Correspondence to: Ramon Latorre, Centro de Estudios Científicos de Santiago, Casilla 16443, Las Condes, Santiago 9, Chile. Fax:Fax: 562-233-8336; E-mail:ramon{at}cecs.cl.
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Abstract |
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Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549568), we calculated a dissociation constant of 2.7 µM for K+ at this lock-in site. We also found that BKCa channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 µM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4 + to the external solution. These results suggest that the BKCa channel stability in solutions of very low [K+] is due to K+ binding to a site having a very high affinity. Occupancy of this site by K+ avoids the channel conductance collapse and the exit of Ba2+ toward the external side. External tetraethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:6977) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivity filter, respectively.
Key Words: KCa channel, multiple occupancy, barium block, tetraethylammonium, lipid bilayer
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Introduction |
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The large conductance Ca2+-activated K+ (BKCa)1 channel has a multi-ion pore (
The large conductance Ca2+-activated K+ channel has a high degree of identity in the pore region with voltage-dependent K+ channels. The crystal structure of a K+ channel from bacteria was recently elucidated (
Despite the similarity with voltage-dependent K+ channels, BKCa channels do not show external K+-dependent phenomena such as C-type inactivation (4 µM) is sufficient to saturate the relevant K+ binding site(s) in the pore. To test this hypothesis, we have lowered the external [K+] below the K+-contamination level using a crown ether that chelates K+ with high affinity. Our results show that when channels are exposed to external solutions containing less than 4 µM, K+ channel electrical activity suddenly ceases, a result that is consistent with our hypothesis.
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Methods |
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Lipid Bilayers and Channel Incorporation
All measurements were performed on planar bilayers with a single BKCa channel inserted. Since depolarizing voltages and cytoplasmic Ca2+ activates BKCa channels, the "internal" side of the membrane was defined according to the voltage and Ca2+ dependence of the channel. Accordingly, the physiological voltage convention is used throughout, with the external side of the channel defined as zero voltage. Bilayers were cast from an 8:2 mixture of 1-palmitoyl, 2-oleoyl phosphatidylethanolamine (POPE) and 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) in decane. Lipids were obtained from Avanti Polar Lipids. Bilayers were formed in 0.01 M 3-[N-morpholino]propane-sulfonic acid-N-methyl D-glucamine (MOPS-NMDG), pH 7. Concentrated KCl and CaCl2 were added to the internal solution to a final concentration of 0.1 M and 125 µM, respectively. The internal [Ca2+] used fully activates the BKCa channel from skeletal muscle (e.g.,
Rat skeletal muscle was used to prepare membrane vesicles containing BKCa channels as previously described (
Data Acquisition and Analysis
Single-channel recordings were acquired using a custom-made current-to-voltage converter amplifier (
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(1) |
where kin is the dissociation rate constant toward the internal side and kext is the dissociation rate constant toward the external side of the channel when the lock-in site is empty, and KdK is the dissociation constant for K+ from the channel containing a K+ and a Ba2+ simultaneously. We used a nonlinear least-square fit procedure to find the values of kin, kext, and KdK, where the statistical weight of each point was the number of observations on each decade (
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Solutions
Determination of the free K+ concentration in solutions containing low K+ and crown ether requires knowledge of the [K+] of "K+-free" solutions and the dissociation constant of the K+-crown ether in the presence of 0.11 M MOPS-NMDG. The [K+] was determined using an ion-specific electrode (Orion 9319BN; Orion Research, Inc.) that is linear in the [K+] range between 1 µM and 1 M. The average K+ contamination of the MOPS-NMDG solutions used in the present study was 4.4 µM. The contaminating [K+] of the stock of MOPS-NMDG and EGTA-NMDG solutions was also determined by atomic absorption spectrophotometry. A crown ether (a gift from Dr. Jacques Neyton, Laboratoire de Neurobiologie, Ecole Normale Supérieure, Paris, France), (+)-18-Crown-6-tetracarboxylic acid (18C6TA) from Merck, was used to chelate the contaminating external K+ and contaminating Ba2+ in the internal solution. The 18C6TA:cation stoichiometry is 1:1 (e.g.,
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Results |
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Lowering External [K+] Modifies Slow Ba2+ Block, Induces the Appearance of a Flickering Ba2+ Block, and Alters the Channel-gating Kinetics
Figure 1 shows K+ currents from single BKCa channel recordings with 70 nM internal [Ba2+] and different external K+ concentrations along with the corresponding closed dwell time histograms. Three different features are evident from the figure. (a) There is a slow internal Ba2+ block described previously2 (
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Slow Ba2+ block.
