Internal blockade of a Ca2+-activated K+ channel by shaker B inactivating “ball” peptide

Neuron,

Vol.

9, 237-245,

August,

1992,

Copyright

0

1992

by Cell

Press

Internal Blockade of a Ca2+-Activated K+ Channel by Shaker B Inactivating “Ball” Peptide 1. Toro,*

E. Stefani,* and R. Latorret

*Department Molecular Physiology and Biophysics Baylor College of Medicine Houston, Texas 77030 +Centro de Estudios Cientificos de Santiago Casilla 16443 and Department Biologia Facultad de Ciencias Universidad de Chile Santiago Chile

Summary Shaker B inactivating

peptide (“ball peptide”, BP) interacts with Ca2+-activated K+ (KcJ channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of Kc. channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to Kca channels in a bimolecular reaction (dissociation constant of 95 pM at +40 mV). The binding site is probably located in the internal “mouth” or conduction pathway, since both external K+and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KG channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.

tntroduction Despite the immense variety and the different roles that K+ channels play in different cells and tissues, they share a number of ion conduction properties. Voltage-dependent K’ channels and Ca2+-activated K+ channels are blocked by Cs+, Baz+, the quaternary ammonium ion tetraethylammonium (TEA), and TEA analogs (Latorre and Miller, 1983; Yellen, 1987; Rudy, 1988; Hille, 1992). It is clear at present that similarities in ion conduction imply closely related structures and that the amino acid sequence thought to form the pore of K+ channels is quite conserved (MacKinnon and Yellen, 1990; MacKinnon, 1991; Yellen et al., 1991; Hartmann et al., 1991). Our present view of the ion conduction system in K+ channels, largely due to the elegant work of Armstrong (e.g., Armstrong, 1971, 1975), is one in which two large vestibules are connected by a tunnel, where the process of ion selection takes place. In both the delayed rectifier and the large conductance Caz+-activated K+ (Kca) channels, the internal mouth binds TEA, and its potency of blockade increases as theTEA analog becomes more hydrophobic (Armstrong, 1971; French and Shoukimas, 1981; Swen-

son, 1981; Villarroel et al., 1988). Internally applied TEA also blocks the Shaker A-type K+ channels (Yellen et al., 1991). Moreover, a combination of site-directed mutagenesis of the SSI-2 region and studies of TEA binding to the external and internal sites indicate that 80% of the electric field falls in a stretch composed of only 8 amino acid residues, probably forming a b-hairpin turn (Guy and Conti, 1990; MacKinnon, 1991). In contrast to delayed rectifier or KG channels, however, wild-type Shaker K’ channels show a fast inactivation process similar tothe inactivation present in Na+channels. In both cases inactivation is removed when these channels are internally treated with proteolytic enzymes (Armstrong et al., 1973; Hoshi et al., 1990). Hoshi et al. (1990) identified the inactivation gate as a cytoplasmic domain of the channel-forming protein located in the amino terminus. A systematic study of a number of different mutations strongly suggested that the first 20 amino acid residues from the amino terminus interact with the open pore, causing inactivation, and that the following 60 or so residues are crucial in determining the speed of inactivation. These results have been interpreted in terms of the “ball-and-chain” model proposed by Armstrong and Bezanilla (1977) to explain Na’ c:hannel inactivation. The first 20 amino acids (the ball) “hanging” on the other 60 (the chain) are able to swing into and occlude the pore once this opens. Furthermore, once synthesized, the peptide forming the ball, when added to the internal side, was able to restore fast inactivation in a mutant A-type K+channel that does not inactivate with a fast time course (Zagotta et al., 1990). W e thought it important to test the effect of this ball peptide (BP) on Kca channels, since it will answer the question whether the receptor for this peptide has been conserved through theevolution of K+channels. If there exists some sort of interaction between the channel and the peptide, the latter may become a much needed probe for the internal vestibule of the channel, as charybdotoxin has been for the external mouth. W e found that BP modifies Kca channel activity from porcine coronary smooth rnusc:le when added to the internal side. The changes in channel gating kinetics can be most simply interpreted in terms of a voltage-dependent binding of the peptide to the internal mouth hindering ion flow through the channel. Similar results have been obtained by Foster et al. (1992) for the interaction of BP with a rat brain Kc-, channel.

