Correspondence to: Christopher Miller, Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454. Fax:(781) 736-2365 E-mail:cmiller{at}brandeis.edu.
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Abstract |
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Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K+ > Rb+, NH4+, Tl+ >> Cs+, Na+, Li+) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl+ > K+ > Rb+ > NH4+ >> Na+, Li+). Determination of reversal potentials with submillivolt accuracy shows that K+ is over 150-fold more permeant than Na+. Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 1001,000 mM. These properties are analogous to those seen in many eukaryotic K+ channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K+ channels.
Key Words: ion conductivity, selectivity
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INTRODUCTION |
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Investigations of K+ channel mechanisms have been marked by functional feast and structural famine. For nearly 50 yr, K+ channels in eukaryotic cells have been subjected to detailed electrophysiological analysis for two basic purposes: (1) to understand their varied biological roles in ion transport and membrane electrical behavior, and (2) to reveal their underlying molecular characteristics. The electrical properties of K+ channels have been unusually informative toward both of these ends, and indeed accumulated work from many labs at the purely functional level has led to a remarkably detailed molecular picture of K+ channels. The high resolution structure of KcsA, a prokaryotic K+ channel, now provides a direct structural rationale for the functional behaviors long studied in eukaryotic K+ channels (
Two features of eukaryotic K+ channels are considered universal: (1) high selectivity among monovalent cations and (2) multiple ion occupancy in the pore. These channels permit permeation by K+, Rb+, NH4+, and Tl+ (1,000-fold (
In this study, we examine ion-permeation properties of KcsA with a classical, high resolution electrophysiological approach. Purified KcsA protein was reconstituted into a chemically defined planar lipid bilayer system, and single-channel properties were observed under varying conditions of ion concentration, ion type, and voltage. We show that KcsA is readily permeable to K+ and its close analogues Rb+, NH4+, and Tl+, whereas permeability for Na+, Li+, and Cs+ is undetectable. The experiments show that in its ion-conduction properties KcsA behaves according to familiar principles long established in eukaryotic K+ channels.
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MATERIALS AND METHODS |
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Materials
Chemicals were of reagent grade or higher. High purity KCl (99.999%), NaCl (99.5%), LiCl (99%), CsCl (>99.5%), RbCl (99+%), TlNO3 (99.999%), Tl(I) acetate (99.99%), and NH4Cl (99.99%) were obtained from Sigma-Aldrich. Dodecylmaltoside was purchased from Calbiochem, and Chaps was purchased from Pierce Chemical Co. Lipids (Avanti Polar Lipids) were 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) and phophatidylglycerol (POPG) stored in sealed ampules at -80°C.
Solutions used for planar bilayer recording contained the desired salt and an appropriate anionic buffer at a concentration of 10 mM: HEPES for pH 7.0 and succinate for pH 4. Internal and external solutions were adjusted to pH 4.0 and pH 7, respectively, with calibrated solutions of hydroxides of the appropriate cations; this pH gradient is used to eliminate activity of channels inserted "backward" into the bilayer (
Purification and Reconstitution of KcsA
The KcsA construct, under control of a tetracycline promoter (model pASK-IBA2; Sigma-Genosys), was as described previously (25% lower than that of COOH-terminal construct. The overall shapes of the I-V curve and of the K+ conductance-concentration curve are identical for these two variants.
KcsA was expressed at 37°C in Escherichia coli (strain JM-83) and purified in 1 mM dodecylmaltoside as described previously (
Single-channel Recording and Criteria for Channel Analysis
The horizontal planar bilayer system (0.5 µL of lipid solution (7.5 mg/ml POPE + 2.5 mg/ml POPG in n-decane) to the hole and drying in air for 20 min. The chambers were then filled with solution, and bilayers were formed by "painting" the hole with a glass rod dipped in the lipid solution. Channels were inserted by adding
1 µl of KcsA-containing liposomes over the hole and painting a new bilayer. Typically, several channels were incorporated, but because of the channel's low open probability and long-lived closed intervals, it was straightforward to record stretches of data containing only single-channel openings. The recording system was oriented such that the upper chamber containing pH 7.0 solutions was equivalent to the extracellular side of the channel, and the lower chamber containing pH 4.0 solutions acted as the intracellular solution. Voltages and currents are reported according to electrophysiological convention, with the extracellular solution defined as zero voltage. Current was sampled at 1050 kHz and low-pass filtered at 210 kHz, depending on the requirements of the experiment. Most analysis was performed on raw data, but in certain cases, digital filtering (Gaussian, 0.251 kHz) was used to resolve low amplitude openings. Typically, single-channel current values report the mean of 30100 individual measurements taken from at least three separate bilayers. Zero-voltage conductances were calculated from the linear coefficients of fifth-order polynomial fits of the I-V curves. Under bi-ionic or mixed-ionic conditions, permeability ratios (with K+ as reference ion) were conventionally defined from the measured reversal potential Vr:
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(1) |
This definition is only useful for reporting permeability ratios, but carries no meaning beyond the raw reversal potential values.