Figure 1, right, shows that the distribution of dwell times in the closed state is multiexponential. Note that mean block times of the slow component became shorter and the number of events increased as the external [K+] was decreased. Upon decreasing the external [K+] from 24 to 0.09 µM, the mean Ba2+ blocked time decreased from 660 to 50 ms. Figure 2 shows a fit to the o-Ba-[K+]ext data using Equation 1. The best fit was obtained with kext = 7.6 ± 1.7 s-1, kin = 0.11 ± 0.02 s-1, and KdK = 2.7 ± 0.4 µM. The value of KdK found indicates that BKCa channels bind K+ fivefold tighter than previously thought (
Our value of kext was determined at 0 mV applied voltage.
KdK is also voltage dependent and eV/kT) with z
= 0.18 and a KdK (0) = 2.7 µM, we find that KdK (50) = 3.9, a value fivefold lower than the KdK of 19 µM determined by
Fast component of the closed dwell-time distribution.
The fast component of the closed dwell-time distribution was also modified by external [K+]. As in the case of the slow Ba2+ block, the number of events increased as the external [K+] concentration was reduced (see dwell time histograms in Figure 1). However, in contrast to the slow component, the mean fast blocked time of the fast component of the closed time histogram is almost unchanged by a 10-fold reduction in the external [K+]. Is the difference in slow and fast dwell-time dependence on [K+] due to a modification of channel gating proper or is it the manifestation of a Ba2+ flickering block? (e.g.,
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(2) |
where kon is the association rate constant for Ba2+ binding, and Po the probability of opening. Therefore, a 14-fold decrease in [Ba2+], considering the Pos before and after the addition of Ba2+, should induce a 90% decrease in NB. The theoretically expected decrease in NB after lowering [Ba2+]int is much more pronounced than the one found experimentally. This analysis suggests that upon diminishing [K+]ext, the increase in NB is only partly due to a Ba2+ flickering block and that the reduction in external K+ also induces the appearance of fast closed events.
The long-lasting closed state.
Occupancy of the outer mouth of the pore of Shaker K+ channels by K+ slows the rate of C-type inactivation (
The single-channel current recorded at 0.09 µM K+ in Figure 1 shows a closed state of very long duration. Figure 3 A shows that the channel enters this nonconducting state of very long duration when the [K+]ext is reduced from the contaminating level (4.4 µM; Figure 3 A, top) to 0.01 µM by the addition of crown ether to the external solution (Figure 3 A, middle). After spending several minutes in the quiescent state, normal channel activity was recovered by adding K+ to the external side to a final concentration of 10 µM (Figure 3 A, bottom). The recovery of channel activity after a drastic reduction in external [K+] occurred in 15 of 22 trials. It appears then that the BKCa channel conductance collapses at external [K+]s much lower than those necessary to arrest other K+ channels.
Lithium, Na+, Rb+, Cs+, and NH4+ were also tested for their abilities to recover the BKCa channel from its long-lasting nonconductive state. Rubidium (20 mM), Cs+ (20 mM), and NH4+ (3.550 mM) were able to recover the channel from the nonconducting state. Figure 3 B shows an example of recovery from the quiescent state when NH4 + is added to the external solution to a final concentration of 10 mM. Sodium (2040 mM) and Li+ (20100 mM) were not able to recover channel activity, suggesting that only permeant cations are able to recover the channel from the conformation it adopts at very low [K+].