Results Mode of Action of the BP In membranes containing a single Kc, channel with a high fractional open probability (P,, >, 0.9, -20 to +80 mV), addition of BP (MAAVAGLYCLGEDRQHRKKQ) to the cytoplasmic side of the channel induced a dimi-

B

A Control

40

Figure 1. ShB lnactivattng Peptide minishes Kc,, Channel Activity

mV

Traces in (A) show channel activity betore (Control) and after addition of 80 FM BP to the internal side of the channel (V,, = f40 mV). Dashed lines indicate the section ot the trace shown at higher time resolution. Filter, 2 kHr; sampling frequency, 100 tts per point. (B) Corresponding P,, versus time plots. BP diminished channel P,, from 0.99 to a mean value of 0.48. During the recording (1.7 min), the channel closed only for several seconds twice (at 58 sand 98 s). Total transitions, 11,032. P,, was calcutdted every 205 ms. P,, reduction after BP was observed in all channels tested fn = 19). Arrows mark the closed state of thr channel.

3+

,

80

[II’,,’

(BP) DI-

‘,

pM BP

0

41

82

Time, s f

5 pA 1 200 ms

nution in P,, (n = 19). In general, this effect was characterized by an increase in the number of short (2-10 ms), well-resolved closing periods and the appearance of closed states of “intermediate” duration (20100 ms). BP also gave rise to less frequent, longer quiescent periods that could last several hundreds of milliseconds (Foster et al., 1992), or even several seconds. An example of this response is shown in Figure 1, in which addition of 80 PM BP to the cytoplasmic face of a Kca channel diminished the initial P,, from 0.99 to a mean of 0.48. Notice that the closing events longer than 1 s were scarce during the experiment. Figure IB shows that during the recording period, only two slow (>I s) closing events were seen. The structure of the single-channel current records obtained in the presence of BP is reminiscent of the flickering blockade induced by some hydrophobic quaternary ammonium ions (Villarroel et al., 1988). As the result of the paucity of long closed events induced by the peptide, in the present study we focus solely on the short-lived channel transitions induced by the peptide. Figure 2 demonstrates that the inhibitory effect of BP on KC-,channels occurs only when the cytoplasmic side of the channel is exposed to the peptide. Moreover, BP does not cause an irreversible effect on channel activity, but it can be easily washed out upon returning to the original recording solution. Examples of channel recordings from the same channel are depicted in Figure 2A, and the corresponding total open channel current histograms are shown in Figure 2B. Addition of external BP to a final concentration of 100 PM did not modify channel activity (holding potential [V,] = 0 mV). However, subsequent addition of 100 and 200 PM BP to the internal side of the channel reduced channel activity from a P, of 0.97 to 0.85 and 0.66, respectively. Because the action of BP from the

internal side is voltage dependent (stronger as voltage is made more positive; see below), we also tested the action of external BP at -40 mV. At this voltage, IOO200 PM BP did not alter channel activity (control P,, = 0.87; after 200 PM BP, P,, = 0.82; n = 2). Concentration and Voltage Dependence of BP Inhibition of Kca Channel Figure 3A shows the effect of increasing BP concentration on channel P,,. A fit to the data using an adsorption isotherm of the form P,, = W(K,I

