should
a p p r o a c h zero. A possible explanation for this is that an additional e n h a n c e m e n t
site exists on the inner side o f the Ba2+-blocking site, and that as this site becomes
filled at very high internal K +, Ba ~+ is p u s h e d o u t to the external solution. We have
no direct evidence for such a fifth binding site, and are presently searching for
experimental conditions suitable for observing internal e n h a n c e m e n t effects.
An a p p a r e n t oddity a b o u t the p h e n o m e n a r e p o r t e d here is the different effects o f
K + o n the association and dissociation rates for Ba ~+. K + decreases b o t h these rates,
but over different c o n c e n t r a t i o n ranges. H o w can this be, if K § exerts its effect at
the same site in b o t h places? The resolution o f this p a r a d o x lies in the fundamental
difference in K + interaction with the channel in its c o n d u c t i n g and blocked states.
As pointed o u t above, in the blocked channel, K + binds to its site in a true equilibrium reaction; the blocking Ba ~+ ion effectively isolates o n e side o f the channel f r o m
K + ions on the o t h e r side. I n contrast, the c o n d u c t i n g channel, which allows K + to
enter f r o m b o t h sides, is a m u c h m o r e complicated system. Here, the effect o f external K +, for instance, o f r e d u c i n g the Ba 2+ on-rate does not measure the o c c u p a n c y
o f the lock-in site, but is the c o n s e q u e n c e o f a large n u m b e r o f rate processes in this
multi-ion pore. We have n o inclination to model such a complicated process with so
m a n y free parameters.
M u l t i - I o n Conduction by the B K C h a n n e l
O u r picture o f K+-binding sites within this channel's c o n d u c t i o n pathway has
e m e r g e d f r o m experiments carried o u t o n the n o n c o n d u c t i n g , Ba~+-blocked channel. Nonetheless, these results i n f o r m us a b o u t the mechanism o f K + p e r m e a t i o n
582
THE JOURNAL OF GENERAL PHYSIOLOGY 9 VOLUME 92 9 1 9 8 8
through the conducting channel. Assuming that the Ba2+-blocking site is designed to
interact with K + u n d e r normal conducting conditions, we conclude that this channel
contains at least four sites that may all be simultaneously occupied during K + conduction. We further propose that these four K§
sites are of inherently very
high affinity for K § and selective for K + over Na +, although with different degrees
of selectivity. We will argue that this proposal leads to a plausible picture of ion
conduction through this channel.
How can we reasonably argue that all the sites are of inherently high affinity for
K +, when we observe an enormous range o f affinities for these sites? The external
lock-in site shows a K D of 20 tzM, while the KDs of the internal lock-in and enhancement sites are on the o r d e r of 10 and 300 mM, respectively. To answer this objection, we must distinguish between the inherent affinity of a site and its observed affinity. By the inherent affinity o f a site in this channel, we mean the affinity of the site
_C
13kcollrnoI
Empty Chonnel
:)-Occupied
SiteAffinity Chonges with Occuponcy
4-Occupied
]
F]GURE 11. Destabilization of ion binding by multiple occupancy. This cartoon qualitatively
represents the effect of ion occupancy on the free energy profile of a multiply occupied channel. The channel has four identical sites of high affinity. The free energy profile for the unoccupied channel is drawn ~8 kcal/mol below the level of the external solution (1 molal standard state), which is equivalent to KD = 1 uM for the first K+-binding step. Also drawn are the
doubly occupied channel (free energy profile seen by the third ion binding step) and the fully
occupied channel. Free energy scale is drawn for heuristic purposes only, and to correspond
roughly to the range of K, values seen in this study.
for a K + ion, given that all the other sites are unoccupied. In other words, the inherent affinity refers to the binding o f an ion to the empty channel. In general, the
observed affinity of sites in a multi-ion channel will be lower than the inherent affinity because of electrostatic repulsion between ions (Levitt, 1978).