External TEA+ Traps K+ Inside BKCa Channels
In Shaker K+ channels, one specific amino acid location in the pore-forming region (position 449) is crucial in determining sensitivity to external TEA+ (
Since we expected TEA+ to increase Ba2+ mean blocked time, we reduced [K+]ext to begin the experiment with short mean Ba2+ block time. The crown ether concentration was adjusted to decrease the potassium concentration from the basal level down to values where the channels would not enter into the long lasting nonconducting state. Furthermore, TEA+ seems to protect the channel from falling into the long lasting closed state since we observed stable channel activity with [K+]ext as low as 0.007 µM. In the experiment shown in Figure 4, we reduced the external [K+] concentration from 6 to 0.03 µM by adding 0.9 mM crown ether to the external solution. The figure shows the effect of external TEA+ on the nonconducting dwell times induced by the presence of internal Ba2+. TEA+ reduces the open channel current and also increases the duration of the closed dwell times. In the absence of TEA+, the measured mean block time was 160 ms; after increasing the external TEA+ to 900 µM, the mean block time increased to 1,700 ms.
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Surprisingly, if external [K+] is reduced from 0.06 to 0.007 µM by the addition of crown ether, in the presence of external TEA+, the mean block time is decreased (Figure 5). Figure 5, middle and bottom, shows that channel conductance is not affected by the addition of crown ether. Therefore, the complexing agent does not affect TEA+ concentration.
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Figure 6 shows that the effect of TEA+ on Ba2+ block strongly depends on external K+ concentration. In the presence of 130 µM external K+, 2 mM TEA+ brings the mean Ba2+ blocked time to ~20 s. Therefore, the blocked time is even longer than the maximum value expected for Ba2+ leaving toward the internal side of the channel in the presence of high external [K+](compare Figure 2). However, in the presence of 0.04 µM K+, the same TEA+ concentration induces a mean Ba2+ blocked time of only 2 s.
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A Possible Model
The ability of external TEA+ to slow down Ba2+ dissociation cannot be reconciled with the idea that TEA+ and K+ compete for the same binding site in the channel or with the simple picture of ionion repulsion within the pore. In both cases, it is expected that TEA+ should behave less effective as a lock-in ion in the presence of K+.
To interpret our results quantitatively, we propose the model illustrated in Figure 7. The channel is viewed as having three sites: a Ba2+-blocking site, a K+-binding site located externally to the blocking site, and the external TEA+ site. As shown by o-B
:
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(3) |
where PBa, PBa-K, PBa-TEA, and PBa-K-TEA are the probabilities of finding the channel occupied by Ba2+ only, by Ba2+ and K+, by Ba2+ and TEA+, or by Ba2+, K+, and TEA+, respectively. These probabilities are given by the following relationships:
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(4a) |
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(4b) |
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(4c) |
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(4d) |
where KdK is the dissociation constant for K+, Kd1TEA is the dissociation constant for TEA+ from the triply occupied state, and Kd2TEA is the dissociation constant for TEA+ from the doubly occupied state. There are five different rate constants for Ba2+ exit: kext is the rate of exit to the extracellular side when the channel is occupied only by a Ba2+ ion, kin is the rate constant of exit to the intracellular side when the channel is occupied only by a Ba2+ ion, and kin(K), kin(TEA), kin(K, TEA) are the rate constants of exit toward the extracellular side when the channel is occupied by Ba2+ and K+, by Ba2+ and TEA+, or by Ba2+, K+, and TEA+, respectively.