+ WI”),

(1)

where Kd is the apparent dissociation constant for the channel-BP complex, n is the Hill coefficient, and [BP] is the concentration of BP, yields K,I = 95 2 4 trM and n = 0.9 + 0.04 (n = 8 channels). In Figure 3B we see that the blocking effect of BP on Kca channels is voltage dependent. At a fixed peptide concentration, the channel P, decreases as the voltage is made more positive. It should be emphasized here that in the absence of the peptide, P,, changes in the opposite direction (PO increases as voltage is made more positive). Figure 3B shows the mean P,, values in the absence (control, n = IO) and presence (80 PM, n = 2) of BP at different voltages. It is evident that the inhibitory effect becomes increasingly pronounced as the membrane is depolarized. This is the polarity of the effect to be expected if BP acts as a cationic blocker entering the conduction system of the Kc.L channel from the internal side (e.g., Woodhull, 1973). Alternatively, the voltage dependence of the peptide binding could be due to a voltage-dependent conformational equilibrium of the peptide receptor, as has been found to be the case for the binding of tetrodotoxin and saxitoxin to the batrachotoxin-modified Na*

ShB Inactivating 239

Peptide Blocks Kc. Channels

B

A Control

OmV

Figure 2. The InhIbition Channels Is Internal

x1$ 8

Channel recordings from the same channel (A) and corresponding total point histograms (B). Addition of BP (100 PM) to the external side did not alter channel activity (P,, remained as in the control, 0.96) (n = 4). On the contrary, BP added to the internal side reduced P,, to 0.85 and 0.66 at 100 and 200 vM, respectively. The inhibitory effect was completely reversed after washing out the peptide from the internal milieu with fresh solution (P,, = 0.96~ W = 21. In the total point histograms, the peak around 0 pA corresponds to the closed channel, and the peak at about 5 pA to the open channel. V,, = 0 mV. Cis chamber: 250 m M K’; trans chamber: 5 m M K’, 245 m M Na’. Filter = 500 Hz; sampling frequency = 400 ps per point. Arrows Intjlcate the closed states of the channel.

4 0

BP, [ 100 pM]ext 2

L-. ‘BP, [lo0 PM] int tieL

1 0 3

6:

(-

BP, [200 pM]int tie L.Washout

500 ms

Current, pA

channel (French et al., 1984; Moczydlowski et al., 1984). Regardless of the actual mechanism of peptide action, we can assume that the Kd is voltage dependent: Kd(V) = K(O)exp(-AV),

(2)

where K(0) is the dissociation constant at zero applied voltage and A is a constant determining the voltage dependence of the binding reaction. Replacing Equation 2 into Equation 1 we have P,, = (1 + [[BP]/K(O)Jexp(AV))-I.

Site for BP in Kc,

(3)

A fit to the data positive to -20 mV from Figure 3B using Equation 3 yields K(0) = 666 f 125 PM and A = 0.027 mV-I. Assuming that the site of BP blockade is at a fractional distance, 6, down the electrical field, A = zGF/RT, where z is the effective valence of the blocking ion. The value of A given above corresponds to zS = 0.66, which, assuming z = 3, gives 6 = 0.22. Inhibition Kinetics for BP The observation of flickering behavior for BP allows in principle the measurement of the kinetic constants of the inhibition reaction at the single-channel level (e.g., Neher and Steinbach, 1978). Figure 4A shows an example of the distribution of open and closed dwell times in the absence and presence of 20 and 80 PM internal BP, at +40 mV. Using a 2 kHz filtering, we found that the dwell times in the open state are distributed as a single exponential, but the distribution of ‘closed dwell times is clearly multiexponential. The

fastest closed component (characteristic time constant for the distribution; ~~~ = 0.48 ms) can be seen in the distribution of closed dwell times both in the control and after addition of BP and correspond to the fast closings seen in the control records (e.g., Figure IA). Addition of BP to the cytoplasmic side of the channel induced a marked increase in the number of short events (T,~ = 4 ms). For example, 20 PM BP increased the number of events of the ~~~ kinetic component from 3.7% to 36.9%. In addition, a new distribution of longer closed dwell times was evident, in this case with a characteristic time constant, T,?, of 69 ms (0.6%). These changes in the closed dwell times may be explained as the appearance of two new closed or blocked states (one with the same time constant as in the control), or as the stabilization of the short closed times (T(J and the appearance of a new blocked state. Figure 48 shows that the mean open time varies inversely with [BP]. The slope of the curve gives an on rate constant for the BP binding to the channel of 2.8 + 0.3 x IO6 M-‘s-’ (n = 7), at least two orders of magnitude below a diffusion controlled rate (e.g., Schurr, 1970). This value is similar to the on rate found for the interaction of the peptide with the Shaker B (ShB)-type K’channel (Zagottaet al., 1990). In addition, neither rCL nor ~~~ varied as a function of peptide concentration. We know from Figure 3A that the equilibrium binding of BP to the channel is enhanced as the applied voltage becomes more positive. The results shown in Figure 5 (80 PM BP) indicate that most of this effect arises as a consequence of the voltage dependence of the mean open time. Both the mean open (T,,) and the

