Counterion-Assisted Cation Transport in a Biological Calcium Channel

Jul 28, 2014 - How ions pass through the lumen of an ion channel is a key question underlying cellular communication. The effects of water and pore-li...
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Counterion-Assisted Cation Transport in a Biological Calcium Channel Hao Dong, Michael L. Klein,* and Giacomo Fiorin Institute for Computational Molecular Science, Temple University, Philadelphia, Pennsylvania 19122, United States ABSTRACT: How ions pass through the lumen of an ion channel is a key question underlying cellular communication. The effects of water and pore-lining residues (channel design) have been extensively explored. However, the role of counterions is less well understood. The pore subunit of the calcium releaseactivated calcium channel, Orai, provides a useful model to explore the effect of anions on the permeation of cations. Herein, we employ computer simulations molecular dynamics (MD) to explore ion permeation through the V174A Orai mutant, a constitutively open pore, in a phospholipid membrane hydrated by a 150 mM NaCl solution. When an external voltage is applied in the MD simulations, it induces only a moderate conformational change to the channel, which facilitates the passage of ions. Infrequent sodium permeation is observed on the microsecond time scale, which is consistent with experiment. In contrast, the chloride counterions exhibit higher mobility in the channel and are actively involved in sodium transport. The anion-assisted cation permeation identified here likely highlights a more general functional role for counterions, especially in ion channels with medium pore-size. Experiments with a variety of counterions might further illuminate the nature of their active role. extracellular to the intracellular region: (i) “direct” cation permeation, in which cations pass through the hydrophobic region of the pore, probably in a partially dehydrated state, and meet anions at the basic region; (ii) “anion-assisted” cation permeation, in which anions effectively screen the charges lining the pore, and therefore help the cations permeation through the central portion of the channel. Seemingly, the presence of a relatively long basic region as found in Orai is unprecedented in a cation channel. It is possible that the Orai channel was crystallized in a posture that deviates from the native-like structure at physiological conditions: besides the potential impact of the missing residues 1−132, the crystallization conditions may bias the protein’s structural equilibrium.19 Moreover, the oligomeric state of Orai is still debatable.20 Nowadays, there is a growing awareness of how membrane mimics can induce small perturbations of the structure of membrane proteins.21,22 Therefore, it is possible that in the closed-state Orai structure, instead of forming the quite compact basic region at the intracellular side, this segment splays to a more expanded conformation in the plasma membrane.19 However, since the M1 helices are tightly wrapped by the surrounding M2 and M3 helices,17 they are more likely to have limited space for expansion before the binding of STIM (stromal interaction molecule), the activator of the CRAC channel.23,24 An alternative possibility is that the reported Orai crystal structure represents the native-like conformation. In previous

1. INTRODUCTION Counterions in biological systems have many functions, one of which is to maintain electric neutrality and help stabilize watersoluble proteins and nucleic acid structures, mainly by reducing the electrostatic repulsion among charged groups.1−3 The role of counterions in biological channels, the pore-forming membrane proteins whose function includes regulating the flow of ions across cell membranes, has been rarely studied directly. A possible reason is that many channels are selective for a specific ion (typically a cation), and counterions do not seem vital to the conduction mechanism. A few exceptions to this paradigm are available, including the chloride channel,4 the OmpF porin,5 the KAT1 channel (a voltage-gated K+ channel from Arabidopsis thaliana),6 and the glycine receptor,7−9 as well as synthetic cation-selective nanotubes mimicking a biological ion channel.10 Among these few examples, the permeability of counterions has been partially characterized, suggesting a possible functional role. Because of its unique architecture, Orai, the pore subunit of a CRAC (calcium release-activated calcium) channel,11−13 the major signaling pathway for T-cell activation with store-operated Ca2+ influx,14−16 provides a good model to explore the effect of counterions on ion permeation. As disclosed by the X-ray crystal structure of Orai from Drosophila melanogaster at 3.5 Å,17 a negatively charged Glu-ring (at the extracellular entrance) and a positively charged basic region (at the intracellular side) delimit the putative ion permeation pathway of the central pore. The two regions are separated by a hydrophobic region of ∼10 Å length, showing distinct electrostatic potential distributions, and thus having a theoretical capacity to accommodate both cations and anions in the pore simultaneously.18 Seemingly, there are two possible permeation mechanisms for cations to go from the © 2014 American Chemical Society

