a, Open channel I–V at 100mM K⁺ (black circles), with 10mM (red), and 20mM (blue) intracellular Li⁺. Data are the mean of at least 3 measurements with S.E.M. error bars. Black circles are fit with a sigmoidal curve (black line). Red and blue lines represent fits with Eq.1 26 in the Supplementary Discussion and the following parameters (± s.e. from fit) for 10 and 20mM Li⁺, respectively: K_B^ap(0)=268.4±33.8 and 345.4±45.9 mM, z =0.40±0.02. b, Apparent Li⁺ blocking affinity over a range of K⁺ concentrations. Data points are from fits as in a. Each symbol is mean ± S.E.M. of at least three different experiments. The red line represents a linear fit to equation Eq. S3, with a slope of 2.3. For comparison, a black dashed line (slope of 5.2 from ref. 26) is shown, for Na⁺ block. c, Voltage dependence of Li⁺ block is constant over a range of K⁺ concentrations. Red circles represent the mean ± S.E.M. from at least six experiments at each K⁺ concentration (from fits as in a). For comparison, the black dashed line shows the increase in voltage dependence for Na⁺ block26.

a, MD system consisting of KcsA (yellow) embedded in a DPPC lipid bilayer (gray tails) and 150mM KCl solution (water - red/white sticks, K⁺ and Cl⁻ are green and gray spheres, respectively). b, Probability density of ions in the cavity obtained from corresponding free energy profiles such as in Fig. 4c. c, Free energy profile across the S4–S3 region from the 8-carbonyl coordinated “cage” to the 4-carbonyl coordinated “plane” obtained from analysis of FEP simulations. The insets illustrate the free energy minimum locations in the S4 region for K⁺, Na⁺ and Li⁺ ions (red, green, and blue circles respectively). Because each free energy profile is determined only to within a constant, the curves for Na⁺ and Li⁺ have been shifted, relative to K⁺, based on relative free energies in the S4 cage, given in Supplementary Table 2. An almost identical result was obtained when based on relative free energies in the S4 plane (not shown). The RMSD errors in the curves are 0.8, 0.4 and 1.0 kcal mol⁻¹ for K⁺, Na⁺ and Li⁺, respectively (explained in the Supplementary Methods). The z-coordinate (x-axes in Fig. 4 and 6) maps positions along the pore as shown in Fig. 1a and 5e and is relative to the center of mass of the selectivity filter.

a, Scheme of possible multi-ion permeation configurations where two K⁺ ions are inside the filter (red) and a K⁺, Na⁺ or Li⁺ ion attempts to enter from the cavity (grey). Path I describes the cavity ion (K⁺, in this case) entering the filter with two K⁺ ions in S1/S3, followed by the movement of the two K⁺ ions to S0/S2 depicted in Path I′. Path II describes two K⁺ ions in the filter moving from S1/S3 to S0/S2, and subsequently allowing the cavity ion to enter the filter, depicted in Path II′ (we note that for paths II and II′ the grey ion can bind either in the cage, as pictured, or in the plane). Path III describes the cavity ion entering the filter in a concerted motion with the K⁺ ions moving from S1/S3 to S0/S2. b, Free energy profiles along Path I for K⁺, Na⁺, and Li⁺ (red, green, and blue respectively in b–d) where the zero has been set arbitrarily at z=−12 Å in the cavity, independently for each ion (one could alternatively add the small sub-kcal/mol offsets from Supplementary Table 2 to enforce free energies relative to bulk solution). c, Free energy profiles along Paths II (solid lines) and III (dashed lines) where we arbitrarily set the zero at the minimum corresponding to a S1/S3 configuration. d, Free energy profile along Path II′, where the zero was set at z=−12 Å. The z coordinate in b and d is the position of the ion along the pore (see Fig. 1a and 5e). The z₁₂ coordinate in c is the position of the center of mass of the two K⁺ ions relative to the filter. RMSD errors in these free energy profiles range from 0.4 to 1.6 kcal mol⁻¹ (see Supplementary Methods and Supplementary Fig. 4).

a, Two opposing KcsA subunits (ribbon), with oxygens (red stick) shown in the selectivity filter. The K⁺ binding sites (purple) and their z-coordinate relative to the center of mass of the filter atoms (defined by the backbone atoms of residues 75–78, in Å) are indicated. b, Single-channel KcsA recordings in 100mM symmetric K⁺ at indicated voltages, before and after addition of 5mM intracellular Li⁺ and Na⁺, as indicated in the figure.

Decrease in burst durations as a function of Na⁺ (a) and Li⁺ (b) concentration. Decrease in mean open time as a function of Na⁺ (c) and Li⁺ (d) concentrations. Each plot represents data from a single bilayer perfused to all indicated blocker concentrations. The data in a and c, and the data in b and d, are from the same datasets, respectively. Different colors indicate different voltages: +50 mV (black), +100 mV (red), and +200 mV (blue). Solid lines in a and b represent global fits of the data with Eq.1, where k_Na+(0)=0.11 M⁻¹s⁻¹, k_Li+(0)=0.07 M⁻¹s⁻¹, z_Na+= z_Li+=0.5. z_Li+ was constrained to be equal to z_Na+ due to the difficulty in fitting the Li⁺ data with a unique set of parameters. Solid lines in c and d have no theoretical significance. e, Representative closed dwell time distributions from control and with intracellular blocker conditions indicated from data at +200 mV. The dashed lines mark the critical time used (15 msec) to separate intraburst from interburst closed times. The lines are fits with sums of two exponential components with the following parameters: control (τ₁=0.84±0.08, τ₂=1714±3.02, A₁=0.97±0.06, A₂=0.03±0.05), Li (τ₁=1.3±0.11, τ ₂=512±0.7, A₁=0.87±0.07, A₂=0.13±0.07), and Na⁺ (τ₁=0.75±0.23, τ ₂=2177±0.78, A₁=0.81±0.13, A₂=0.19±0.1). All experiments were repeated at least 3 times with similar outcomes. The data in this entire figure were not averaged due to substantial differences between individual bilayers, as previously reported34.

a, Composite omit electron density maps contoured to 1.25σ for KcsA in 150 mM Li⁺ (NoK-Li⁺, PDBID:3GB7), b, KcsA in 150mM Li⁺ and 3mM K⁺ (LowK-Li⁺, PDBID:3IGA), c, KcsA in low K⁺ (PDBID:1k4d7), and d, KcsA in Na⁺ (NoK-Na⁺, PDBID:2itc8). Waters (red), K⁺ (purple), Na⁺ (blue), and Li⁺ (green) e, Root mean squared (RMS) deviations of pair-wise comparisons of C-α atoms between NoK-Li⁺ and LowK-Li⁺ (blue), Low-K⁺ (red, PDBID:1k4d7) or High-K⁺ (grey, PDBID:1k4c7). The black bar indicates the selectivity filter. The RMS deviation of the NoK-Li⁺ structure to the Low-K⁺ structure excluding residues 75–80 is 0.22Å, and of residues 75–80 alone is 0.56Å. The RMS deviation of the NoK-Li⁺ structure C-α atoms within the filter decreases to 0.19Å when compared to the conductive form of the filter in High-K⁺. f, Overlay of the backbone residues of NoK-Li⁺ (yellow) with Low-K⁺ (red), high-K⁺ (grey), and LowK-Li⁺ (blue). g, The S3 and S4 sites of NoK-Li⁺ showing coordination (numbers in Å) of Li⁺ ion and water molecules within the filter. Waters (red) and Li⁺ (green).