A

Specificity of the BP-Binding Site Following the strategy of Zagotta et al. (1990), we synthesized a 20 amino acid peptide with the same sequence as BP, with the exception that the amino acid residue in position 7, a leucine, was changed to a glutamate. This peptide (ShB-L7E) is unable to induce fast inactivation in a mutant ShB K+ channel lacking this gating process (Zagotta et al., 1990). We found that the noninactivating ShB-L7E peptide was indeed unable to inhibit Kca channel activity when added to the cytoplasmic side (n = 3). Figure 6 shows an example of such an experiment, in which addition of 200 PM ShB-L7E peptide to the internal side did not produce any appreciable change in channel activity. On the contrary, subsequent addition of 80 PM BP to the samesidecaused aclear inhibition of channel activity.

PO 1.0 0.8 0.6 0.4 0.2 0 1

10

1000

loo

W ’l, PM B

PO

0 i ~ ~ ~~~0 -100

~~ ~ ~I 100

200

Holding potential, mV Figure 3. BP Inhibits and Voltage-Dependent

Kc, Channel Manner

Activity

in a Concentration-

(A) Dose-response curve obtained at +40 mV (bars represent the standard deviation of the mean; n = 8). The fitted curve gives a Hill coefficient of near l(O.9 + 0.04) and Ka of 95 + 4 PM. (B) Plot of P, as a function of voltage before (triangles) and after (circles) 80 PM BP. The inhibitory effect of BP was stronger at depolarized Values were potentials, at which P, - 1 under control conditions. fitted toa Boltzmann distribution (continuous line) (control, n = lO;BP,n = 2).Thevoltageatwhich50%oftheinhibitionoccurred was 82 f 8 mV, and the slope factor was 45 f 9 mV.

mean

tially

closed

with

were found to vary exponenand are well described by

(T,) times

voltage

r,(V)

= r,(O)exp(-BV)

(4)

r,(V)

= r,(O)exp(CW,

(5)

and

where B = 0.019 mV-’ and C = 0.012 mV-‘. In other words, to decreased e-fold per 53 mV and T‘ increased e-fold per 82 mV. The sum of B + C should predict the voltage dependence of Kd(V), since detailed balance demands that A = B + C. The parameter A obtained for the equilibrium behavior was 0.027 mV’, in reasonable agreement with the sum B + C = 0.031 mV-‘, obtained from kinetic measurements.

