Correspondence to: Christopher Miller, Department of Biochemistry, Brandeis University, 415 South Street, HHMI, Waltham, MA 02254-9110. Fax: 781-736-2365; E-mail:cmiller{at}brandeis.edu.
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Abstract |
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Basic electrophysiological properties of the KcsA K+ channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na+ to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.
Key Words: potassium channel, permeation, gating, block
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INTRODUCTION |
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The high-resolution structure of the KcsA K+ channel has invigorated current approaches to the molecular foundations of cellular electrical excitability (
As a prelude to a full ion selectivity study of KcsA, we sought to establish a planar lipid bilayer system in which single purified KcsA channels may be recorded accurately and to survey several basic pore properties of the channel. Single KcsA channels can be observed at 5 kHz bandwidth in a low-noise planar bilayer system. We document functionally asymmetric characteristics of KcsA and use several of these to show that protons gate this channel from the cytoplasmic, not the external, side of the membrane.
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MATERIALS AND METHODS |
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Materials
General chemicals were of reagent grade or higher. High-purity (>99.997%) KCl was obtained from Alfa Inorganics. [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET, Br salt)1 and 2-(sulfonatoethyl)methanethiosulfonate (MTSES, Na salt) were obtained from Anatrace. Dodecylmaltoside was from Calbiochem Corp. and CHAPS from Pierce Chemical Co. Lipids (Avanti Polar Lipids) were 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) and phosphatidylglycerol (POPG), stored in sealed ampules at -80°C. Charybdotoxin (CTX) was expressed in Escherichia coli and purified as described (
Two slightly different constructs of KcsA were used. Most experiments employed a synthetic gene coding for the natural KcsA polypeptide sequence with a hexahistidine tag added to the NH2 terminus. This was derived by truncating the previously described "SliK" construct (
Solutions used for planar bilayer recording are coded according to the convention: nKm, where n and m are numbers denoting the concentration (mM) of K+ ion, and the pH, respectively. The solutions also contained an appropriate anionic buffer. Thus, solution 200K7 consists of 195 mM KCl/5 mM KOH/10 mM HEPES, adjusted to pH 7.0 with HCl, and 20K4 consists of 15 mM KCl/5 mM KOH/10 mM succinic acid, adjusted to pH 4.0 with HCl.
Purification and reconstitution of KcsA.
KcsA was expressed in E. coli and purified on Ni2+ affinity columns as described (
Single-channel recording in planar lipid bilayers.
Single-channel recordings of KcsA were performed in a horizontal planar lipid bilayer, with the following improvements over the system's previous description (
After sealing the partition between cis and trans chambers with a worm of Vaseline or silicone grease, the hole was pretreated with ~0.5 µl of phospholipid solution (15 mg/ml POPE, 5 mg/ml POPG in n-decane) and was allowed to air dry for ~30 min. The trans chamber was then filled with 20K4 solution and the cis with 200K7 or 100K7 solution. A bilayer was spread on the hole with a glass or plastic rod wetted with phospholipid solution kept at room temperature. Capacitances were typically 2540 pF, and resistances were in the 1 T range.
For channel insertion, a day's supply of reconstituted vesicles was prepared by thawing an aliquot, transferring the suspension to a glass test tube, and sonicating in a cylindrical bath sonicator for ~10 s. Vesicles were kept at room temperature throughout the day's use, and then discarded. A newly formed bilayer was ruptured by physical violence, ~1 µl of reconstituted vesicles were added to the cis solution directly above the open hole and a new bilayer was immediately spread. Current was monitored at 100200-mV holding voltages, and if channels failed to appear within 5 min, the bilayer was ruptured and the procedure was repeated. Typically, channels were observed in ~50% of such attempts. After channel insertion, recording conditions were established by perfusion with desired solutions or by dilution of stock solutions into the bilayer chamber with mixing. In all experiments reported here, 100 mM K+ was present on both sides of the membrane. All data reported and all standard errors displayed are based on three to seven independent experiments.
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RESULTS |
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An essential requirement for structurefunction analysis of any ion channel is a firm knowledge of its orientation in the membrane under study. Most ion channels are studied in their native cellular membranes, where orientation is obvious. However, the KcsA channel is best investigated as a purified protein reconstituted in biochemically defined membranes. In such a system, transmembrane orientation of the channel is not assured and must be empirically established. This study proceeds towards a single goal: to assign specific functions to defined sides of the KcsA protein. We approach this goal in two steps. First, we show that several of the channel's fundamental properties of gating, permeation, and blockade are asymmetric with respect to the "cis" and "trans" sides of the reconstituted membrane. Then, the channel's absolute orientation is established by assigning specific residues in the KcsA structure to the corresponding sides of the bilayer.