Received: June 16, 2014 Revised: July 24, 2014 Published: July 28, 2014 9668

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work, we carried out microsecond-long time scale molecular dynamics (MD) simulations on the wild-type Orai structure and its V174A mutant, respectively.18 No significant conformational changes were observed, indicating high stability of the structures. We found that the Glu-ring binds some Na+ ions, while several Cl− ions occupy the basic region: both groups of ions greatly reduce the electrostatic repulsions between protein subunits, and thus stabilize the pore structure.18 The structure of the basic region disclosed by X-ray crystallography17 seems to be stable in a membrane environment, and thus further indicates its possible functional role in CRAC channel gating. For the constitutively open V174A mutant (equivalent to the V102A Orai1 mutant from Homo sapiens),17,25 the calculated free energy barrier height along the ion permeation pathway is ∼7 kcal/mol.18 However, we did not observe the free diffusion of Na+ ions through the pore, largely because of the low unitary conductance of Na+ in Orai, ∼700 fS, which is equivalent to conducting 1 Na+ ion per 2 μs.26 Ca2+ ions were measured to have an even lower conductance of ∼30 fS.27 Presumably, the presence of external polarity greatly alters the diffusion rate of ions in the pore. In measurements using the patch-clamp technique, a potential difference is imposed across the membrane with embedded ion channels, by inserting electrodes on both sides of the membrane, to facilitate ion permeation and amplify the signals for recording. Here, we revisited the ion permeation in the V174A Orai mutant pore under the more realistic scenario by exploring the effects of applied membrane potentials with MD simulations. With the −500 mV bias of polarity, permeation of one Na+ ion and several Cl− ions was observed in 500 ns molecular simulations. The two species moved in the opposite directions, which effectively screened the charges from the pore-lining residues, indicating an active role of the counterions (Cl−) in cation permeation. In the control simulation, no ion permeation was detected under the positive bias of +500 mV, which is consistent with what we found in the previous simulations without applied field.18 The active role of monovalent Cl− ions was further evident when half of them were replaced with the methyl phosphate dianions, in which no cation permeation was observed with a −500 mV applied field. The present work offers an explanation for the polybasic design of the Orai pore, the resulting occurrence of anions in the pore, and the nature of cation permeation via a hitherto elusive mechanism.

Figure 1. Structure of the V174A Orai mutant and its central pore at different voltage conditions. (a) Shown is the Orai structure embedded in a fully hydrated lipid bilayer. The protein is shown in cartoon, with the central pore highlighted in orange. Ions are shown as blue (Na+) and green (Cl−) spheres. The lipids are represented by purple sticks. The polarity of the applied field is shown on the left. (b) Shown is the structure of the pore: key pore-lining residues are represented as stick. The Glu-ring, hydrophobic and basic regions are colored in red, cyan, and blue, respectively. (c) Pore radius profiles of three systems, S−500 (green), S+500 (red), and S0 (blue). Under the −500 mV hyperpolarized potential, the pore in S−500 experienced a moderate expansion. The pore in S+500 resembles more closely that of S0. Radius profiles were plotted using HOLE.71 The distributions of Na+ (d), Cl− (e), and water molecules (f) along the pore identify the major consequence of pore expansion. The error bars represents the standard deviation. The color code is the same as shown in panel c.

initial configuration, in which 25% of Cl− ions was replaced with methyl phosphate dianions and 25% was removed. Therefore, the molar ratio between the monovalent Cl− ions and the divalent methyl phosphate groups is 2:1. After careful equilibration with gradually reduced harmonic restraints on the protein structure to relax the ionic groups, the simulation was accumulated for another 740 ns with the applied field of −500 mV, and was denoted as S−500,P. In electrophysiological experiments with CRAC channels, the highest absolute magnitude of the voltage used is typically 120 mV. However, owing to the difficulty in exploring long time scales (milliseconds to seconds) in molecular simulations of transmembrane (TM) channels, it is routine setup to apply high voltages (up to 800 mV).30,31 We introduced a ±500 mV voltage in the present study, with the purpose of accelerating the slow permeation events without distorting the channel structure. In our simulations, the structure of the pore was well maintained under such conditions, justifying the high voltage applied. It can be expected that the pore only experiences similarly small changes at lower voltages. Therefore, the present study is physiologically relevant with strong biological implications. To calculate the potential of mean force (PMF) for Na+ ion permeation in a more realistic scenario, the −500 mV applied field was kept in the free energy calculations. However, to compare directly the permeation pathway of a Cl− ion with the

