Correspondence to: Roderick MacKinnon, Howard Hughes Medical Institute, Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Fax:(212) 327-7289 E-mail:mackinn{at}rockvax.rockefeller.edu.
Released online: 14 February 2000
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Abstract |
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X-ray diffraction data were collected from frozen crystals (100°K) of the KcsA K+ channel equilibrated with solutions containing barium chloride. Difference electron density maps (Fbarium - Fnative, 5.0 Å resolution) show that Ba2+ resides at a single location within the selectivity filter. The Ba2+ blocking site corresponds to the internal aspect (adjacent to the central cavity) of the "inner ion" position where an alkali metal cation is found in the absence of the blocking Ba2+ ion. The location of Ba2+ with respect to Rb+ ions in the pore is in good agreement with the findings on the functional interaction of Ba2+ with K+ (and Rb+) in Ca2+-activated K+ channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.
Key Words: potassium channel, barium, ion channel, ion selectivity, ion conduction
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INTRODUCTION |
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To a K+ channel, the alkaline earth metal Ba2+ is like a divalent K+ ion. Barium's size allows it to fit into the selectivity filter, but its charge apparently causes it to bind too tightly. When it is bound in the filter, Ba2+ prevents the rapid flow of K+.
The K+ channel inhibitory properties of Ba2+ have been analyzed in detail (
The membrane voltage dependence of lock-in and enhancement effects exerted by K+ on Ba2+ provided an estimate of the electrical distance that K+ must travel through the channel to reach a specific site. Ions in the pore presumably move in a somewhat concerted fashion, making the electrical distance a complicated quantity without a simple structural interpretation. Nevertheless, electrical distances for the K+ sites were informative. The external lock-in and enhancement sites appeared to reside ~15 and 50%, respectively, of the way across the membrane electric potential difference relative to the outside, and the internal lock-in site appeared to reside ~70% of the way (i.e., 30% of the electrical distance from the internal solution). These experiments supported the picture in Fig 1, right. Following the
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X-ray structure determination of the KcsA K+ channel showed that it contains multiple permeant ions in its pore (
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METHODS |
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The KcsA K+ channel was purified and crystallized as described (, where Fbarium and Fnative are the observed structure factors for the Ba2+-containing and native crystals, respectively, and
is the experimental (MIR, solvent-flattened, averaged) phase. Due to crystal damage during the soaking procedure, data were useful only to 5.0-Å resolution.
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RESULTS |
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Crystals of the KcsA K+ channel were equilibrated in solutions containing 100 mM BaCl2. Ionic strength was kept constant by replacing CaCl2 present in the crystal growth solution with BaCl2. At the same time, 150 mM KCl was replaced by an equal amount of NaCl. It was necessary to lower the K+ concentration to observe Ba2+ in the pore. We assume that K+, at high concentrations, displaces Ba2+ from the pore.
The location of Ba2+ in the pore is well defined in a difference Fourier map (Fbarium - Fnative) calculated at 5.0-Å resolution (Fig 1, left and center, green mesh). Electron density for Ba2+ is compared with that for Rb+ ions determined in a separate experiment (red mesh). The Rb+-difference Fourier map, calculated at 3.8-Å resolution, shows two ions in the selectivity filter and one in the cavity at the membrane center, below the selectivity filter (
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DISCUSSION |
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The consistency of structural data on the KcsA K+ channel with functional data on Ca2+-activated K+ channels is quite remarkable. The external lock-in effect implied the existence of a first ion binding site external to Ba2+. This site had to be highly selective for permeant alkali metal cations and very near the extracellular solution, because external lock-in is a weakly voltage-dependent process. The outer ion in the KcsA selectivity filter fulfills these criteria. The external enhancement effect implied a second site, also external to Ba2+ and highly selective for permeant ions. The stronger voltage dependence of its apparent occupancy suggested that the enhancement site is deeper in, closer to the Ba2+ ion. The effective Kd for achieving the enhancement configuration of two K+ ions external to Ba2+ is nearly 500 mM, indicating that it is a relatively unstable configuration. In the structure, the outermost peak of the inner ion is too close to Ba2+ to be the precise site of the enhancement ion. However, the structure of the selectivity filter provides several potential ion binding sites external to Ba2+.
The internal lock-in effect required a site internal to Ba2+ that, in contrast to the external sites, exhibits little ion selectivity. The cavity ion in the crystal structure, being fully hydrated and not in direct contact with protein functional groups, fulfills the requirements of the internal lock-in site completely. Even the relatively weak voltage dependence of internal lock-in is consistent with the idea that a large fraction of the membrane voltage falls across the narrow selectivity filter (
Given the high affinities of the external and internal lock-in sites, it seems likely that they would contain a K+ most of the time at physiological ion concentrations. Therefore, the picture of the pore with three ions (two in the selectivity filter and one in the cavity), derived from the crystal structure, is probably not very different from a snapshot of what the conducting channel looks like in the membrane. The external enhancement effect is interesting because it suggests that a fourth ion enters the pore (that is, a third ion in the selectivity filter) to push the queue along (and Ba2+ out). The affinity for the enhancement site is low, and corresponds reasonably well to the concentration range over which the conductance increases as K+ concentration is raised (0.31.0 M, depending on the K+ channel). This enhancement configuration would not have been abundant in the crystal bathed in only 150 mM KCl (or RbCl). Further experiments are needed to test this possibility.
The idea that K+ channels have multiple ions in their pore is as old as the ion channel field itself (
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Acknowledgements |
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We thank Joao Morais Cabral for helpful discussions and Christopher Miller and Jacques Neyton for manuscript criticism.
This work was supported by National Institutes of Health grant GM47400. R. MacKinnon is an Investigator in the Howard Hughes Medical Institute. Y. Jiang is a Postdoctoral Associate in the Howard Hughes Medical Institute.
Submitted: 3 December 1999
Revised: 19 January 2000
Accepted: 20 January 2000
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References |
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