The BP-Binding Site Is Located in the Internal Mouth of the Kcl Channel An important issue to determine was whether the BP-binding site was located in the internal mouth of the channel. To this end, we used TEA. TEA blocks KCa channels by binding to a receptor located in the intracellular channel mouth, thus hindering the movement of K+ (Latorre et al., 1982; Yellen, 1984; Blatz and Magleby, 1984; Villarroel et al., 1988). Moreover, if BP is actually occluding the pore, it is expected that its effect might be relieved by external K’, but a nonpermeant ion such as Na’ should not be able to relieve BP inhibition. Figure 7 shows that both internal TEA and external K’ are able to relieve the inhibition of Kc ,, channels caused by BP. To demonstrate the relief by TEA, we used millimolar concentrations of TEA known to block the coronary KCd channel from the internal side (Toro and Stefani, unpublished data). In Figure 7A we show examples of channel recordings at +20 mV, at which 100 uM BP reduced channel activity from a P, of 0.93 (control) to 0.5. Under these conditions, addition of TEA to the internal side of the channel clearly overcame the previous BP-induced inhibition. Notice that besides relieving the flickery behavior, TEA also diminished channel amplitude, as expected, from a rapid equilibrium between the blocker and its site in the channel. Figure 7B shows that the ratio of the mean open time before (BP) and after TEA addition increases linearly with TEA concentration ([TEA]), while the characterisconstant in the same tic time constant rcL remains range of [TEA]. In these experiments, the intermediate (described by r,J and long-lived closed events were too few (<25 events) to determine reliably the effect of TEA. In any event, it is clear that TEA could compete with the short-lived interaction of BP with the internal mouth of the Kca channel. Moreover, the ratio of the open channel currents before and after TEA addition as a function of [TEA] closely follows the mean open time ratio versus [TEA] relationship (Figure 7B, triangles), further suggesting a competitive interaction between TEA and BP for the internal mouth of the channel. A similar strategy has also been used to locate the

ShB inactivating 241

Peptide Blocks Kc, Channels

Open

B

Closed 12 8

r( I

4 0

0.6 /’

Open /-/-i

3 T;’- 0.4

‘/’

3 zlE 0.2 s

Time, ms Figure 4. Kinetics

of Kc, Channel

Inhibition

Dwell-time histograms for a typical experiment at +40 mV (A). Open-time histograms (left) were well fitted by a single exponential before and after addition of 20 and 80 uM BP. The open time constant changed from 288 ms to 24and 4 ms, respectively. Closed-time histograms were well fitted by two (control) and three (after peptide) exponentials, and demonstrate that BP increased the number of short events (TJ and induced longer closings of intermediate duration (T,& Values were as follows: control, T,, = 0.48 ms (96%) and rcl = 5.1 ms (3.7%); after 20 uM BP, T,, = 0.5 ms (62.5%), r,) = 3 ms (36.9%), and rrl = 69 ms (0.6%); after 80 uM BP, T,, = 0.7 ms (53.3%), rt2 = 3.6 binned (total events: control = 690; after 20 uM BP = 6576; after 80 PM ms (45.8%), and 5, r = 59 ms (0.8%). Data were logarithmically BP = 13,562). Bars are the experimental data; continuous lines represent the best fit to the probability density function. Peak values correspond to the time constants. Data were filtered at 2 kHz and sampled at 100 bs per point. (B) The reciprocal of the mean open time increases linearly with peptide concentration (circles), while the short (rJ and intermediate (TV,) closed time constants (triangles and squares) remain unchanged.

binding site of charybdotoxin to the external mouth of Kc, channels (Miller, 1988). Figure 7C exemplifies the results obtained when external K+ is increased from 5 to 250 m M after inhibiting Kc, channel activity with 100 PM BP added to the internal side (applied voltage20 mV). Figure 7D shows that K+ is able to increase in a linear fashion the mean open time, while the mean closed times remain essentially constant. Furthermore, in another experiment in which the nonpermeant ion Na’ in the external side was increased from Oto 245 mM, the inhibition caused

by 100 PM BP was not modified. These results imply that a cation can modify the channel-blocking action of BP only by going through the conduction system of the channel. Discussion In the present paper we have demonstrated that BP interacts with the internal mouth of Kc, channels, producing a flickery inhibition of channel activity. This interaction was voltage and dose dependent. Further-

A

Figure 5. Voltage Dependence Open and Closed Times

0 50 100 Holding potential, mV

20 40 60 80 Holding potential, mV

of the Mean

The mean open times (A) were obtained directly from the dwell-time histograms, and the mean closed times (B) were calculated as r, = (1 -. P,)T,,/P,. These measurements give the kinetic parameters of a twostate channel or the mean of a multistate channel. In the presence of BP, the mean open time was more voltage dependent (decreased e-fold per 53 f 2 mV) than the mean closed time (increased e-fold per 82 f 3 mV) (BP, 80 PM; n = 2).