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The model accommodates rate constants for Ba2+ exit toward the internal side that are different (compare Figure 2 and Figure 6) in the absence and presence of TEA+. Experimentally, we found that when the quaternary ammonium ion and K+ are present in the external solution, kin(K,TEA) is approximately twofold slower than in the absence of TEA. On the other hand, the fit to the data with the model shown in Figure 7 indicates that in the absence of K+, the rate constant for Ba2+ exit, kin(TEA), is approximately four times larger than kin(K,TEA) (see Figure 6). We have tested our model by comparing the measured mean Ba2+ blocked times at different [K+] and [TEA+] from 26 different single-channel membranes with the calculated mean blocked times obtained using Equation 3. The model proposed in Figure 7 describes the data rather well (Figure 8 A). The model is unable to predict the experimental results if triple occupancy is not allowed (Kd1TEA = ) (Figure 8 B) or if TEA+ is unable to bind the channel unless it is occupied by K+ (Kd2TEA =
) (Figure 8 C). Figure 8 B shows that if triple occupancy is not allowed, the model predicts an attenuated effect of TEA+ on the mean Ba2+ blocked time relative to that found experimentally. On the other hand, Figure 8 C illustrates that if TEA+ can only bind to the Ba2+-occupied channel in a triple occupancy configuration (when the lock-in site for K+ is full), then the model fails to account for the data obtained at very low [K+]. The best correlation between model generated and experimental values of the mean Ba2+ block time was obtained when TEA+ binding was allowed in any configuration of the model with rate constants of Kd1TEA = 180 µM and Kd2TEA = 67 µM. It is very interesting that the ratio between these two dissociation constants (2.5) reveals a K+TEA+ repulsion of ~0.6 kcal/mol. This very low repulsion energy implies that the bound TEA+ ion is essentially shielded from the K+ ion occupying the external lock-in site.
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The kinetics of block by TEA+ are rapid, operating in the time scale of microseconds (i
, is a measure of the channel occupancy by TEA+ at its blocking site:
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(5) |
Since in this case TEA+ blocks a channel containing only K+ ions in its conduction machinery, it is pertinent to ask whether the dissociation constant for TEA+, KdTEA, is similar to that obtained from its effect on the mean Ba2+ block time.
Figure 9 illustrates the dependence of the channel current on TEA+ concentration at 0 mV and in different [K+] (each symbol represents a different [K+]). There is a linear relationship that is well described by Equation 5 with a KdTEA = 106 µM (Figure 9, solid line). Notice that the fit to Equation 5 is reasonably good for all the [K+] tested, indicating that there is not a single hint of competition between K+ and TEA+ for a site(s). Moreover, the value of KdTEA+oß
sigma}">
sigma}">
µ
o
oß
ß
o
> +'°° +-'' µM) under symmetrical 100-mM KCl conditions.
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Discussion |
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The crystal structure of the K+ channel pore from Streptomyces lividans revealed two binding sites for potassium in the selectivity filter that are ~0.75-nm apart ( helical structures pointing their partial negative charge toward the cavity where the ion is located. We hypothesize that the Ba2+ flickering block originates from Ba2+ entering and leaving the pore from the pore cavity.
In the case of potassium channels, it is clear that permeating ions within the conduction pathway affect some of the structural changes associated with gating (
The data presented here shows that occupancy of a very high affinity site for K+, most likely the lock-in site, controls ion permeation in the BKCa channel. Emptying the channel of K+ ions could lead to the equivalent of the C-type inactivation or to the K+ conductance collapse phenomena described for other K+ channels. When the lock-in site is empty, the channel clearly undergoes structural changes that lead finally to the long-lasting inactivated state. These changes are probably triggered by electrostatic repulsion of the carbonyl groups, which makes the selectivity filter atoms move apart. Figure 2 shows that the KdK for the lock-in site is 2.7 µM, which corresponds to an energy well of -13 kT. Considering that this value of KdK is for the double occupied [K+Ba2+] channel, this energy is an upper limit that indicates that the binding of K+ to BKCa channels as tight as the binding of Ca2+ to Ca2+ channels (e.g.,
Although we do not know the details of the molecular mechanism that governs C-type inactivation, it is known that external TEA+, K+, and other monovalent cations inhibit it. Point mutations in Shaker K+ channels have also shown that the rate of C-type inactivation and the K+ permeability properties can be altered simultaneously (
The effect of external K+ on the ability of TEA+ to lock Ba2+ into the channel was explained using a model in which Ba2+, K+, and TEA+ can simultaneously occupy the channel. The analysis of our results demonstrated that TEA+ binding to the Ba2+-blocked channel is essentially the same whether or not a K+ ion is bound and that the binding constant is not very different from the one obtained measuring the current amplitude in the presence of different [TEA+] and [K+]. This result implies that there is little electrostatic repulsion between the K+ in the external lock-in site and the TEA+ bound to its external receptor. The crystal structure of the K+ channel from Streptomyces lividans showed that the distance separating the K+ ion located in the external site of the selectivity filter and the TEA+ ion is 0.8 nm (
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Footnotes |
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1 Abbreviations used in this paper: BKCa, large conductance Ca2+-activated K+; MOPS, 3-[N-morpholino]propane-sulfonic acid; NMDG, N-methyl D-glucamine; TEA+, tetraethylammonium.
