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Molecular Mechanism of Conductance Enhancement in Narrow Cation Selective Membrane Channels Williams Ernesto Miranda, Van A. Ngo, Ruiwu Wang, Lin Zhang, S. R. Wayne Chen, and Sergei Yu. Noskov J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b01005 • Publication Date (Web): 10 Jun 2018 Downloaded from http://pubs.acs.org on June 10, 2018

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Molecular Mechanism of Conductance Enhancement in Narrow Cation Selective Membrane Channels Williams E. Miranda,† Van A. Ngo,† Ruiwu Wang,⧧ Lin Zhang,⧧ S.R. Wayne Chen,⧧ and Sergei Yu. Noskov† †

Centre for Molecular Simulations and Department of Biological Sciences, University of Calgary, Alberta, Canada of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary

⧧Department

ABSTRACT: Membrane proteins known as Ryanodine Receptors (RyRs) display large-conductance of ~1 nS and nearly-ideal charge selectivity. Both properties are inversely correlated in other largeconductance but non-selective biological nanopores (i.e. alpha-hemolysin) used as industrial biosensors. Although recent Cryo-EM structures of RyR2 show similarities to K+ and Na+-selective channels, it remains unclear whether similar ion conduction mechanisms occur in RyR2. Here, we combine microseconds of all-atom molecular dynamics (MD) simulations with mutagenesis and electrophysiology experiments to investigate large K+ conductance and charge selectivity (cation-vs-anion) in an open-state structure of RyR2. Our results show that a water-mediated knock-on mechanism enhances the cation permeation. The polar Q4863 ring may function as a confinement zone amplifying charge selectivity, while the cytoplasmic vestibule can contribute to the efficiency of the cation attraction. We also provide direct evidence that the rings of acidic residues at the channel vestibules are critical for both conductance and charge discrimination in RyRs. Understanding selective ion transport via integral membrane proteins, which function as cation or anion selective channels, porins, and solute transporters has provided rich grounds for the rational design of nanodevices that mimic biological properties such as ion-selectivity, current rectification, and gating.1-4 These bio-inspired synthetic nanopores are widely used for industrial applications as molecular filters for water desalination, biosensors for DNA sequencing, and energy converters (e.g. salinity gradient electricity).3-8 Although the ideal nanoporous membrane should combine large unitary conductance and high selectivity, both properties are usually anticorrelated.3-5, 9-12 Biological nanopores for sensory and DNA sequencing applications, such as alpha-hemolsyin, MspA, and VDAC-based systems, display relatively large unitary conductance of 1 to 5 nS and relatively low charge selectivity of ~ 2 to 5 -fold preference for the charge carrier.13-16 Conversely, systems showing ~1000-fold

selectivity such as bacterial K+-selective channels (i.e. KcsA17-18, MthK19) or their mammalian counterparts (i.e. voltage-gated potassium channels like hERG120 and Kv1.221-22) display relatively low unitary conductance ranging from only 10-15 pS to 100-150 pS in hERG1 and KcsA, respectively.23 Such a low ion conductance also hampers the performance of ion-selective fabricated nanoporous membranes.9-12 For example, recently developed synthetic polymeric membranes (PET Lumirror®) have shown promise for nanotechnology applications as molecular sieves.12 The pores comprising this nanomaterial are distributed over the membrane with an average density of ~ 5x1010 pores/cm-2, have a diameter of ø ~10 Å, and are functionalized with acidic moieties to enable charge-selective transport. Their average single-pore conductance is in the range of femto-Siemens,12 which is around 3 orders of magnitude lower than small conductance of K+-selective channels.12, 24-25 1


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The recently solved Cryo-EM structures of ion channels from the Ryanodine Receptors (RyRs) family may finally provide a fresh impetus to understand the molecular principles governing the combination of large unitary conductance and high charge selectivity.26-29 The RyRs show unitary K+ conductance up to 1 nS, comparable to alpha-hemolysin, while preserving near-ideal charge selectivity. 24-25 The open-pore of RyR2, the cardiac isoform, is shown in Figs. 1 and S1.26 The overall pore-domain (PD) organization and dimensions are reminiscent of canonical Na+- and K+-selective channels. Although mutagenesis studies have mapped key residues involved in fast and selective cation permeation in RyR2, the detailed molecular mechanisms underpinning the enhanced cation conduction in RyR2 are still unclear. In this work, we combined micro-second all-atom molecular dynamics (MD) simulations and experimental electrophysiology approaches to investigate K+ permeation and charge selectiv-

ity (K+ vs. Cl–) in RyR2 open-channel structure (details on MD simulations and experiments are provided in SI).26 For a comparative study, we performed mutations in the PD for luminal(D4829A, D4829N) and cytoplasmic- (D4868N, G4871H) vestibules (Fig. 1). To simulate ion permeation, we added [KCl]= 810 mM at both sides of the membrane containing each RyR2variant and applied a constant electric field30 Ez corresponding to a transmembrane potential (V) of 400 mV along the z direction (Fig. S2).31-37 We collected permeation events of ions, compared conductance with experiments, and examined the ion-permeation energetics and mechanism. We also computed electrostatic maps and channel radius-profile in order to extract key principles of design for large conductance and near-ideal charge selectivity observed in RyR2 in comparison with other Na+- and K+-selective biological channels and synthetic nanopores.

