Mechanism for Variable Selectivity and Conductance in Mutated NaK

Sep 20, 2012 - Mechanism for Variable Selectivity and Conductance in Mutated NaK. Channels. Rong Shen and Wanlin Guo*. Institute of Nano Science, Stat...
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Mechanism for Variable Selectivity and Conductance in Mutated NaK Channels Rong Shen and Wanlin Guo* Institute of Nano Science, State Key Laboratory of Mechanics and Control for Mechanical Structures, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, China S Supporting Information *

ABSTRACT: Na+ conduction has been demonstrated in a few K+ channels and has been widely used to characterize the physiological selectivity and C-type inactivation in K+ channels. By using molecular dynamics simulations and free-energy calculations, we found that K+ and Na+ have distinct preferable binding configurations in the conductive filter of two highly K+ selective channels, which are mutated from the nonselective NaK channel and can conduct Na+ upon removal of K+. Disruption of a conserved hydrogen bond interaction between residues in the filter and the pore helices can significantly decrease the free-energy differences and barriers between the K+ binding configurations, whereas it has little effect on the free-energy landscape for Na+. We propose that the enhancement of the fluctuation of the filter structure decreases the affinity and conducting barrier of K+ and therefore the ability of K+ to block Na+ currents, predominantly responsible for the reduced K+ selectivity. SECTION: Biophysical Chemistry and Biomolecules he selectivity filter of K+ channels is the central structural element essential for catalyzing rapid and selective conduction of K+ across cell membranes.1 Despite having the highly conserved signature sequence of GYG,2,3 K+ channels show varying selectivity and structural stability of the filter, mainly attributed to the different protein interactions surrounding the filter.4−8 For instance, in the absence of K+, some naturally existing9−15 and mutated16−18 K+ channels can display a significant Na+ flux, which is blocked by the adding of low concentrations of K+, whereas others do not conduct Na+ even upon removal of K+.18,19 The high-resolution structure and molecular dynamics (MD) simulations of a prokaryotic K+ channel, KcsA, revealed four K+ binding cage sites (S1−S4) in the filter (Figure 1), consisting of two planes of four oxygen atoms, as well as two additional K+ binding sites at the extracellular entrance to the filter, S0 and Sext.20,21 Under physiological conditions, two K+ ions are proposed to reside in the filter, predominantly at sites S1 and S3 or at sites S2 and S4.20,22,23,33 In contrast, Na+ was found to bind at planar sites in the filter of K+ channels (S01−S34), made up of a single plane of four oxygen atoms (Figure 1).24−29 However, little is known about the multi-ion configurations and conduction mechanism of Na+ in K+ channels. By double mutations in the filter region, Jiang and colleagues recently converted the nonselective NaK channel to two highly K+ selective channels NaK2K (NaK_D66Y/N68D) and NaK2K_Y66F (NaK_D66F/N68D).30−32 The permeability ratio (PK/PNa) of NaK2K_Y66F is 3-fold smaller than that of NaK2K, despite their virtually identical filter structure (Figure 1C and D).32 In addition, NaK2K can maintain a conductive filter and conduct Na+ in the absence of K+, as was observed for

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© 2012 American Chemical Society

Figure 1. Selectivity filter structures of (A) KcsA, (B) NaK, (C) NaK2K, and (D) NaK2K_Y66F. Hydrogen bond interactions surrounding the filter are highlighted by red lines. K+ (S0 to S4) and Na+ (S01 to S34) binding sites are labeled from the extracellular to intracellular side.

the MthK K+ channel,14 albeit with an approximately 12-fold lower conductance than K+ at 150 mM.32 Therefore, it is of considerable interest for us to use the two channels as model systems to investigate the mechanism of Na+ permeation and Received: August 21, 2012 Accepted: September 20, 2012 Published: September 20, 2012 2887

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Figure 2. PMFs as a function of the z coordinates of the two K+ (top) and Na+ (bottom) ions in the filter of NaK2K. Contours are drawn at every 1 kcal/mol. The minimum-energy paths connecting local energy minima are represented as white lines. Snapshots taken from the umbrella sampling simulations are used to illustrate the configurations of some specific energy minima. K+ and Na+ ions are shown as yellow and orange spheres, respectively.