2. METHODS We followed the simulation protocols used in our previous studies,18,28,29 and only provide a brief description here. The initial configuration of the system was taken from the last snapshot of our previous MD simulations without applied membrane potential, in which the V174A Orai mutant was embedded in a hydrated POPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine) lipid bilayer (Figure 1a), with the physiologically relevant NaCl concentration of 150 mM.18 Both positive and negative potentials were investigated in this work (Figure 1a); S+500 and S−500 are used to represent systems with applied constant electric field set at +500 and −500 mV, respectively. After full equilibration, trajectories of the S+500 and S−500 systems were accumulated for 335 and 500 ns, respectively. As a control, data from a MD simulation without an external potential were also used and are denoted as S0.18 For the sake of investigating the possible role of polyvalent anions in the pore, divalent phosphate groups were also used. The last snapshot from the S−500 trajectories was used as the 9669

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Figure 2. Positions of the ions in the V174A Orai mutant channel in the S−500 system. The transmembrane positions of Na+ (a) and Cl− (b) ions within 10 Å of the pore axis are plotted versus time (shown in red). Some representatives are highlighted with empty circles. (a) Only one Na+ ion (highlighted in green) is able to permeate through the channel during a 500 ns MD simulation. (b) Multiple Cl− ions cross the membrane: three representatives are highlighted in green, blue, and black, showing that a few Cl− ions have two or three successive permeations during the same simulation run due to the periodic boundary conditions. The coordination numbers of Na+ (c) and Cl− (d) throughout the pore disclose the role of Cl− counterions in Na+ permeation. The unsaturated coordination of the Na+ ion around −12 Å further demonstrates that this region forms the peak of the free energy profile for Na+ permeation in the V174A Orai mutant. The coordination number with Cl− ions also peaks in this region, appearing as the key factor in decreasing electrostatic repulsions between the permeant Na+ ion and the polybasic region, and thus facilitates cation diffusion. Bin size for ion coordination is 2 Å. Three representative snapshots (e,f,g) show the different positions of Na+ along the permeation pathway in S−500. Na+ and Cl− ions are represented as blue and green spheres.

data we previously obtained for Na+,18 free energy calculations were performed in the absence of the external field. For Na+ ions under an applied field, by taking a snapshot from the aforementioned simulations with applied field where ions have been fully relaxed, steered molecular dynamics (SMD)32 was employed, in which an ion was driven by an external force to move in a predefined direction. We defined the pulling speed of 10 Å/ns along the transmembrane position (z-axis) of the target ion, with a force constant of 5 kcal/mol. Quasi-equilibrium trajectories generated by adaptive biasing force (ABF)33 were then used to calculate the PMF of ion permeation along the same reaction coordinate. The motion of the ion in z direction has a range of approximately −60 to +40 Å. We used 25 evenly distributed windows, with the width of 4 Å, to fully cover this range of the reaction coordinate. A force constant of 100 kcal/ mol was imposed at the boundary of each window, and bins of width 0.1 Å were used. Each window was accumulated until uniform sampling was achieved for its values of the reaction coordinate. For Cl− ions without applied field, the initial configuration was taken from our previous simulations.18 We followed the same protocol described above, except for the range of the reaction coordinate. Part of the channel, with the major barrier included, was explored, ranging from the polybasic region to the extracellular region (−18 to +30 Å along the z-axis). Thus, 12 windows were calculated.

All of the simulations and free energy calculations were carried out with NAMD version 2.9.34 Lipids were described with the CHARMM36 force field,35 and all other molecules were described with the CHARMM27 force field.36 The TIP3P model was used for water.37

3. RESULTS AND DISCUSSION 3.1. Influence of the External Electric Field on the Orai Pore Structure. We monitored the changes in the pore structure of the V174A mutant under the different external fields, using the structure without external voltage (S0) as a reference (Figure 1b). The hyperpolarized potential (S−500) introduces minimal structural perturbations to the pore (Figure 1c): even though the pore radius profiles under different conditions share many similarities, the entire pore in the S−500 experienced expansion during the MD simulations. Notably, pore radii at both constricted regions (−12 and 12 Å) increase by 0.5−1.0 Å. The moderate expansion of the pore is sufficient to allow the channel to remain in a “fully open” state (further described later), with the diameter of the most constricted part being 3.3 Å, very similar to the size of the STIM1-gated pore (3.8 Å).38 Therefore, the presence of the hyperpolarized potential gently alters the ion permeation pathway by widening it. No significant distortion of the pore-lining M1 helices was observed, even though the residue G170 at the center of the pore may introduce flexibility in the pore.39 9670