A common hazard of single-channel analysis arises from the extreme sensitivity of the technique and the inherently anecdotal character of the raw recordings. Therefore it is imperative to establish criteria demonstrating that a given experimental record actually represents the channel protein intended for studyrather than denatured protein, peptide fragments, residual detergent, lipid components, or other minor contaminants, all of which produce channel-like activities (
Supplemental Material
Supplemental I-V curve data are available at http:www.jgp.org/cgi/content/full/118/3/303/DC1.
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RESULTS |
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Permeation by K+
Single-channel current traces for KcsA in symmetrical 100-mM K+ solutions are illustrated in Fig 1 A. The voltage- and pH-dependent gating of this channel is complicated (
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Fig 2 A shows representative single-channel openings for KcsA in the presence of different symmetrical concentrations of K+. Not only does the current amplitude increase with K+ concentration, but open times and open probability greatly decrease as K+ increases, an effect localized to the intracellular side of the membrane. Amplitude histograms (from data taken exclusively within single-channel bursts and therefore not reflective of overall open probability) show well-defined peaks devoid of "subconductances." A series of I-V curves at different concentrations of K+ is shown in Fig 2 B. Outward rectification decreases as K+ concentration is lowered, such that at 20 mM K+, the I-V curve is nearly symmetrical.
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KcsA conductance increases in a sublinear fashion with symmetrical K+ concentration over the range 51,600 mM, as shown in Fig 3 for both zero-voltage slope conductance and 200-mV chord conductance. Although the absolute values differ, the shapes of the conductance-concentration curves are identical for the two conductance measurements. The conductance increases with concentration in two stages, a low concentration "burst" phase (020 mM) followed by a much slower "creep" in conductance that fails to reach full saturation even at 1,600 mM K+. The curve shows that the lower limit for half-saturation of the conductance lies above 450 mM K+. As expected for multi-ion single-file channels, the conductance-concentration curve cannot be even crudely fit by a rectangular hyperbola (demanded of a simple single-ion conduction mechanism) or by a linear function (demanded of an independent, constant-field electrodiffusion mechanism).
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Conduction by K+ Analogues
To assess ion selectivity under symmetrical solution conditions, we first recorded KcsA channels in K+ solutions, and then perfused both sides of the bilayer with solutions of each of six different monovalent cations at 100 mM: Rb+, Tl+, NH4+, Li+, Na+, and Cs+. Single-channel fluctuations were detected for only the first three of these, in addition to K+ itself (Fig 4 A). The channels' gating characteristics are strikingly dependent on the conducting ion (e.g., note long open-times in Rb+ and NH4+), and open-channel amplitudes are well-defined in all cases. As with K+ (
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Conductance-concentration curves for all permeant ions are plotted in Fig 5, displayed for clarity on two different scales. As with K+, the curves have bimodal shapes, with a low concentration increase in conductance followed by a creep, which is far more prominent for K+ than for the other ions. For the K+ analogues, half-saturation concentrations are 90 mM for Rb+, 120 mM for NH4+, and 45 mM for Tl+, as if all three bind
510-fold more strongly than does K+.