2 Barium is effective from either side of the membrane, but is much more potent when applied to the internal solution. At zero applied voltage, the association rate constant for internally applied Ba2+ is ~50x higher than that for external Ba2+, while the dissociation rate does not depend on the side of application (
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Acknowledgements |
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We thank Joan Haab and Dorine Starace for helpful comments on the manuscript.
This work was supported by Chilean grants FONDECYT 197-0739 (R. Latorre), 198-1053 (C. Vergara), and Cátedra Presidencial and a group of Chilean companies (AFP Protection, CODELCO, Empresas CMPC, CGE, Gener S.A., Minera Escondida, Minera Collahuasi, NOVAGAS, Business Design Association, and XEROX Chile) and a grant from Human Frontiers in Science Program (to R. Latorre).
Submitted: January 20, 1999; Revised: June 26, 1999; Accepted: June 29, 1999.
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References |
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Almers, W., Armstrong, C.M. (1980) Survival of K+ permeability and gating currents in squid axons perfused with K+-free media. J. Gen. Physiol. 75:61-78[Abstract].
Alvarez, O., Villarroel, A., Eisenman, G. (1992) Calculation of ion currents from energy profiles and energy profiles from ion currents in a multibarrier, multisite, multioccupancy channel model. Methods Enzymol. 207:816-854[Medline].
Baukrowitz, T., Yellen, G. (1996) Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. Science. 271:653-656[Abstract].
Blatz, A.L., Magleby, K.L. (1984) Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle. J. Gen. Physiol. 84:1-23[Abstract].
Candia, S., Garcia, M.L., Latorre, R. (1992) Mode of action of iberiotoxin, a potent inhibitor of the large conductance Ca2+-activated K+ channel. Biophys. J. 63:583-590[Abstract].
Cecchi, X., Wolff, D., Alvarez, O., Latorre, R. (1987) Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle. Biophys. J. 52:707-716[Abstract].
Dang, T.X., McCleskey, E.W. (1998) Ion channel selectivity through stepwise changes in binding affinity. J. Gen. Physiol. 111:185-193
Díaz, F., Wallner, M., Stefani, E., Toro, L., Latorre, R. (1996) Interaction of internal Ba2+ with a cloned Ca2+-dependent K+ (hslo) channel from smooth muscle. J. Gen. Physiol. 107:399-407[Abstract].
Dietrich, B. (1985) Coordination chemistry of alkali and alkali-earth cations with macrocyclic ligands. J. Chem. Ed. 62:954-964.
Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., MacKinnon, R. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 280:69-77
Eisenmann, G., Latorre, R., Miller, C. (1986) Multi-ion conduction and selectivity in the high-conductance Ca++-activated K+ channel from skeletal muscle. Biophys. J. 50:1025-1034[Abstract].
Gómez-Lagunas, F. (1997) Shaker B K+ conductance in Na+ solutions lacking K+ ions: a remarkably stable non-conducting state produced by membrane depolarizations. J. Physiol. 449:3-15.
Heginbotham, L., MacKinnon, R. (1992) The aromatic binding site for tetraethylammonium ion on potassium channels. Neuron. 8:483-491[Medline].
Hodgkin, A.L., Keynes, R.D. (1955) The potassium permeability of a giant nerve fiber. J. Physiol. 128:61-88.