The point is illustrated in Fig. 11, which shows the free energy profiles we propose for this channel at various degrees of occupancy by K +. The empty channel
contains four identical sites of very high affinity, equivalent to a KD in the submicromolar range. A K + ion alone inside this channel would be stuck in a very deep
energy well; it would be able to move rapidly a m o n g the four sites, but not to leave
the channel easily. As K + concentration is raised, additional sites begin to become
significantly occupied. A K + ion about to enter the doubly occupied channel would
see a much higher energy profile, equivalent to an observed K D of 1 mM, for exam-
NZYTONANDMILLER K + Block of Baz+ Permeation in Ca2+-aaivated K + Channels
583
pie. The last K + ion would bind with an even lower affinity to the triply occupied
channel. In this way, all the ions in the fully occupied channel would mutually destabilize each other, so that the rates o f escape f r o m the pore would be high enough
to account for the high conductance actually observed in this channel. This argument must remain qualitative, however, since we do not know the physical separation between the sites, and thus cannot calculate the energies o f mutual destabilization. Experimental and theoretical studies o n gramicidin A, (Levitt, 1978), however,
show that identical sites separated by 1 n m can easily produce KD values differing by
factors of 100-1,000. Since these are the sorts of destabilization factors we observe
here, it is reasonable to propose that these four sites span a distance of ~3 nm. This
estimate is in harmony with the length o f the channel measured by bis-quaternary
a m m o n i u m blockers, 3.5 nm (Villarroel et al., 1988).
We find a very low K D of 20 #M for K + binding to the external lock-in site. This is
the highest affinity yet reported for any K+-specific protein binding site. Since this
m e a s u r e m e n t was made with a divalent Ba 2+ ion in the blocking site and a K + in the
O
--K
-- Bo2+
Bo-Block
OUT g / / / / / / I / / / / / I / I / / / / / / / l
0
IIlIIIIIIIIIIIIIIIIIIIIA
8 = 0
I
0.15
t
0.5
I
0.7
I
Lock-in
Enhoncement
Lock-in
1.0
I
FIGURE 12. A model of the
pore of the BK channel. This
cartoon summarizes our conclusions from this and the
companion study. The channel
is pictured as containing four
IN K*-binding sites located at the
indicated electrical distances,
measured from a series of 12
experiments as in Fig. 10. The
Bae+-blocking site is constrained to be located between
the internal lock-in and enhancement sites.
internal lock-in site, the inherent affinity of this site must be even higher than this.
Likewise, the observed KD (8 mM) of the internal lock-in site was measured in the
presence of Ba ~+ and a single K § already in the channel. The enhancement site
begins to fill only above 50 mM K +, but this occurs in a channel already occupied by
a Ba l+ and two K+ions. Moreover, the enhancement site is located closer to the Ba ~+
site, as j u d g e d by the voltage dependence o f its binding (Fig. 10), so ionic repulsion
here is expected to be particularly strong. For these reasons, we consider it plausible
to propose the simple picture here of four structurally similar, high-affinity
K+-binding sites.
According to this view, significant K + conduction occurs only when the channel is
occupied by at least three K + ions. For lower occupancies, K § conduction is
retarded because the resident ions bind too tightly to the pore. Only at higher ion
occupancies do the K § ions mutually destabilize each other and rapidly permeate
the channel; this mutual destabilization raises the K § dissociation constants by factors on the order of 10 s, thus lifting the free energy profile up by ~7 kcal/mol (Fig.
584
T H E J O U RN A L OF GENERAL PHYSIOLOGY 9 VOLUME
92
9 1988
1 |). Since this channel allows K + conduction at rates approaching diffusion limitation (Latorre and Miller, 1983), there cannot be significant barriers to K + movement within the pore itself. Instead, we picture the p o r e as a structure that allows
thermodynamically favorable specific liganding o f K + over an extended region of
~3 nm, as pictured in Fig. 12. As a speculative interpretation o f o u r results, this
cartoon cannot be taken seriously in detail; but the essential point expressed in this
proposed structure, the existence o f a long pore containing numerous sites suited to
interact strongly with the K § ion, follows inescapably from o u r results. This model is
consistent with this channel's unusual relation between single-channel conductance
and symmetrical K + concentration (Moczydlowski et al., 1985), which can be fit in
the high concentration range (where local surface potentials are largely screened) by
a rectangular hyperbola of Km = 130 mM. While this relation cannot be expected to
correspond precisely to the binding steps described here, the increase in conductance in the range o f 100-1,000 mM probably represents the filling o f the fourth
site identified in this study.