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scenario of conduction through a cylinder pore with 3 Å radius, Cl− ions were observed to have a larger diffusion constant than Na+ ions.41 Another example comes from carbon nanotubes with wider pores, in which the discrepancy is enlarged with the presence of a large axial electric field.42 More importantly, Cl− anions cross the channel gate at the central pore to meet Na+ cations prior to their passage in the opposite direction. This implies that in the V174A Orai channel, Cl− can overcome the free energy barrier of the central pore more easily than the Na+ traveling in the opposite direction. To further understand the possible functional role of Cl− ions on cation transport, we analyzed the species around the permeant Na+ ions. Figures 2 panels c and d show the correlation between the position of an ion and its coordination. At the exterior of the pore, Na+ predominantly binds to water molecules. However, multiple Cl− were found bound to Na+ at the interior of the pore: two peaks can be identified with a coordination number between 3 and 4 Cl− ions. The first peak (at ∼6 Å) is located just below the Glu-ring, where cations start to experience the hydrophobic region. Coordination by Cl− efficiently protects the permeant Na+ from the hydrophobic mismatch with those pore-lining nonpolar residues. This effect is quite similar to the increased hydration in this region due to the V174A mutation.18 The second peak (at −12 Å) is located nearby the residues K163, which form the narrowest region along the permeation pathway (Figure 1b,c). When protected by multiple counterions, the permeant Na+ experiences greatly reduced electrostatic repulsion from the positively charged residues (K163, K159, and R155), and thereby surmounts the major barrier along the permeation pathway more easily. Similarly, the extensive binding of Cl− by Na+ could be a reason for the former’s fast upstream diffusion through the negatively charged Glu-ring region. Therefore, the tight binding between Na+ and Cl− ions demonstrates that counterions in the pore are actively participating in cation permeation (Figure 2e−g). Therefore, the “anion-assisted” permeation of Na+ represents a more likely scenario in the constitutively open V174A mutant channel than the “direct” mode. 3.3. Pore-Water Distribution. The average number of pore waters is plotted in Figure 1f. The hydrophobic region in the S−500 system is slightly wetter than in the other two systems. A likely explanation is that the additional waters protect the additional ions (both Na+ and Cl−) at the center of the pore in the S−500 system. Aside from this difference among the three systems, the population is quite conserved. However, water molecules in the pore have quite different orientations. We analyzed the angle between the dipole of each water molecule and the pore axis and plotted its distribution along the pore in Figure 3. When compared with S+500 and S0, pore-waters in S−500 show nearly random orientations. Because the effects of the inherent membrane potential created by the protein are counterbalanced by the applied hyperpolarized potential in the opposite direction, the pore waters are less driven by the electric field. Therefore, they have higher rotational freedom and present a more favorable environment to stabilize ions crossing the hydrophobic region. Again, the orientation of pore waters is identified to be a key factor in regulating channel conductance. This is consistent with what was found in the wild type Orai and its V174A mutant in the absence of an applied field.18 On the other hand, in the S+500 system, pore waters are arranged in an orderly pattern. Especially those at the center of the pore are preferentially oriented with their dipole moments