selectivity in K+ channels and characterize the effect of protein interactions surrounding the filter on the permeability ratio. We first performed MD simulations of atomic systems of NaK2K and NaK2K_Y66F to explore the binding configurations of K+ and Na+ in the filter. For each system, a total of five initial configurations were considered here with ions (K+ or Na+) being positioned at sites S0 and S2 (C02), sites S1 and S3 (C13), sites S1 and S4 (C14), sites S2 and S4 (C24), and sites S0, S2, and S4 (C024), which are proposed to be the preference configurations in K+ channels.20,22,33−36 In NaK2K, the K+ ions remained in their initial sites in the 10 ns simulations of C02, C13, C24, and C024, while a concerted ion transition occurred in the simulation of C14, moving from sites S1 and S4 to sites S2 and S5 (beneath the lower edge of S4) within 300 ps, respectively, and residing there for the rest of the simulation (Figure S1, Supporting Information). In contrast, all of the Na+ ions left their initial cage sites for an adjacent planar site. In the simulation of C02, the Na+ ions had moved to sites S01 and S23 in the equilibrium stage and remained there stably. However, the Na+ ions in the other four simulations all finally resided in sites S12 and S34. It should be noted that in the simulation of C024, the outermost Na+ moved to S01 initially and then hopped to the extracellular solution about 5 ns later because the Na+ ion in S4 reached S34. The MD simulation results of Na2K_Y66F are similar to that of NaK2K, except in the K+ simulations of C13 and C024. In C13, the K+ ions resided in sites S1 and S3 for about 2 ns and then moved to sites S2 and S4, respectively, while in C024, the outermost K+ ion resided in S0 for about 6 ns and then moved up to the extracellular solution as the outermost Na+ ion in the C024 configuration (Figure S2, Supporting Information). The MD simulations show that K+ and Na+ ions have distinct binding configurations in the filter of NaK2K and NaK2-

K_Y66F, and the presence of the hydrogen bond interaction between Tyr66 (in the filter) and Thr60 (in the pore helix) reduces the fluctuation of the filter structure of NaK2K (Figure S3, Supporting Information). To get further insights into the mechanism for the difference in selectivity and conduction in the two channels, free-energy profiles for the translocation of two K+ or Na+ ions in the filter were calculated using umbrella sampling simulations.37 Potentials of mean force (PMFs) as a function of positions of the outer (Z1) and inner (Z2) ions along the pore axis of NaK2K, together with typical configurations of the filter with bound ions and nearby water molecules, are shown in Figure 2. The free-energy profile for K+ conduction displays five minima (a−e), and the minimumenergy path connecting the local energy minima (a) and (e) is indicated with a white line. In minimum (a), the K+ ions reside in sites S3 and S5 with a single water molecule between them. It is a new configuration not found in the MD simulations. Minima (c) and (d) are associated with the two lowest freeenergy configurations [S2, S4] and [S1, S3], respectively, and the free energy of minimum (c) is 3.9 kcal/mol lower than that of minimum (d). The free-energy barriers between the two minima (c) and (d) are 6.7 and 2.8 kcal/mol in the outward and inward directions, respectively. The free energy of minimum (e), associated with the [S0, S2] configuration, is 2.3 kcal/mol higher than that of minimum (d). The barrier for the outward movement from (d) to (e) is 3.7 kcal/mol, while it decreases to 1.4 kcal/mol for the opposite movement (Figure S4, Supporting Information). The free-energy profile for Na+ conduction is markedly different from that for K+. It displays a single global free-energy minimum (g) with Na+ binding in sites S12 and S34, which is deeper and broader than the free-energy minima observed in the case of K+. This difference means that Na+ in the filter of 2888

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To make a more convenient comparison, we calculated reduced one-dimensional PMFs W(Z1), depending on the position of the outermost ion, by numerical integration of the Boltzmann factor of the two-dimensional PMFs W(Z1,Z2), exp[−W(Z1,Z2)/kBT],29 as shown in Figure 4. The PMFs of K+

NaK2K is predominantly in the specific configuration [S12, S34], as shown in the MD simulations. This configuration was also observed in the crystal structure of KcsA_E71A with a high Na+ environment,18 and site S34 has been proposed to be the most Na+ selective site in KcsA,27,28 The free-energy profile also reveals two local minima (h) and ( f) at the extracellular and intracellular sides, associated with the [S01, S23] and [S23, S45] configurations, respectively. The free-energy differences between the two local minima and the lowest minimum (g) are higher than 6.5 kcal/mol, and the barriers opposing the outward and inward translocation from the lowest minimum (g) are 9.4 and 7.3 kcal/mol, respectively, (Figure S4, Supporting Information). As seen in Figure 3, the number and location of free-energy minima for K+ and Na+ in the filter of NaK2K_Y66F are similar

Figure 4. Reduced one-dimensional potentials of mean force for the z coordinate of the upper K+ (top) and Na+ (bottom) ions in NaK2K (red lines) and NaK2K_Y66F (blue lines).