Control

40 mV

addition is actually representing a blocked state, the presence of a single-exponential distribution of open times and two exponential distributions of blocked times strongly suggests the presence of more than one peptide-bound configuration of the channel. It is worthwhile to point out that Demo and Yellen (1991) found in the Shaker K+ channel a double-exponential time course for the recovery from the inactivated state in low K+. In this case the recovery represents the exit of the inactivating gate from the conduction system. It is tempting to suggest, similar to the effect promoted by BP in Kc d channel, that the double-exponential time course corresponds to recovery through two inactivated states mediated by the same gate. However, other alternative explanations are possible for this phenomenon (see Demo and Yellen, 1991). We may, therefore, approximate the BP binding reaction by the scheme

--. fl.

200 ,nM ShB-L7E mT

peptide

80 PM BP -s

0

c$!$

OBP,

&

OBPL

15pAl d”lwillmun, 200 ms Figure 6. The Noninactivating K, d Channel Activity

Peptide

ShB-L7E

Does

Not Alter

Typical records before (Control; P,, = 0.86) and after subsequent additions of noninactivating (200 PM ShB-L7E; P,, = 0.8) and inactivating (80 PM BP; P,, = 0.4) peptides to the internal side of the channel. Only the inactivating peptide was able to reduce channel activity. VH = 40 mV; filter = 2 kHz; sampling frequency = 100 ps per point. Arrows mark the closed states of the channel.

more, the interaction between the peptide and the internal mouth of Kca channels can be qualified as specific, since BP inhibited channel activity only from the internal sideand the noninactivating peptideShBL7E, with a single amino acid difference, was unable to induce any appreciable blockade when added to the cytoplasmic side of the channel. Similarities with the ShB-type K‘ Channel One of our objectives was to test the hypothesis that Kca channels share properties of their internal mouth with other K+channels, such as the ShB-type K+channel. Some of the results consistent with this idea are the following: BP but not ShB-L7E peptide produces channel inhibition. A similar specificity is found for ShBtype K+channel, in which the ShB-L7E mutation of the BP does not produce inactivation (Zagotta et al., 1990). The dose of BP needed to produce inhibition of KC,, channels is within the concentration range needed to produce inactivation of ShB-type K’ channel (Zagotta et al., 1990). Both the dissociation and the association rate constants are similar in both channels. Finally, as in MB-type K’ channels (Demo and Yellen, 1991), BP behaves like an open Kc,channel blocker (see below). Mechanism of Action Assuming that the distribution of dwell scribed by the characteristic time constant

where 0 represents the open state and O&P, and OBPL are the two forms of blocked states. Here we are assuming interaction of BP only with the open channel, and we are not considering the closed-open equilibrium. We think these are safe assumptions, since in these experiments the effect of BP was tested at positive potentials at which P,, 2 0.9. Whether BP is able to bind to the closed channel will be discussed in a separatecommunication. Intheabovekineticscheme the reaction is strictly bimolecular, since the equilibrium results indicate that the Hill coefficient of the reaction is never larger than 1 (Figure 3) and only the mean open time is dependent on BP concentration (see below). This scheme predicts that the mean open time, T(,, is related to the second order rate constant k, by the equation l/r,,

(b)

where ~~ is the mean open time obtained from the logarithmic open dwell-time histograms (Figure 4A). This prediction is in agreement with the experimental results. We recall that Figure 4B shows that the mean open time varies inversely with [BP] as demanded by Equation 6. This scheme also predicts that the mean blocked times must remain constant with [BP]. These predictions are borne out by the data shown in Figure 4B, which indicates that neither TUJ (?
times deTQ after BP

= k,[BP],

s

IlKI OTEA

OPB,

+

OBP,

ShB Inactivating

Peptide Blocks Kc,
243

A Control -+ 100 UM BP

20 mV i4 ’ $ 3u I .