The motion of each M1 helix is associated with the neighboring subunits, among which K157 at the N-terminal side forms a stable salt bridge with E245 on the M3 helix from the same subunit, with an average Nξ(K157)−Oε(E245) distance of 2.85 Å. Presumably, charge reversal at K157 inhibits the splaying of the N-termini of the M1 helices. This could explain why the K85E mutation in the human protein Orai1 (equivalent to K157 in Orai) removes its store-operated activation by STIM1, while Orai1-STIM1 binding is unaffected.40 Theoretically, depolarization inhibits the influx of cations. In our MD simulations, the depolarized potential in S+500 results in a pore size of the restricted region that is quite similar to the one in S0,18 and thus cannot further facilitate cation permeation by regulating the pore size. Moreover, the direction of the depolarized potential operates against the downstream flow of cations. Thus, no ion permeation was observed in the S+500 simulation. 3.2. Ion Distributions. We monitored the redistribution of ions within the pore in response to the applied voltage. As shown in Figure 1d,e, the distribution of ions along the pore in S+500 resembles that in S0,18 in which Na+ cations bind to the negatively charged residues (E178, D182, etc.) at the extracellular entrance of the pore, and Cl− anions appear at the basic region on the intracellular side. However, the ions in the S−500 system exhibit quite different distributions. One of the prominent characteristics in S−500 is that both Na+ and Cl− are able to access more space in the pore: Na+ ions move downward, and have notable population in most of the hydrophobic region (above −5 Å along the z-axis) (Figure 1d); Cl− ions move upward and accumulate abundantly at the Glu-ring region and above (Figure 1e). Plotting the time evolution of ion positions along the z-axis discloses more details about the ion motion in the pore in the S−500 system, where spontaneous ion diffusion happens under the hyperpolarized potential. As shown in Figure 2a, Na+ ions could easily reach the center of the hydrophobic region (z = 0 Å) in S−500. However, very few can occasionally access the residue K163 (z = −10 Å) at the top of the basic region, which we previously identified to have the highest barrier height for Na+ along its permeation pathway in the V174A Orai mutant.18 Only after ∼500 ns of MD simulation does one Na+ ion surmount this positively charged residue and then relocate rapidly to the intracellular side of the system. This low mobility of Na+ is consistent with its known small conductance in the CRAC channel.26 On the other hand, Cl− ions exhibit much higher mobility in the pore than Na+ and relocate frequently from the basic region to the Glu-ring via the central hydrophobic section, and then escape to the extracellular region (Figure 2b). In our MD simulations, the efflux but not the influx was observed for Cl− anions. In contrast, Na+ ions only form inward flow. Besides the multiple Cl− ions crossing the membrane region in the 500 ns simulations, we found that some of them have two or three successive permeations in one simulation run. The use of periodic boundary conditions means that when an ion passes through the top boundary of the simulation cell, it reappears at the opposite boundary with the same velocity. The conduction cycle for a Cl− ion crossing the TM can be achieved on a time scale of ∼100 ns, and there were 40 permeation events observed for Cl− ions within a 500 ns trajectory. One of the main reasons for the higher conduction is an intrinsic higher mobility of Cl− than Na+ in confined environments, which is not unique to the Orai channel. In a simple 9671

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Figure 3. Distributions of water dipole moments along the pore in the S+500 (left) and S−500 (right) systems. The average dipole in S+500 resembles that in S0,18 which is asymmetric in the hydrophobic region, and adopts the oxygen down−hydrogen up orientation. Water molecules in the same region of the S−500 system are able to change orientations freely, and thus facilitate the permeation of ions.

tilted by about 30° from the axis of the pore. This alignment complies with both the inherent membrane protein potential and the applied positive external field. Similar arrangements were observed in the wild type and the V174A mutant in the absence of an external field.18 As a direct consequence of the applied field, waters in the present S+500 system are more constraint-aligned with the pore axis, whereas the dipole moments of waters in S0 are tilted by about 45° from the axis of the pore.18 3.4. Polyvalent Anions in the Pore. On the basis of the crystallographic analysis of the Orai channel structure,17 it is conceivable that larger anions such as phosphate might also occupy the pore. We set up a simulation, indicated as S−500,P, using a mixture of Cl− and monomethyl phosphate, CH3PO42−, with the molar ratio of 2:1. We observed the mixed mono- and divalent anionic groups populating at the basic region (Figure 4). During the simulation, the boundary between the basic and the hydrophobic region was always occupied by a methyl phosphate dianion. Interestingly, the methyl tail of this phosphate group reoriented toward the central hydrophobic region after 100 ns to get an optimal configuration in the pore, while the negatively charged headgroup formed extensive interactions with the positively charged K163. As the result, no translocation of phosphate groups from the basic region to the Glu-ring region was observed. Therefore, the only apparent role of the phosphate group is to bind to and stabilize the basic region, especially the internal gate of pore. Cl− ions mainly occupied the interstitial regions between K159 and R155 (Figure 4b). More importantly, the presence of phosphate groups near the K163 is likely to hinder the efflux of Cl−. In the 740 ns trajectory of S−500,P, only 1 permeation event of Cl− ion was observed. Consequently, no Na+ diffusion to the intracellular region was observed, presumably due to the lack of screening effect from the counterions along the permeation pathway. 3.5. Potentials of Mean Force (PMFs) of Cl− without an External Electric Field. To better characterize the high mobility of Cl− ions in the V174A mutant channel, we carried out PMF calculations for Cl− translocation from the basic region (K159) to the extracellular end of the pore (Figure 5). To compare directly the permeation pathway of a Cl− ion with the data we previously obtained for Na+,18 the free energy calculations were performed without the external field. Without applied field, for both Na+ and Cl−, the major free energy barriers against ion diffusion are relatively broad, but located in different sections of the pore. The barrier height for

Figure 4. Mono- and divalent anions in the pore in S−500,P. (a) Shown is the flipping of the phosphate group nearby the K163 to adopt a more favorable orientation. The orientation was defined as the angle between the P1···O1 (C1) vector and the z-axis. The inset shows the structure of methyl phosphate group used as a divalent anion. (b) Shown is the occurrence of Cl− (in blue) and P from the methyl phosphate dianions (in red) in the pore. Two representative snapshots show the orientation of phosphate group nearby the K163 before (c) and after (d) flipping. The Cl− ions are shown as green spheres, and the phosphate groups are shown as stick (phosphate in orange, oxygen in red, carbon in cyan, and hydrogen in white).

