Polyamine blockade and binding energetics in the MthK potassium channel

Figures

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  Figure 1.. MthK channel blockade by SPD3+. (A) Mean normalized macroscopic MthK currents. Currents were obtained over a range of [SPD3+] (≤1,000 µM) using a voltage-ramp protocol and normalized to the current amplitude at −100 mV for the same bilayer with 0 SPD3+. (B) Fractional current versus [SPD3+] plotted at five representative voltages. Data were fitted with Hill equations (smooth curves) to estimate Kapp over a range of voltages (parameters are in Table S1). (C) Kapp versus voltage, fitted with Eq. 1 (solid blue line; Kapp(0) = 860 ± 20 µM, zδ = 0.67 ± 0.03 e0).

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  Figure 2.. MthK channel blockade by SPM4+. (A) Mean normalized macroscopic MthK currents. Currents were obtained over a range of [SPM4+] (≤100 µM) using a voltage-ramp protocol and normalized as described in Fig. 1. (B) Fractional current versus [SPM4+] plotted at five representative voltages. Data were fitted with Hill equations (smooth curves) to estimate Kapp over a range of voltages. (C) Kapp versus voltage, fitted with Eq. 1 (solid blue line; Kapp(0) = 77 ± 6 µM, zδ = 0.88 ± 0.05 e0).

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  (Scheme 1).

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  Figure 3.. SPD3+ and SPD4+ blockade described with Scheme 1. (A) Mean fractional MthK current over a range of [SPD3+], with Scheme 1 predictions superimposed (solid red curves). This model predicts that MthK currents approach 100% blockade with very strong depolarization, and thus provides a poor description of the data over the observed range of voltage and [SPD3+]. (B) Mean fractional MthK current over a range of [SPM4+], with Scheme 1 predictions superimposed. As with SPD3+, Scheme 1 predicts that MthK currents approach 100% blockade with very strong depolarization and is thus inconsistent with the data over the observed range of voltage and [SPM4+].

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  (Scheme 2).

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  Figure 4.. SPD3+ and SPM4+ blockade described with Scheme 2. (A) Fractional MthK current over a range of [SPD3+] with Scheme 2 predictions superimposed. Scheme 2 predicts that depolarization drives the channel toward a receptive open state (O’) with enhanced blocker affinity, accounting well for the experimental data. Fit parameters for individual experiments are provided in Table 3. (B) Fractional MthK current over a range of [SPM4+] with Scheme 2 predictions superimposed. Scheme 2 accounts for the major features of SPM4+ blockade over a 1,000-fold range of [SPM4+]. Fit parameters for individual experiments are provided in Table 4.

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  (Scheme 3).

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  (Scheme 4).

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  Figure S1.. SPD3+ blockade described with an alternative three-state model (Scheme 4). Mean fractional MthK current (I/I0) over a range of [SPD3+], with Scheme 4 predictions superimposed (solid red curves). (A) Scheme 4 (left), with equations describing fractional current (right). (B) Mean fractional MthK current (I/I0) over a range of [SPD3+] as indicated, with Scheme 4 predictions superimposed (solid red curves). The best fit with this model (χ2 = 35.4) predicts that MthK currents will ultimately reach 100% blockade with very strong depolarization, although the apparent voltage dependence of blockade is less steep than that of Scheme 1. (C) An alternative fit representing a local minimum (χ2 = 97.6) predicts that MthK currents can increase with very strong depolarization.

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  Figure 5.. Structure and dimensions of the MthK pore. Two of the four pore-forming subunits from the crystal structure of the MthK pore (PDB accession no. 3LDC) are shown side by side, to illustrate the transmembrane ion conduction pathway. Distances represented in the z-axis scale in subsequent computational experiments (corresponding to Figs. 5, 6, 7, 8, and 9) are shown at the left. K+ ions in the SF are represented by purple spheres, and selected main chain and side chain atoms are shown as indicated (red, oxygen; blue, nitrogen; gray, carbon).

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  Figure 6.. Correlated movement of K+ ions and polyamine in the MthK pore. (A) Simulation trajectory showing the positions of the NL of SPD3+ (blue) and K+ ions in the SF (shades of purple), relative to oxygen atoms of SF residues (red, as indicated to the right) with 0 mV transmembrane voltage. (B) Simulation trajectory for SPD3+ with 500 mV transmembrane voltage. (C) Simulation trajectory for SPM4+ with 0 mV transmembrane voltage. (D) Simulation trajectory for SPM4+ with 500 mV transmembrane voltage. These data illustrate that movement of NL to positions within hydrogen-bonding distance of T59 oxygen atoms depends on outward displacement of K+ ions.

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  Figure S2.. Time series of RMS fluctuations for SPD3+ and SPM4+ during simulations at 0 and 500 mV. (A–D) For each condition, the RMS fluctuations varied by <1 Å over the course of the simulation.