120 mM TEA

2++B 1 1

t

4

0

500 ms

0

40

80

F’EAl,

120

mM

C Control, 250/5 K m+

20mV

100 pM BP -e 250/250 K 12pAi

Figure

-* 500 ms

U

0

150

300

External [K], mM

7. TEA and External

K’ Relieve

BP-Induced

Inhibition

(A) Examples of channel activity before additions (Control, P,, = 0.93), after addition of BP (100 PM, P,, = 0.5). and after subsequent addition of 120 m M TEA to the internal side of the channel. TEA increased the P,, of the channel (P,, = 0.89) and decreased the open channel current amplitude (n = 2). Arrows mark the closed states of the channel. (B) Mean open times (circles) and short closed times (T,> = 10 ms; squares) after TEA (~~~~1normalized to the corresponding dwell times in the presence of BP (T&. The ratio of the open channel current before and after TEA (triangles) is also shown. V ,. = 20 mV. Cis chamber: 250 m M K’; trans chamber: 5 m M K, 245 m M Na’. Filter = 500 Hz; sampling frequency, 400 pts per point. (C) Examples of channel records before additions (control), after addition of 100 nM BP (at 5 m M external KCI), and following addition of external KCI to a final concentration of 250 mM. VH = 20 mV; filter = 500 Hz: sampling frequency = 400 ps per point. (D) Mean dwell times (open times, circles; closed times, triangles) as a function of external K’ concentration of another experiment. BP concentration = 40 nM; V+, = 0 mV; filter = 2 kHz; sampling frequency = 100 ns per point

where K, is the dissociation constant for the complex OTEA. In this second scheme the mean open time is not related to k, through the simple Equation 4, but now depends on the channel TEA occupancy. Noticing that the state OBP, can be entered only from 0, the mean open time in the presence of TEA, r,,(TEA), is given by

IIT,,(TEA)= kdBPlPw, orOX,,,,

(7)

where P,o,o or orEA) is the conditional probability that the channel is open given that is in one of two states, open or blocked by TEA. Since the TEA blocking reaction is essentially in equilibrium with respect to the BP-channel reaction, P1O,OaroTLAjis given by (1 + [TEA]/ K,))‘. Therefore, combination of Equations 6 and 7 yields (Miller, 1988) r,(TEA)/r,

= (1 + [TEA])/K,.

Notice that the right-hand equal to i,/ (e.g., Latorre

(8)

side of Equation 8 is also and Miller, 1983; Villarroel

et al., 1988), where is the average channel current measured in the presence of TEA, and i0 is the value of the unblocked channel current. The predictions of the second scheme were tested experimentally, and as demanded by Equation 8, the ratio of time constants is a linear function of [TEA]. As also predicted by this scheme, we found that the mean blocked times 7oBp1 and roBPL were independent of [TEA]. These findings are suggestive of a simple competitive relationship between BP and TEA. Moreover, the K, value of 30 mM, obtained from the slope of the straight line fitted to the mean open time data of Figure 78 (circles), is almost identical to that obtained from the current depression induced by 26 m M TEA (triangles).

Origin of the Voltage Dependence of the Peptide Blockade in Kca Channels In Shaker K’ channels, the inactivation process has been postulated to be inherently voltage independent (Zagotta and Aldrich, 1990; Demo and Yellen, 1991). Demo and Yellen found that hyperpolarization in-

creased the rate of opening from the inactivating state. They argued, however, that this apparent voltage dependence actually reflects the fact that hyperpolarized voltages favor the movement of K+ through the pore from the external side, clearing the internal mouth from BP. They based their arguments on the observation that increasing external K’ increases the rateofexitofthe ballfromthechannel,asifthiscation pushes the inactivation out of the conduction system. in this regard, BP blockadeof the Kca channel is different, and we believe that in our case voltage dependence can be explained by a positively charged particle (BP) moving in the electric field. In addition, the location of the BP site within the membrane electric field may also contribute to the reported voltage dependence of blockade. We based this statement on observations that, contrary to what is observed in the Shaker channel, external K+ does not affect the rate of exit, but solely the rate of entry of the peptide to the channel, as expected from competition for a site; and both the rate of exit and entry are voltage dependent and their voltage dependence appears to be independent of the range of voltages studied. If the movement of external K+ is the origin of the voltage dependence, one would expect only the rate of exit to be voltage dependent. Experimental