Figure 5. Shown are free energy profiles for ion permeation through the V174A Orai mutant channel under different conditions. Na+ permeation under a −500 mV external field is shown in red, and Cl− ion permeation without an external field is shown in blue. For comparison, the free energy profile for Na+ permeation without an external field obtained in our previous work18 is shown in green. In the absence of the external field, Cl− permeation experiences a lower free energy barrier height than Na+, which is consistent with their higher mobility. The hyperpolarized potential at −500 mV alters the energetics of Na+ permeation through the central pore by lowering the major free energy barrier height. The blue and green curves only covered part of the permeation pathway along the pore. However, the most constricted region in the pore had already been explored.

Cl− is ∼6 kcal/mol, which is ∼1 kcal/mol lower than that for Na+. The peak of the free energy profile for Cl− corresponds to the position of F171 and L167, while K163 was identified as the main obstacle for Na+.18 Moreover, the extracellular entrance of the channel provides the other two barriers for Cl−, at ∼10 Å and ∼18 Å, respectively, which corresponds to the Glu-ring (the internal/central binding sites for Na+), and the negatively charged residues on the M1-M2 loops (the external binding site for Na+).18 9672

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could be obtained. Therefore, the available experimental observations regarding Cl− flux through the Orai channel under experimental conditions are ambiguous. In this work, by using molecular simulations to study the V174A Orai mutant in a fully hydrated lipid bilayer environment, we identified an “anion-assisted” cation permeation mode as a possible mechanism. Different coordination shells are adopted by Na+ to adapt to the changing environments during its permeation, in which Cl− ions are instrumental in making the cations diffuse more smoothly. More importantly, the coupling between ions appears to be a factor in effecting the apparent single-channel conductance determined experimentally. Therefore, the results presented herein provide evidence for the functional role for the counterions, which were rarely discussed in the literature. In the V174A mutant channel, considering the low Cl− concentration (∼4 mM) and Na+ (∼12 mM) concentrations in the cytosol and their much higher concentrations (>100 mM) in the extracellular fluid,49 the concentration gradient is likely to enhance Na+ influx and inhibit Cl− efflux, and thus counterbalance the effects of the hyperpolarized potential observed here. However, given the relatively broad hydrophobic region (10 Å or more) at the center of the Orai channel and the subsequent compact polybasic region, the contributions from counterions to Na+ conduction should not be negligible. Moreover, the higher charge density of Ca2+ ions, and thus the larger hydrophobic mismatch with the hydrophobic region as well as the electrostatic repulsion with the basic region are likely to make the passage of Ca2+ through the pore even more dependent on the presence of Cl− anions along the permeation pathway. The role of counterions is very likely to depend upon the size of the pore. The KcsA potassium channel, especially the selectivity filter with the pore radius of less than 1 Å,50 is far too narrow to accommodate cations and counterions simultaneously, especially considering the electrostatic repulsions between anions and backbone carbonyl groups. On the other hand, connexin 26 gap junction channel, a much wider pore with the smallest radius of 7 Å,51 is expected to enable a more active role of counterions. The intermediate-size pore of Orai therefore seems to be right at or above the critical point for counterions to be involved in cation permeation. 3.8. Role of the M1 N-Terminal Segment. The structure of the N-terminus of the M1 helix in Orai (located further inside the cell with respect to the central hydrophobic region) is critical for the CRAC channel function. This is not only because the pore-facing polybasic residues in the cation channel may significantly affect ion permeation but also because of the possible engagement of the M1 N-terminal segment in OraiSTIM binding,52−58 and thus mediation of the CRAC channel activation. Along the pore’s axis, the Cα−Cα distance from the first positively charged residue (K163) to the C-terminal end of the M1 helix (Q180) is ∼27 Å,17 approximately the thickness of the hydrophobic section of the membrane. Presumably the K163 always resides within the TM region regardless of the conformation and orientation of M1. Even if the N-terminal half of M1 stays in an expanded configuration in the open state at physiological conditions, this basic residue is still capable of forming a positively charged intracellular entrance for anions. Therefore, the major role of K163 in the V174A mutant is to form a channel gate to regulate cation influx.18 The interstitial regions between K163 and K159, and between K159 and R155 are two favorable sites for Cl− ions to reside.17,18