The planar bilayer system consists of two experimentally accessible aqueous phases: the cis solution to which KcsA-reconstituted liposomes are added, and the opposing trans solution. According to the electrical polarity convention used here, the cis chamber is the zero-voltage reference. Single KcsA channels were inserted into planar lipid bilayers under asymmetric salt conditions, and both sides of the bilayer were then flushed with the desired recording solutions.
Asymmetry of Gating by Voltage and pH
Like other two-transmembrane helix K+ channels, KcsA lacks an S4 voltage sensor. However, its gating shows a definite, though weak, voltage dependence. Figure 1 compares KcsA channel activity at opposite voltage polarities in a multiple-channel membrane. At high positive voltage (175 mV), channel activity is marked by frequent openings throughout the duration of the record, a pattern that changes dramatically when voltage is reversed in polarity. At high negative voltage (-175 mV), the channel open probability is much lower. Most channels (>80%) insert into the planar bilayer with this orientation; a minority show reversed orientation, with frequent openings at negative voltages. Thus, KcsA channels preferentially orient in the reconstituted membrane, but the results do not even hint at their absolute orientation; i.e., which side of the bilayer is equivalent to the cytoplasmic or external face of the channel protein.
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KcsA is a proton-activated channel. As described by
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The majority of channels incorporate into the membrane with sensitivity to trans pH, only a minority appearing with reversed sensitivity. (The channels sensitive to cis pH also show reversed voltage sensitivity.) We exploited this asymmetric pH sensitivity to ensure that all channels observed have a single orientation; all subsequent experiments were performed with trans pH 4 and cis pH 7, a maneuver that enforces a perfectly oriented set of active channels by silencing any channel inserting in the "minority" direction.
Asymmetry of Ion Permeation
In symmetric 100-mM K+ solutions, KcsA shows open-channel rectification, as seen in the raw recordings and the open-channel currentvoltage (IV) curve of Figure 3. Channels are well defined in amplitude and do not display the substate behavior reported previously (
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Asymmetry of Tetraethylammonium Block
Many eukaryotic K+ channels are reversibly blocked by tetraethylammonium (TEA), which can bind to two distinct sites located near the two ends of the narrow selectivity filter (
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Na+ Block: A Hint of Absolute Orientation
A biological imperative of K+ channels is to prohibit permeation by Na+. However, far from being inert to K+ channels, Na+ is known to interact with them in a sided fashion, blocking K+ currents in nerve and muscle membranes exclusively from the intracellular side (
This Na+ blocking behavior seen in eukaryotic K+ channels is echoed in KcsA (Figure 5). Addition of 30 mM Na+ to the cis side has no effect on the open-channel IV relation in symmetrical K+. In contrast, 10 mM Na+ added to the trans side reduces the channel amplitude with voltage dependence strong enough to produce a negative conductance, as seen originally in squid axon K+ channels (
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The Absolute Orientation of KcsA
This conclusion is uncertain, however, because of its reliance on an analogy to only a few carefully studied K+ channels. We therefore scrutinized the absolute sidedness of KcsA by combining measurements of the functional influences of specific residues with the channel's known structure (Figure 6).
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Location of the external TEA binding site.
It is well established in eukaryotic K+ channels that the affinity of external TEA block is enhanced by an aromatic residue at the position equivalent to Y82 in KcsA (
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Covalent modification of Y82C.
The introduction of cysteine at position 82 offers an independent means of assessing the channel's orientation. Devoid of cysteine, KcsA is an ideal target for analysis by site-specific modification. In eukaryotic K+ channels, ion permeation is affected by substitutions at this position (
The Y82C substitution preserves the basic KcsA properties of trans pH sensitivity and voltage-dependent gating, but it alters the shape of the open-channel IV curve, nearly eliminating the rectification normally observed (Figure 8). The IV curve was unchanged immediately following addition of 70 µM MTSET to the cis compartment, but ~3 min of exposure to the reagent led to a distinct asymmetry. This rectification resulted from a decrease in cis-to-trans current while leaving current in the reverse direction unchanged, as expected from an electrostatic influence of a positive charge near the channel's cis entryway. This effect persisted after removal of the MTSET by perfusion, and it was not reversed by several minutes of exposure to 5 mM dithiothreitol. Since MTSET is membrane impermeant (
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The modification of Y82C provoked a supplementary experiment. We have already seen that mutations at Y82 affect external TEA block, and we anticipate from experiments on the equivalent position in Shaker channels (
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Charybdotoxin sensitivity of KcsA.