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Selectivity under Bi-ionic Conditions
Ionic selectivity of KcsA was further assessed by observing channels under bi-ionic conditions, i.e., with K+ in the external solution and an equal concentration of test cation in the internal. Fig 6 displays traces in which voltage was increased linearly from -200 to 200 mV over 1 s; in these "voltage-ramp" experiments, 100 sweeps were accumulated in each set of ionic conditions, and a single representative sweep is highlighted in the figure. Reversal potentials determined from I-V curves measured at constant holding voltages are indicated by arrows. According to electrophysiological convention (Equation 1), we define permeability ratios from these measured reversal potentials: Tl+(3.2) > K+(1) > Rb+(0.8) > NH4+(0.2). Thus, all conductive ions (Fig 6 A) show substantial bi-ionic permeability, and under these ionic conditions the least conductive ion, Tl+, emerges as the most "permeant," a circumstance often associated with tightly binding permeating ions in both cation and anion channels (
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Because KcsA currents were not observed in symmetrical solutions of Li+, Na+, or Cs+, these ions were also tested in bi-ionic conditions (Fig 6 B). In these experiments, we placed K+ in the internal solution and the test cation in the external, since internal (but not external) Na+ is a strong voltage-dependent blocker of KcsA (
The absence of Na+ current in bi-ionic conditions provoked a more sensitive test of Na+ permeation. Here, in the presence of a K+ gradient (100 mM internal/20 mM external), we examined the degree to which addition of 100 mM external Na+ perturbs the K+-dominated reversal potential. The single-channel I-V curve is shown in Fig 7, along with raw records near the reversal potential (-30 mV in this experiment). Because of uncertain liquidjunction corrections, unstirred layer effects, and other imbalances, the relation of the measured reversal potential of the recording system to the absolute reversal potential across the planar bilayer is uncertain within 10 mV. To obtain a more accurate value, as required for our purposes here, we used valinomycin, an ideally K+-selective ionophore, to determine the true equilibrium potential for K+ in each experiment, as in previous submillivolt precision measurements of single-channel reversal potentials (
100-fold, and determined the reversal potential of the valinomycin-mediated current within 200 µV. As shown in Fig 7, this value is indistinguishable from the KcsA reversal potential. Considering that for valinomycin conductance PNa/PK
10-4 (
Vr, between the reversal potentials of valinomycin-mediated and KcsA-mediated currents is:
which, given the small measured value of Vr, simplifies to:
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Our value of Vr determined in four independent experiments (180 ± 250 µV) puts a very conservative upper limit on the Na+/K+ permeability ratio for KcsA of
0.006. This upper limit is consistent with measurements in certain eukaryotic K+ channels that offer opportunities for more precise selectivity determinations (e.g.,
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DISCUSSION |
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It is widely appreciated that ion selectivity of eukaryotic K+ channels is both biologically necessary and mechanistically informative. These proteins have evolved to pass K+ efficiently, and at rates 1,000-fold greater than for Na+, the physiologically relevant competitor cation. The structure of KcsA, despite its prokaryotic origin, provides a rich model illuminating the mechanism by which K+ channels achieve rapid permeation along with strong ionic selectivity. Under physiological ionic conditions, KcsA is occupied by up to three K+ ions constrained to move in single file (
In the ion conduction properties reported here, KcsA is reminiscent of eukaryotic K+ channels (Table 1). At physiological K+ concentrations, the conductance of KcsA falls solidly within the range of values measured for Kv, Kir, and BK-type channels. Moreover, ions known to permeate eukaryotic K+ channels also permeate KcsA with a similar selectivity sequence, and those known to be impermeant are excluded from this prokaryotic channel.
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Beyond this familiar behavior, several characteristics of KcsA are notable. First, this channel's gating is extraordinarily sensitive to the type and concentration of conducting ion. The classic "foot-in-the-door" mechanism, by which ion occupancy of the pore inhibits closing, is customarily invoked to explain coupling of ion permeation to gating in K+ channels (
A second noteworthy feature of KcsA is the common shape of the conductance-concentration curves for all four permeant ions. These curves show two distinct phases: a burst of conductance below 20 mM, and a subsequent creep as concentration increases up to 1 M and above, as has also been observed in mammalian Ca+-activated K+ channels (
Another point worth mentioning is our failure to detect Cs+ conduction through KcsA despite the well-known interactions of Cs+ with K+ selectivity filters. In macroscopic measurements, Cs+ permeation has been observed for eukaryotic K+ channels (
Our results conflict with suggestions that the high resolution KcsA structure is inadequate as a basis for understanding K+ permeation (
Our experiments lead to a simple conclusion: KcsA is a conventional K+ channel. All ion permeation properties common to eukaryotic K+ channels are recapitulated in KcsA: conduction by K+, Rb+, NH4+, and Tl+, exclusion of Li+, Na+, and Cs+, and complex variation of conductance with concentration, as expected for multi-ion occupancy in the pore (
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Footnotes |
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The online version of this article contains supplemental material.
The present address of L. Heginbotham is Department of Molecular Biophysics and Biochemistry, Yale University New Haven, CT 06520.
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Acknowledgements |
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We are grateful to Dr. C. Nimigean for criticisms on the manuscript, to Dr. Z. Lu for suggestions on the experiments, and Dr. R. Blaustein for unceasing mathematical consultations.
This study was supported by a National Institutes of Health grant GM31768 to C. Miller.
Submitted: 26 June 2001
Revised: 2 August 2001
Accepted: 3 August 2001
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