The kind of picture offered here is a wholly conventional view of K + channel
structure. Hodgkin and Keynes (1955) originally proposed a "long p o r e " model for
the squid axon delayed rectifier, and Hille and Schwarz (1978) pointed out that
multi-ion, single-file conduction applies in many K + channels. Estimates of pore
occupancies of two or three K + ions have been made from flux ratio measurements
on squid axon-delayed rectifier K + channel (Hodgkin and Keynes, 1955; Begenisich
and deWeer, 1980), the inward rectifier o f skeletal muscle (Spalding et al., 1981),
and a Ca2+-activated K + channel from h u m a n erythrocytes (Vestergaard-Bogind et
al., 1985). The only novel aspect o f o u r structure is the enhanced detail that we can
plausibly propose. O u r data force us to two unexpected conclusions: (a) that a large
n u m b e r of ion-binding sites, at least four, can be simultaneously occupied inside the
confines of this highly selective pore, and (b) that the inherent affinity o f this pore
for K + is extremely high, many orders o f magnitude higher than the affinity
deduced by conduction properties alone. O f course, the high inherent binding
affinity is required for multiple occupancy here; it is the large favorable free energy
of binding that allows several ions to overcome their mutual repulsion and cohabit
the pore.
These results lead us to a less conventional view of the mechanism o f K + selectivity. Traditionally, K+-selective channels have been considered to select against Na +,
not by favoring the binding of K +, but by placing large barriers to Na § movement at
the channel's "selectivity filter" (Armstrong, 1975; Coronado et al., 1980), although
Hille and Schwarz (1978) have pointed out that multi-ion channels are so complicated that their ionic conduction properties tell us almost nothing about the
underlying energy profile experienced by a permeating ion. O u r results show that
the channel contains binding sites that interact strongly with K + and much less
strongly with Na § For example, the K + / N a + binding affinity ratio is > 1,000 for the
external lock-in site, and at least 10 for the enhancement site. (The internal lock-in
site is less selective, with K + favored over Na + only by a factor o f 5.) We imagine that
Na + fails to permeate the channel simply because most of the cation-liganding
region has a low inherent affinity for this ion. The presence of even a minute concentration o f K + will effectively exclude Na + from the channel, exactly as Ca ~+
NEYTONAND MILLER K + Block of Baz+ Permeation in Ca2+-activated K + Channels
585
blocks m o n o v a l e n t cation c o n d u c t i o n t h r o u g h the "L-type" Ca ~+ c h a n n e l (Almers
a n d McCleskey, 1984; Hess a n d Tsien, 1984). T h e fact that the i n t e r n a l lock-in site
can a c c o m m o d a t e a Na + ion with a Ba 2+ a n d a K + in the c h a n n e l may m e a n that the
i n h e r e n t affinity o f this p a r t i c u l a r site is actually quite high for N a +. I n d e e d , this
may be the site o f i n t e r n a l N a + block o f this c h a n n e l (Marty, 1983; Yellen, 1984). A
c o m b i n a t i o n o f a single high-affinity N a + site in series with several o t h e r low-affinity
N a + sites w o u l d explain this c h a n n e l ' s failure to c o n d u c t N a § appreciably in the
total a b s e n c e o f K +.
We are grateful for Dr. Rod MacKinnon's suggestions and provocations throughout the course of
this investigation.
Support was provided by a National Institutes of Health grant GM-31768. Dr. Neyton was supported by a Fogarty International Fellowship TWO 3898, a European Molecular Biology Organization Fellowship ALTF90-1986, and by the Centre National de la Research Scientifique.
Original version received 1 April 1988 and accepted version received 27 June 1988.
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