Overall, the free energy calculations justified the more favorable permeation pathway of Cl− in the V174A mutant channel, quantifying its higher mobility and possible functional role in cation permeation. So we now consider the permeation of Na+ in a more realistic situation. 3.6. PMF of Na+ with −500 mV Electric Field. In our previous studies, the permeation of Na+ ions through the V174AOrai channel was not observed in free MD simulations without an external electrical potential,18 although the central pore is determined as constitutively open based on electrophysiological measurements.17 Therefore, a biasing force was used to drive a single Na+ along the pore, and the free energy cost for this cation permeation was estimated from the biasing force.18 In this work, cation permeation through the channel was observed in the presence of a −500 mV hyperpolarized-potential, which mimics an electrophysiology experiment. To quantify the magnitude of this bias, we calculated the PMF experienced by Na+ in the channel with a −500 mV electric field (Figure 5). The overall PMF profile resembles the one without external field, as the key features are conserved: a major barrier spread over a relatively broad region ranging from A174 (5 Å) to R155 (−30 Å). The highest peak is located at −8 Å, corresponding to the position of K163, the first positively charged residue in the basic region. Two local minima were found at 10 and 18 Å, corresponding to the Glu-ring, and the negatively charged residues on the M1-M2 loops. More importantly, the peak value of the free energy barrier associated with Na+ transport through the pore in the presence of the hyperpolarized potential has been reduced ∼4 kcal/mol compared to the one in the absence of the external field. The free energy values of ion on both sides of the membrane are not identical by virtue of the hyperpolarized potential: the intracellular side is 2 kcal/mol lower than the extracellular side. Assuming a linear proportion between the applied field and the induced local field at the interior of the channel, the external field should contribute ∼1 kcal/mol. This indicates that the structural relaxation of the pore under the hyperpolarized potential contributes an additional ∼3 kcal/mol toward lowering the major barrier’s height. 3.7. Counterions in the Channel. The apparent ionselectivity in a channel and the possible counterion conductance through the pore are not necessarily mutually exclusive, as multiple examples are available where the transition from a cation channel to an anion channel is made possible by molecular engineering, including α7-nicotinic receptor,43 5-hydroxytryptamine3A (5-HT3A) receptor,44 and channelrhodopsin.45,46 This suggests that the passage of ions through the central pore is controlled in variable ways, where either switching ion selectivity or coupled fluxes between cation and anions are possible. In Orai pore, the distinct electrostatics of both ends strongly implies that the coexistence of both cation and anion occurs naturally. The presence of the Cl− flux in Orai revealed from our simulations could be confirmed experimentally with different protocols. For example, appreciable diffusive Cl− flux through the channel could be detected as a lower ratio of Ca2+ influx to current, with part of the inward current reflecting Cl− efflux.47 Another feasible scheme is to use impermeant cations to eliminate their contributions to the apparent current and isolate Cl− efflux specifically.48 There are however potential issues with either protocol. For the first one, it could be difficult to decompose the current from the coupled fluxes of ions and counterions. For the second one, it is possible that the pore becomes occluded by the impermeant cations, and no current 9673

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simulations and experiments. For example, the ion selectivity of the constitutively open V102A or V102C mutant in Orai1 was found to be dependent upon STIM1,25 suggesting that the two activated structures differ slightly. Moreover, the V102I mutant of Orai1 was found to have different voltage sensitivity than the wild type.70 We thus propose that further studies of constitutively open mutants and wild-type Orai under an external voltage are likely to provide useful information to understand activation of the wild-type CRAC channel.