Figure 1. The pore domain of RyR2 in the open state. Left: The pore radius measured along the z-axis for all RyR2variants analysed in this work. Right: The RyR2-WT with only two of the four monomers shown for clarity. The black dotted surface and the blue line represent the permeation pathway and the central axis, respectively.

We did not observe any substantial conformational changes due to the high voltage in the submicrosecond simulations, suggesting that the instantaneous interactions and high-statistical permeation events could be used to get some insights into the molecular mechanism of RyR2. Indeed, our simulations reproduce the relative changes in potassium conductance (݃௄ା ) experimentally observed for the RyR2-variants, show-

ing a correlation coefficient of R2= 0.93 (Table 1 and Figs. S3-4), even though 400 mV exceeds physiological values. Our experiments show that D4829A/N mutations reduce the RyR2 conductance to values commonly found in relatively small-conductance bacterial K+-channels like KcsA. Although our molecular model overestimates conductance values for both mutants, as 2



we will show, it does reveal many interesting properties of the RyR2 channel. Table 1. Permeation events (P), currents, and conductance at Vsim = 400 mV, calculated from a combined 1 µs simulation time in the luminal to cytosol direction (see Fig. S2). ≠Xu et al.38 ⁑Chen et al.39 ⁂Gao et al.40 Variant

P (K+/Cl-)

ࡵതsim/pA

ഥ sim/pS ࢍ

ࢍࢋ࢞࢖ /pS

WT

1586/40

254 (±9%)

635 (±9%)

763

G4871H

1390/60

223 (±14%)

557 (±14%)

516

D4868N

1292/80

207 (±9%)

518 (±9%)

514

D4829A

1110/26

178 (±16%)

445 (±16%)

160⁑

D4829N

972/30

156 (±24%)

389 (±24%)

145⁂

≠

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(PMFEB) shows minima at the SF, CZ, and CV, which are 1.5 - 2.0 kBT lower than the bulk (Fig. 2 D), suggesting that these regions are weak cation binding sites. Furthermore, the barriers separating these minima are less than 1.0 kBT (Fig. 2D). According to Kramer’s theory,41-42 such small energy barriers between different positions along the pore may lead to fast transition rates. Therefore, the small energy barriers in the PMFEB resulting from multiple-ion effects,37, 43-46 may give rise to the observed large K+ conductance (Table 1). Our multi-ion PMF(EB) differs from single-ion unbiased PMF recently reported for RyR1 isoform,32 which shares 93% sequence identity with RyR2 at the PD (Fig. S1). The RyR1 PMF shows the deepest energy well at the CV of the channel. We will show that the CV of RyR2 is critical for selecting K+ over Cl–. Electrophysiology studies have shown that RyRs cannot discriminate among cations with large differences in hydration energies.25, 47 The cross-sectional radius of the channel at the SF and the CZ (the narrowest sections of the pore) is

Figure 2. Permeation of particles through the RyR2 pore. A) - C) Average iso-density surfaces for K+, water, and Cl-, respectively, in RyR2-WT. The labels a, b, and c in A) indicate regions of high K+ density. D) Calculated PMF(EB) profiles for K+ permeation in RyR2-variants (error bars in Fig. S6).

To understand the large-conductance in RyR2WT, we first analysed the ion-density profiles and energy barriers. Figure 2 shows that the pore domain displays high K+ density (ρ(K+) ~ 2.0 - 3.0 E-03 ions/Å3) at the SF, CZ, and CV (labels a, b, and c in Figs. 2A and S5A), which on average bind 3 ±1, 1 ±1, and 5 ±1 cations, respectively. These sections of the pore comprise carbonyland carboxyl- oxygens (Fig. 1), which are known to coordinate K+ in other potassium channels.18 The effective-biased potential of mean force

~ 2 - 3 Å (Fig. 1), allowing the permeation of hydrated cations (Figs. 2 A, B, 3, and S5 B) in our simulations. Furthermore, we observed drive-by and water-mediated knock-on events among the permeant cations in the SF and CZ (Fig. 3 and Video S1). This permeation mechanism has been observed in canonical Na+-selective channels.37, 46 Moreover,

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Figure 3. Representative single- (A) and double(B) occupancy states for cations at the constriction zone of the channel. The chloride ions and POPE lipids are omitted for clarity. C) Close-up of the double occupancy state, showing the constriction zone formed by a ring of four Q4863 residues (licorice). The plane formed by three water molecules positioned between both K+ ions is highlighted by green lines.