for both mutated channels have free-energy minima at the positions of cage sites S0, S1, S2, and S3. However, it is obvious that K+ has to overcome a higher barrier to make the outward transition from S2 to the extracellular solution in NaK2K, and the relative free-energy differences between the local minima at S1 and S0 and the global minimum at S2 are larger for NaK2K than those for NaK2K_Y66F. In contrast, the two onedimensional PMFs for Na+ coincide much better with each other, having a deep free-energy well at S12 and two neighboring shallow free-energy minima at S01 and S23. We have employed MD simulations and free-energy calculations to explore the underlying mechanism for variable ion selectivity and conductance between the two newly determined Na+-permeable potassium ion channels, NaK2K and NaK2K_Y66F. We find that K+ has two preferable binding configurations in the two channels, [S1, S3] and [S2, S4], as observed in KcsA.20−23,33 However, Na+ has only one optimal configuration [S12, S34] with a deep free-energy well, accompanied by two shallow metastable configurations, [S01, S24] and [S23, S45]. The large barriers and free-energy differences between the Na+ binding configurations are essentially responsible for the low single-channel conductance of Na+. Remarkably, the presence of the hydrogen bonding interaction between Tyr66 and Thr60 in NaK2K dramatically increases the barrier opposing K+ conduction through the filter and K+ affinity at cage sites S2 and S4, whereas it has little influence for Na+. As a result, disruption of this interaction can decrease the K+ affinity, the ability to block Na+ conduction, of NaK2K_Y66F and therefore decrease its selectivity for K+. Our results offer a potential explanation for the diversity of K+ selectivity among K+ channels having the same signature sequence.

Figure 3. Two-dimensional PMFs as a function of the z coordinates of the two K+ (top) and Na+ (bottom) ions in the selectivity filter of NaK2K_Y66F. Contours are drawn at every 1 kcal/mol. The minimum-energy paths are denoted by white lines.

to that of NaK2K, indicating that the preferable ion binding configurations in the two channels are the same, as observed in the MD simulations. In addition, in the case of Na+, the freeenergy barriers along the minimum-energy path and the freeenergy differences between the minima are quite similar in the two channels (Figure S4, Supporting Information). However, in the case of K+, remarkable differences in the barriers and relative free-energy differences between the minima have been found between NaK2K and NaK2K_Y66F. Although the lowest and the second lowest free-energy configurations in NaK2K_Y66F are still [S2, S4] and [S1, S3], respectively, like in NaK2K, their free-energy difference has decreased to 1.2 kcal/mol. The largest free-energy barrier along the minimumenergy path has also decreased to 4.7 kcal/mol, which is 2.0 kcal/mol lower than that in NaK2K (Figure S4, Supporting Information).

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COMPUTATIONAL METHODS The coordinates of NaK2K and NaK2K_Y66F were taken from the Protein Data Bank, entries 3OUF and 3TET, respectively. 2889

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The Journal of Physical Chemistry Letters The channels were inserted into a partially hydrated palmitoyloleylphosphatidylcholine (POPC) lipid bilayer. Two ions (K+ or Na+) and two water molecules were added into the filter, with ions in sites S1 and S3 and water in sites S2 and S4. Eleven K+ (or Na+) and 13 Cl− ions, corresponding to a 200 mM ion concentration, were added to ensure electrical neutrality of the whole system. Four additional structures with different ion (K+/Na+) configurations were built for each system, ions in S0 and S2 (C02), ions in S1 and S4 (C14), ions in S2 and S4 (C24), and ions in S0, S2 and S4 (C024). The rest of the cage sites were occupied by water molecules. Each model was initially minimized for up to 5000 steps, followed by a 1 ns equilibration run with gradually decreasing harmonic constraints being applied to the protein and the ions in the pore. Then, 10 ns of a production run was performed with all restraints being removed. MD simulations were carried out using the program NAMD,38 with the CHARMM27 parameter set including CMAP correction for the protein and lipid,39,40 the TIP3P model for water,41 and optimized parameters for K+ and Na+ ions.42 Periodic boundary conditions were applied in all three directions. Electrostatic forces were calculated using the particle mesh Ewald (PME) method with a grid density of at least 1 Å−3.43 van der Waals interactions were smoothly switched off at 8−10 Å. The temperature was kept at 310 K using a Langevin thermostat, and the pressure was maintained at 1 atm by the Nose−Hoover Langevin piston method.44,45 The time step was 2 fs with the SHAKE and SETTLE algorithms being used.46 Free-energy profiles for ion translocation were calculated by umbrella sampling simulations,37 using a biasing harmonic potential centered on the position along the pore axis of the outer (varying in 0.5 Å steps from S5 to S2) and inner (varying in 0.5 Å steps from S3 to S0) ions, with a force constant of 10 kcal/mol/Å2. The starting configurations for the umbrella sampling simulations were taken from the MD simulations in the C024 configuration, moving the ions to the center of the biasing potentials. The minimum distances between the K+ and Na+ ions were 4.5 and 4.0 Å,29 respectively, allowing the presence of one water molecule between them. In total, 840 simulations were performed, and each simulation consisted of 500 ps, with the first 100 ps being discarded as an equilibration period. Data were unbiased and combined using the weighed histogram analysis method.47 The minimum-energy path connecting the free-energy minima was calculated using the string method.48,49

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ACKNOWLEDGMENTS

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REFERENCES

We thank Benoit Roux and Eduardo Perozo for helpful discussion. Support is acknowledged from the National Science Foundation of China (30970557), the NUAA (BCXJ 08-02), and the National Scholarship Council (No. 2010683025) of China.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures showing ion trajectories in the MD simulations and the free energy along the minimum-energy paths detected from the PMFs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-25-84891896. Fax: +86-25-84895827. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2890

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