Scorpion venom peptides of the charybdotoxin family block eukaryotic K+ channels by binding to a receptor site in the outer vestibule, thereby occluding the conduction pathway (
Figure 10 demonstrates block of single KcsA-Tx channels by CTX added to the cis side. As expected (
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Covalent modification of Q119C. The above experiments all involved manipulations of residues exposed to the cis solution. For completeness, it was desirable to test a residue exposed on the opposite side of the membrane. We substituted cysteine at Q119 and monitored changes in channel conductance in response to covalent modification. While the experiment is identical in motivation to those with Y82C above, the low pH of the trans solution requires two special considerations. First, the reaction rate of cysteine with thiosulfonate reagents is negligible at pH 4, and so MTSES was applied in a pH 7 solution. Since under these conditions the channels were closed, after several minutes of exposure to the reagent, the trans chamber was returned to pH 4 to gauge the effect of the modification. Second, two criteria guided the selection of a target residue: (a) susceptibility to closed-state modification by MTSES, and (b) proximity to the conduction pathway, maximizing the likelihood of an electrostatic influence on conduction.
On the basis of these requirements, we selected Q119 as the target residue. This residue is located in the COOH-terminal domain close to the cytoplasmic opening of the pore (Figure 6). The equivalent residue in the Shaker K+ channel, H486, reacts with MTS reagents in both open and closed states (
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DISCUSSION |
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These experiments exploit a fully oriented reconstituted system in which single KcsA channels, purified after high-level expression in E. coli, can be studied electrophysiologically. We have taken pains to document functional asymmetries in KcsA to assign absolute sidedness to the system. Eight separate asymmetries were examined: voltage-dependent gating, proton activation, open-channel rectification, block by Na+, TEA, and CTX, and covalent modification of channels with cysteines substituted on either side of the membrane. Three independent lines of evidence establish the channel's orientation in the bilayer. The cis solution bathes the extracellular face of the channel protein containing the CTX receptor and the aromatic TEA blocking site. The trans solution bathes the intracellular face containing Q119.
These assignments unambiguously demonstrate that the protonation sites linked to KcsA gating face the intracellular solution. This orientation makes sense from a statistical standpoint: of the 56 carboxylate groups in KcsA (16 asp, 36 glu, 4 COOH termini), 44 are located at intracellular positions; all 44 histidine residues in the His-tagged protein are also intracellular. Previously,
Our results are perplexing from a biological perspective. Indeed, they suggest strongly that this channel is not gated by low pH in its native membrane, since Streptomyces, like most bacteria, tightly regulates cytoplasmic pH near neutrality. If the physiological role of KcsA is in fact to gate the K+ conductance of the bacterial membrane, then it is likely that some factor other than pH, perhaps an as yet unrecognized partner protein, provides control of gating. Questions of this kind must remain unresolved until the physiological purposes of prokaryotic K+ channels are clarified.
In the course of assessing KcsA orientation, we have also shown that pore characteristics of this prokaryotic channel are remarkably similar to those of many well-studied eukaryotic K+ channels. The functional familiarity of KcsA is not surprising, given how closely the structural features proposed for eukaryotic K+ channels match those actually observed with KcsA (
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Footnotes |
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Dr. Heginbotham's present address is Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520.
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Acknowledgements |
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We are grateful to Drs. Rob Blaustein, Merritt Maduke, and Irwin Levitan for suggestions on the manuscript.
Supported by National Institutes of Health grant GM-31768 and training grant GM-07596 (M. LeMasurier).
Submitted: 12 July 1999
Revised: 9 August 1999
Accepted: 10 August 1999
1used in this paper: CTX, charybdotoxin; IV, currentvoltage; MTSES, 2-(sulfonatoethyl)methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; POPE, 1-palmitoyl-2-oleoyl phosphatidylethanolamine; POPG, 1-palmitoyl-2-oleoyl phosphatidylglycerol; TEA, tetraethylammonium
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