Either double mutation of K87 and R83 in the V102A Orai1 (equivalent to K159 and R155 in Orai, respectively), or truncation up to K87 leads to almost nonfunctional channels.57 Thus, it seems that accumulating enough Cl− ions within the polybasic residues network is critical to facilitate cation influx. We further showed that while the divalent phosphate anions can reach the gate of the channel and stabilize the pore by having extensive interactions with the positively charged pore-lining residues, they cannot pass through the gate. Given the size of the pore, presumably only atomic anions are able to pass through the central pore in Orai (comparing S−500 and S−500,P). This allows Cl− anions to actively participate in cation permeation. Moreover, cysteine-substituted residue A8859 or R9139 in Orai1 (equivalent to A160 or K163 in Orai, respectively) can be cross-linked efficiently, indicating relative narrow separations at these positions. Thus, the compact packing of the N-terminal segment, disclosed by the X-ray structure17 and further validated by our molecular simulations,18 is likely to be rationalized. Even though the residues prior to the M1 helix (1−143 in Orai, equivalent to residues 1−71 in Orai1) are missing from the X-ray structure,17 the recombinant Orai1 with residue 1−6454 or 1− 6640 deleted in the presence of STIM1 does not inhibit endogenous current, indicating that this fragment may be poorly involved in Orai-STIM binding. However, by screening the functional motif on the N-terminal segment of the M1 in Orai1 it was found that residues starting from W76 (equivalent to W148 in Orai) are essential for retaining the STIM1-dependent CRAC channel current activation.57,60 Interestingly, W148 in Orai is exposed to the central pore. As shown in PMF (Figure 5), the helical segment prior to the basic region on M1, which protrudes into the cytosol, cannot block ion flow in the V174A mutant. Therefore, instead of forming the channel gate, the role of the segment before the basic region on M1 in Orai is to bind STIM, and W148 may directly get involved in binding. 3.9. Gating Mechanism for the Wild-Type Orai. In our previous work, we demonstrated that even without conformational changes of the pore structure of the V174A mutant, increased hydration at the C-terminal half of the pore is able to alter the local electrostatic profile of the permeation pathway in the pore, and thus lower the energetics of Na+ permeation through the channel.18 Here, we show that a subtle expansion of the pore can facilitate ion permeation even further. This moderate conformational change in the V174A mutant achieved by the applied hyperpolarized potential creates the conditions for anions to participate to the permeation mechanism. The activation of wild-type Orai is triggered by the binding of STIM to its cytosolic strands.61 Despite the recent breakthrough in elucidating their structures17,62,63 and conformational dynamics,64−67 a detailed mechanistic understanding of the STIM-Orai machinery at the molecular level is still obscure.68,69 Because of the high similarity between the wild-type structure of Orai and our computational model of the V174A mutant,18 the aforementioned observations imply that the conformational change triggered by STIM is an expansion of the pore resembling that induced by the hyperpolarized potential applied in this study. Within this scenario, waters and counterions in the pore are expected to act in synergy to help cation permeation. Importantly, the “fully open” structure of the V174A mutant modeled here provides an initial basis to study the open-state structure of wild-type Orai. A detailed description of the differences between the activation mechanism by the hyperpolarized potential and the one by STIM binding will likely come from a comparative analysis of

4. CONCLUSIONS Inward flow of Na+ through the V174A mutant of the Orai channel was observed using microsecond MD simulations in the presence of a −500 mV hyperpolarized potential. The number of permeation events is quantitatively consistent with the conductance rate determined from electrophysiological measurements.26 The applied hyperpolarized potential induces a moderate conformational change of the pore, and further facilitates the ion passage. A concomitant efflux of Cl − counterions was observed. Cl− actively participates in cation permeation by coordinating the permeant Na+ to form an energetically more favorable cluster. This “anion-assisted” cation permeation identified in our simulations highlights a likely functional role for the counterions in conduction through a cation channel. Analysis of the water dipole moment orientation in the interior of the pore reveals that a more favorable local electrostatic environment along the cation permeation pathway is formed in the pore during conduction. We have previously demonstrated that without significant channel-gating motions, a subtle change in the number of pore waters is sufficient to reshape the local electrostatic field and modulate the energetics of ion conduction.18 Here we provide further evidence of how the local environment is reshaped to regulate channel conductance. While observations from the present MD simulations have yet to receive needed experimental validations, they nonetheless help rationalize the conduction and gating mechanisms operating in the CRAC channel. The “fully open” structure of the V174A mutant obtained from our atomic level molecular simulations provides an initial basis to study the open-state structure of wildtype Orai. More importantly, the active role of chloride in cation permeation revealed in the present work suggests that future studies of Orai and other ion channels with wide pores could profitably explore the role of counterions.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Patrick Hogan for insightful comments and discussions. This work was supported in part by the Department of Health of the Commonwealth of Pennsylvania and by the National Science Foundation through Grant CHE-1212416. The computations were performed using the Temple University High-Performance Computing System purchased in part with NSF Grant MRI-R2 0958854 as well as resources from XSEDE (www.xsede.org/high-performance-computing) provided under Grant MCA93S020. 9674

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