the coulombic repulsion resulting from the high cation density at the SF (label a Figs. 2A and S5A) is reflected by ~0.3 V increase in the local electrostatic potential with respect to the bulk (see “bump” under label a in Fig. S7). The accumulation of electrostatic repulsion among cations due to the strong attraction between the SF and cations may contribute to high through-put multiion permeation as observed in canonical channels.37, 43-46 The Q4863 ring at the constriction zone may function as a charge selectivity filter. The K+/Clpermeation ratio of the WT is approximately 40 (Table 1), in agreement with experimental evidence showing that RyRs are cation selective.24, 48 Figure 2C shows no Cl– accumulation above the CZ, suggesting that the Q ring of the CZ may impose an energy barrier for anion permeation in addition to the negatively charged luminal loops, while K+ can be coordinated by oxygen atoms from the carboxamide sidechains and water molecules (Fig. 3C). This Q ring binds one or two K+ (Figs. 3A-B) with 0.40 or 0.04 occupancy probabilities, respectively. These observations indicate that the Q ring may function as a charge selectivity filter, although it is likely a weak cation binding site (energy depth of 1.0 kBT, label b in Fig. 2D). The conductance and selectivity of RyR2 is modulated by the chemistry and geometry of the pore vestibules. The SF and CV have rings of acidic residues (Asp and Glu) facing the aqueous environment (Fig. 1). These residues contribute substantially to the local minima of the electrostatic potential (Figs. S7 and S8). As a result, the cations accumulate in these regions (Figs. 2A and

S5A) with occupancies of 3 ±1 and 5 ±1, respectively. The mutations D4868N and G4871H, which effectively increase the net charge (+|e|/per residue) to the CV, mildly decrease occupancy to 3 ±1 cations (Fig. S8). In contrast, the mutations D4829A/N, which remove the negatively charged residues in the SF (Fig. S8), profoundly decrease the cation density (label a in Fig. S5) and occupancy (1 ±1), make the energy well shallower (label a in Fig. 2D), and reduce the electrostatic “bump” with respect to the WT (label a in Fig. S7 B). Regarding the pore radius, the DN mutations cause no significant change because the residues D and N are isosteric, while all the DA mutations only increase the radius at the luminal side of the SF by ~ 1 Å (Fig. 1). In contrast, the mutation G4871H noticeably decreases the radius of the CV by ~ 3 Å (Fig. 1). Overall, these observations highlight the role of vestibular charges and pore radius to achieve large conductance. Moreover, the mutations G4871H and D4868N approximately double Cl– permeation events (Table 1) and decrease the ion discrimination by half (40:1 to ~ 20:1) compared to WT. This is in agreement with previous reports, where the mutations of acidic residues located at the CV would decrease the conductance and selectivity of the channel.38 The other mutants D4829A/N can further reduce the permeation events of both anions and cations, but display relatively large selectivity ratios (32-43:1). To this end, we can conclude that the CV would be responsible for the charge (cation-vs-anion) selectivity, and interestingly the SF, which would normally select 4



among cationic species, would only control the total rates of both cation and anion permeations. The PD of RyR2 shows distinctive structural and electrostatic features. To better understand the underpinnings of large cation conductance in RyR2, we compared its structure and electrostatic properties with those of biological and synthetic channels with known structure and unitary conductance. Calcium-activated K+ channels like Slo149 and MthK19, 50 display a wide IC and rings of acidic residues at their CVs that establish a negative electrostatic potential (Figs. 4A&E). It has been proposed that both of these features enhance the accessibility and accumulation of cations, leading to relatively large ݃௄ା ~ 250 pS.23, 49, 51-52 Conversely, the small conductance observed in voltage-gated K+ channels like hERG (݃௄ା ~15 pS) is influenced by a narrower IC and the absence of rings of negative charges at the CV (Figs. 4B&F).20, 23, 51-52 However, the diameter of the cytoplasmic access to the IC is not sufficient to explain conductance enhancement. For example, the cytoplasmic access to the IC of RyR2 is narrower than both Slo1 and hERG channels (Fig. 4C), albeit wide enough to allow more permeation of hydrated K+ ions (Figs. 2A&B, 3A&B, and S5B).

