Molecular Mechanism of Conductance Enhancement in Narrow Cation-Selective Membrane Channels

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 large-conductance but nonselective biological nanopores (i.e., α-hemolysin) used as industrial biosensors. Although recent cryo-electron microscopy 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

Molecular Mechanism of Conductance Enhancement in Narrow Cation-Selective Membrane Channels

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 large-conductance but nonselective biological nanopores (i.e., α-hemolysin) used as industrial biosensors. Although recent cryo-electron microscopy 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

Effect of the H29D mutation on the activation of RyR2 by caffeine.

<p>HEK293 cells were transfected with RyR2 WT (A) or the H29D mutant (B). Fluorescence intensity of the Fluo-3 loaded transfected cells before and after additions of increasing concentrations of caffeine (0.025-5mM) was monitored continuously. (C) Ca²⁺ release–Cumulative caffeine concentration relationships in HEK293 cells transfected with RyR2 WT and the H29D mutant. The amplitude of each caffeine peak was normalized to that of the maximum peak for each experiment. Data shown are mean ± SEM (n = 5).</p

Effect of the H29D mutation on the thermal stability of the N-terminal domains of RyR2.

<p>Location of residue H29 in the three-dimensional structure of the N-terminal domains of RyR2 in the top view (A) and side view (B). The central chloride ion is shown as a sphere. Panel A also shows the relative location of the RyR2 N-terminal region within a recent RyR1 cryo-EM map. (C). Representative thermal melting curves of the WT (filled circles) and the H29D mutant (open circles) N-terminal domains of RyR2 (RyR2ABC) (n = 5).</p

Effect of the H29D mutation on the threshold for SOICR in HEK293 cells.

<p>Stable, inducible HEK293 cell lines expressing RyR2 WT or H29D were transfected with the FRET-based ER luminal Ca²⁺ sensing protein D1ER 48 h before single cell FRET imaging. The expression of RyR2 WT and H29D was induced 24 h before imaging. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mM) to induce SOICR. This was followed by the addition of 1.0 mM tetracaine to inhibit SOICR, and then 20 mM caffeine to deplete the ER Ca²⁺ stores. FRET recordings from representative RyR2 WT (A) and H29D (B) cells (total 68 cells each) are shown. The activation threshold (C) and termination threshold (D) were determined using the equations shown in panel A. F_SOICR indicates the FRET level at which SOICR occurs, while F_termi represents the FRET level at which SOICR terminates. The fractional Ca²⁺ release (E) was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal F_max is defined as the FRET level after tetracaine treatment. The minimum FRET signal F_min is defined as the FRET level after caffeine treatment. The store capacity (F) was calculated by subtracting F_min from F_max. Data shown are mean ± SEM (n = 6).</p

Effect of the H29D mutation on the Ca²⁺ dependent activation of [³H]ryanodine binding to RyR2.

<p>(A) [³H]ryanodine binding to cell lysate from HEK293 cells expressing the RyR2 WT or the H29D mutant was carried out at various Ca²⁺ concentrations (~0.2 nM to 0.1 mM), 100 mM KCl, and 5 nM [³H]ryanodine. Amounts of [³H]ryanodine binding at various Ca²⁺ concentrations were normalized to the maximal binding (100%). Data points shown are mean ± SEM from 3 separate experiments. (B,C) Immuno-blotting of RyR2 WT and the H29D mutant from the same amount of cell lysates using the anti-RyR antibody (34c) (n = 3).</p

Effect of the H29D mutation on the propensity for SOICR in HEK293 cells.

<p>Stable, inducible HEK293 cells expressing RyR2 WT and the H29D mutant were loaded with 5 μM Fura-2 AM in KRH buffer. The cells were then perfused continuously with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mM) to induce SOICR. Fura-2 ratios of representative RyR2 WT (A) and H29D (B) cells were recorded using epifluorescence single cell Ca²⁺ imaging. (C) The percentages of RyR2 WT (total 1087 cells) and H29D (total 1161 cells) cells that display Ca²⁺ oscillations at various extracellular Ca²⁺ concentrations. Data shown are mean ± SEM (n = 9–10).</p

Effect of the H29D mutation on cytosolic Ca²⁺ induced Ca²⁺ release in HEK293 cells.

<p>Stable, inducible HEK293 cell lines expressing RyR2 WT (A) or the H29D mutant (B) were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER and their expression were induced using tetracycline. The transfected and induced cells were permeabilized with saponin, washed and perfused with intracellular-like medium plus increasing levels of free cytosolic Ca²⁺ (0.1, 0.2, 0.4, 1.0 and 10 μM) to induce Ca²⁺ release. FRET recordings from representative cells (total 51–93 cells each) are shown. To minimize the influence by CFP/YFP cross-talk, we used relative FRET measurements for calculating the steady state ER Ca²⁺ level (defined in panel A). Dash lines (F_{0.1 –}F₁₀) indicate the steady state FRET levels after perfusion with each Ca²⁺ concentration (0.1, 0.2, 0.4, 1.0 or 10 μM). The maximum FRET signal F_max is defined as the FRET level after tetracaine application. The minimum FRET signal F_min is defined as the FRET level after caffeine application. Data shown are mean ± SEM (n = 4–5).</p

Effect of the H29D mutation on cytosolic Ca²⁺ activation of single RyR2 channels.

<p>Single channel activities of RyR2 WT (A) and H29D (B) were recorded in a symmetrical recording solution containing 250 mM KCl and 25 mM Hepes (pH 7.4). EGTA was added to either the <i>cis</i> or <i>trans</i> chamber to determine the orientation of the incorporated channel. The side of the channel to which an addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytoplasmic face. The Ca²⁺ concentration on both the cytoplasmic and the luminal face of the channel was adjusted to ~45 nM. The cytosolic Ca²⁺ concentration was then increased to various levels by an addition of aliquots of CaCl₂ solution. Recording potential was -20mV. Openings are downward, and baselines are indicated (short bars). Open probability (Po), mean open time (To), and mean closed time (Tc) are shown. The relationships between Po and cytosolic Ca²⁺ concentrations (pCa) of single RyR2 WT (filled circles) and H29D mutant (open circles) channels are shown in panel C. Data points shown are mean ± SEM from 7 RyR2 WT and 5 H29D single channels.</p

The G357S mutation decreases both activation and termination thresholds for SOICR.

<p>Stable, inducible HEK293 cell lines expressing RyR2 WT (A) or the G357S mutant (B) were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER and induced using tetracycline before the experiment. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mM) to induce SOICR. FRET recordings from representative cells (total 72 for WT and 77 for G357S) are shown. To minimize the influence by YFP/CFP cross-talk, we used relative FRET measurements for calculating the activation threshold (C) and termination threshold (D) using the equations shown in A. F_SOICR indicates the FRET level at which SOICR occurs, whereas F_termi represents the FRET level at which SOICR terminates. The fractional Ca²⁺ release (E) was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal F_max is defined as the FRET level after tetracaine treatment. The minimum FRET signal F_min is defined as the FRET level after caffeine treatment. The store capacity (F) was calculated by subtracting F_min from F_max. Data shown are mean ± SEM (n = 5–6) (*, p<0.01 vs WT; NS, not significant).</p