Procedures

Membrane Vesicles Plasma membrane vesicles from pig coronary smooth muscle were prepared from 20-30 arteries as has been already described (Tore et al., 1991). In brief, microsomes were obtained in the presence of protease inhibitors and were subsequently purified in a sucrose gradient. The membrane fractions (IO-30 mg of protein per ml) were frozen in liquid NJ and stored at -70°C until used. Membranes obtained from the 20%:25% and 25%:20% (w/w) sucrose interfaces were used in this study (two preparations). Electric Recording Experiments were performed using reconstituted K,, channels from coronary smooth muscle with high open probability (>0.8) at membrane potentials from -60 to +60 mV (Toro et al., 1991). Lipid bilayers were cast from a phospholipid solution in n-decane containing a 5:2:3 mixture of phosphatidylethanolaminei phosphatidylserinelphosphatidylcholine (25 mgiml). Bilayer capacitances were about 200 pF. The membrane vesicles were applied with a glass rod to one side (cis) of the preformed bilayer. Channel insertion was not always asymmetric, and therefore, channel orientation was checked in each experiment. The laterality of channel incorporation was initially determined by the voltage dependence of channel gating. We also established the compartment to which the Ca?+ sites faced by varying the free Ca?’ concentration. Voltage was applied to the cis chamber, and the trans chamber was virtual ground. Data Acquisition and Analysis Single-channel recordings wereacquired on line using pCLAMP software with 12-bit A/D D/A converters (Axon Instruments, Burlingame, CA) and a custom-made voltage-to-current converter amplifier (Hamilton et al., 1989). The majority of the recordings were filtered at 2 kHz with an &pole Bessel filter and acquired at 100 ps per point for further analysis, or at 500 Hz and 400 )IS per point, respectively. Single-channel analysis was done using TRANSIT, a program developed by A. M. J. VanDongen (1992, Biophys. J., abstract). All data analysis was performed in bilayers

with a single active channel. The 50% threshold-c rosslng technique was used for estimating event durations and for obtaining “idealized” records (Colquhoun and Sigworth, 1983). The idealized records were corrected for dead time due to sampling frequency and filter characteristics. Open and closed dwell-time histograms were logarithmically binned and fitted by a sum ot exponential probabilitydensityfunctions using maximum likelihood. In this type of representation, peaks correspond to the time constant of the distribution considered (Sigworth and Sine, 1987). Solutions The cis chamber contained 250 mM KCI and IO mM KOH-MOPS (pH 7.4; pCa 4). The trans chamber contained 5 mM (before incnrporation) or 250 mM (after Incorporation) KCI and 10 mM KOHMOPS (pH 7.4; pCa 4). Modifications to these solutions are Indicated in the figures. Chemicals BP and ShB-L7E peptide were synthesized and puritled by highpressure liquid chromatography in the Protein Chemistry Facllity of Baylor College of Medicine. Stock solutions were adjusted to pH 7.4. All other chemicals were from Sigma (St. L.ouis, MO) or Aldrich (Milwaukee, WI). Acknowledgments The authors thank Dr. A. M. J. VanDongen tar TRANSIT sottware and Garland Cantrell for buildingthe bilayeramplifier. Thtswork was supported by Grant-in-Aid 900963 from the American Heart Association, by the National Center (L. T.), by grants HD-25616 and HL-37044 from Natlonal Institutes of Health (E. S.), and by NIH grant CM35981 and grant 863-1991 from Fondo National de Investigaciones, Chile (R. L.). We also thank Drs. C. D. Foster, S. Chung, W. N. Zagotta, R. W. Aldrich, and I. B. Levitan for communication of their results prior to publication. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertrsemenf’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

9, 1992; revised

May 13, 1992

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