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Although the SF of RyR2 displays a sequence motif (GIGD) similar to K+ selective channels (GYGD),23, 26 this section of the pore is wider than SF region of the K+ channels (Fig. 4 A-C). This explains, at least in part, the higher occupancy values found for RyR2 at this region (3 ±1 cations) compared to reported values for canonical K+ channels (2 - 3 cations).43, 45 Note that the SF with carbonyl oxygen atoms of canonical K+ channels promote complete cation dehydration (penalty of 5.0 – 7.0 kBT),43, 53 while the SF of RyR2 allows permeation of hydrated cations. There is also a markedly negative electrostatic potential that spreads from the luminal side to the CV of RyR2 (Figs. 4C&G), but only briefly disrupted at the IC for a range of 5 Å. In contrast, the negative electrostatic potentials of Slo1 and hERG channels are mostly focused at the SF (Fig. 4E&F), which may result from the fewer acidic residues localized in the vestibules than RyR2 (Fig. 4A-C). Therefore, the enhanced conductance in RyR2 compared to canonical K+ channels is attributable to the wider selectivity filter and the broader negative electrostatic potentials along the pore. A counterargument to the above comparisons can be posed by examining the PD of sodium-

Figure 4. Structural and electrostatic features of representative large and small conductance channels. Upper panel: channel radius (R) along the permeation pathway for A) Slo1, B) hERG, C) RyR2, and D) NavMs. Each channel and the residues at the constriction zone are shown in tube and licorice representations, respectively, following the same coloring scheme as in Fig. 1. Bottom panel: the corresponding electrostatic potential (kJ/(mol·|e|) maps of the channels.


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selective channels like NavMs54, which display a similar width at the SF and IC regions (Fig. 4C&D). Both channels contain a ring of acidic residues at the SF (Figs. 4C&D), which allows the permeation of hydrated cations and the occurrence of drive-by and water-mediated knock-on events.37, 46, 55 Nevertheless, the Na+ conductance, ݃୒ୟ୴୑ୱ ~50 pS, is ~ 10-fold smaller than RyRs. In this regard, Figure 4 shows some noticeable differences between RyR2 and NavMs: (i) the pore radius at the cytoplasmic IC access of NavMs (I215) is about half the radius at Q4863 of RyR2 (Figs. 4C&D); and (ii) there is a substantial broad barrier of positive electrostatic potentials at the IC of NavMs that would hinder permeation of cations (Figs. 4G&H). Recently reported ultrafast sieving synthetic membranes12 show a conductance ~6 orders of magnitude smaller than that of RyR2. The PET Lumirror® pores contain acidic moieties randomly distributed along the permeation pathway12, which may decrease the efficiency of cations concentrating at the mouths of the pore and reduce the probability of permeation events. Another essential factor contributing to low conductance found in this narrow artificial nanopore is axial resistance arising from its length ~2 µm vs ~4 nm for RyR2. In conclusion, RyRs achieve large conductance and high charge selectivity. These two properties are often inversely correlated in other biological and synthetic channels. This results from the narrow and short pore (ø ~4 – 5 Å, l ~ 40 Å), flanked by rings of polar and negatively charged residues at different locations. The polar Q ring may function as a charge selectivity filter, while the cytoplasmic vestibule can contribute to the efficiency of the charge selectivity. At the selectivity filter and along other channel confinements, the cations undergo drive-by and watermediated knock-on events. This permeation mechanism is enabled by the surface chemistry with multiple rings of acidic residues spreading along the pore domain and the relatively large diameters of the SF and CV. As a result, the electrostatic properties of RyR2 can help to reduce de-solvation penalties and enable the accumula-

tion of coulombic repulsion among multiple cations. CORRESPONDING AUTHOR *Sergei Noskov, CMS, University of Calgary Email: [email protected]; Phone: 403-210-7971 AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Supporting Information Available Includes supplementary materials and methods, supplementary results, figures S1-S8 and captions, and Video S1. ACKNOWLEDGEMENTS This work was supported by research grants from the NSERC (RGPIN-315019), the Alberta Innovates Technical Futures Strategic Chair in Bio-Molecular Simulations S.Y.N); from AIHS and CIHR postdoctoral fellowships (to V.A.N); from AIHS and VANIER graduate studentship (to W.M.); from the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut, the Heart and Stroke Foundation Chair in Cardiovascular Research, and the Alberta InnovatesHealth Solutions (AIHS) to S.R.W.C.; We also thanks to Dr. Valentina Corradi for her useful advice on the figures presented in this manuscript. The authors declare no competing financial interest. References (1) Majd, S.; Yusko, E. C.; Billeh, Y. N.; Macrae, M. X.; Yang, J.; Mayer, M. Applications of Biological Pores in Nanomedicine, Sensing, and Nanoelectronics. Curr. Opin. Biotechnol. 2010, 21, 439-476. (2) Simmel, F. C. Bioelectronics: Wiring-Up Ion Channels. Nat. Phys. 2009, 5, 783-784. (3) Tagliazucchi, M.; Szleifer, I. Transport Mechanisms in Nanopores and Nanochannels: Can We Mimic Nature? Mater. Today 2015, 18, 131-142. (4) Zhang, H.; Tian, Y.; Jiang, L. Fundamental Studies and Practical Applications of Bio6



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