Jåger, H., Rauer, H., Nguyen, A.N., Aiyar, J., Chandy, K.G., Grissner, S. (1998) Regulation of mammalian Shaker-related K+ channels: evidence for non-conducting closed and non-conducting inactivated states. J. Physiol. 506:291-301
Kavanaugh, M.P., Varnum, M.D., Osborne, P.B., Christie, M.J., Busch, A.E., Adelman, J.P., North, R.A. (1991) Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels. J. Biol. Chem. 266:7583-7587
Kiss, L., Immke, D., Loturco, J., Korn, S.J. (1998) The interaction of Na+ and K+ in voltage-gated potassium channels: evidence for cation binding sites of different affinity. J. Gen. Physiol. 111:195-206
Kiss, L., Korn, S.J. (1998) Modulation of C-type inactivation by K+ at the potassium channel selectivity filter. Biophys. J. 74:1840-1849
Kiss, L., Lo Turco, J., Korn, S.J. (1999) Contribution of the selectivity filter to inactivation in potassium channels. Biophys. J. 76:253-263
Khodakhah, K., Melishchuck, A., Armstrong, C.M. (1997) Killing K+ channels with TEA+. Proc. Natl. Acad. Sci. USA. 94:13335-13338
Latorre, R., Vergara, C., Hidalgo, C. (1982) Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle. Proc. Natl. Acad. Sci. USA 79:805-809[Abstract].
Levy, D.I., Deutsch, C. (1996) Recovery from C-type inactivation is modulated by extracellular potassium. Biophys. J. 70:798-805[Abstract].
López-Barneo, J., Hoshi, T., Heinemann, S.F., Aldrich, R.W. (1993) Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels. 1:61-71[Medline].
Melishchuck, A., Loboda, A., Armstrong, C.M. (1998) Loss of Shaker K channel conductance in 0 K+ solutions: role of the voltage sensor. Biophys. J. 75:1828-1835
MacKinnon, R., Yellen, G. (1990) Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels. Science. 250:276-279[Medline].
Miller, C., Latorre, R., Reisin, I. (1987) Coupling of voltage-dependent gating and Ba2+ block in the high conductance Ca2+-activated K+ channel. J. Gen. Physiol. 90:427-449[Abstract].
Moczydlowski, E., Latorre, R. (1983) Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions. J. Gen. Physiol. 82:511-542[Abstract].
Neyton, J. (1996) A Ba2+ chelator suppresses long shut events in fully activated high-conductance Ca2+-dependent K+ channels. Biophys. J. 71:220-226[Abstract].
Neyton, J., Miller, C. (1988a) Potassium blocks barium permeation through a calcium-activated potassium channel. J. Gen. Physiol. 92:549-568[Abstract].
Neyton, J., Miller, C. (1988b) Discrete Ba2+ blockade as a probe of ion occupancy and pore structure in the high-conductance Ca2+ activated K+ channel. J. Gen. Physiol. 92:569-586[Abstract].
Pardo, L.A., Heinemann, S.H., Trelau, H., Ludewig, U., Lorra, C., Pongs, O., Stühmer, W. (1992) Extracellular K+ specifically modulates a rat brain K+ channel. Proc. Natl. Acad. Sci. USA. 89:2466-2470[Abstract].
Shen, K.-Z., Lagrutta, A., Davies, N.W., Standen, N.B., Adelman, J.P., North, R.A. (1994) Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation. Pflügers Arch. 426:440-445.
Sohma, Y., Harris, A., Wardle, C.J.C., Argent, B.E., Gray, M.A. (1996) Two barium binding sites on a maxi-K+ channel from human vas deferens epitheliala cells. Biophys. J. 70:1316-1325[Abstract].
Stampe, P., Begenisich, T. (1996) Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes. J. Gen. Physiol. 107:449-457[Abstract].
Vergara, C., Latorre, R. (1983) Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade. J. Gen. Physiol. 82:543-568[Abstract].
Vergara, C., Moczydlowski, E., Latorre, R. (1984) Conduction, blockade and gating in a Ca2+-activated K+ channel incorporated into planar lipid bilayers. Biophys. J. 45:73-76.
Villarroel, A., Alvarez, O., Oberhauser, A., Latorre, R. (1988) Probing a Ca2+-activated K+ channel with quaternary ammonium ions. Pflügers Arch. 413:118-126.
Yellen, G. (1984a) Ionic permeation and blockade in Ca-activated K channels of bovine chromaffin cells. J. Gen. Physiol. 84:157-186[Abstract].
Yellen, G. (1984b) Relief of Na+ block of Ca++-activated K+ channels by external cations. J. Gen. Physiol. 84:187-199[Abstract].