All experimental protocols were approved by the State veterinary and food administration of the Slovak Republic (Ro-3820/05-221, Ro-2821/09-221) and by the ethical committee of the Centre of Biosciences. All methods were carried out in accordance with the European directive 2010/63/EU. Male rats (Wistar Han, 240–310 g, breeding station Dobra Voda, reg. No. SK CH 24011, Slovak Republic) were anesthetized with sodium pentobarbital (50 mg kg⁻¹ i.p.), the hearts were excised, and cardiac SR microsomes were isolated from rat heart ventricles by differential centrifugation without further purification steps, as previously described (Gaburjakova and Gaburjakova, 2006; Faltinova and Zahradnikova, 2013), snap frozen in liquid nitrogen and stored at −70°C.

1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (cis)—DOPC, 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were from Avanti Polar Lipids (USA).

The peptide DP_cpvtN2, containing amino acids 410–438 of human RyR2 with the sequence HEESRTARVIRSTVFLFBRFIRGLDALSK was synthesized by GenScript (USA). Aliquots of a stock solution of the lyophilized peptide in water (100 μmol/l) were kept at −20°C.

Preparation of bilayers, incorporation of channels and electrophysiological measurements were performed as previously described (Faltinova and Zahradnikova, 2013). In brief, bilayer lipid membranes were formed over a 20–50 μm aperture in a polystyrene chamber. The aperture was first treated with a solution (26 mg/ml) of DOPS, DOPC and DPPC (3:5:5) in N-decane (Aldrich, USA). After evaporation of N-decane, the cis and trans compartments of the bilayer chamber were filled with the solutions (in mmol/l): cis, 250 HEPES, 50 KCl, 1 EGTA, 125 TRIS, 0.5 CaCl₂, pH 7.35, [Ca²⁺] = 90 nM; trans, 30 HEPES, 50 KCl, 1 Ca(OH)₂, 7 Ba(OH)₂, pH 7.35, and the bilayer was formed using a mixture of DOPS and DOPE (3:10; 26 mg/ml in N-decane). After formation of a bilayer, RyR2 channels were incorporated from a suspension of microsomal vesicles of the SR, added to the cis compartment. RyR identity was checked at the beginning of the experiment at high Ca²⁺, when it was characteristic by its single-channel amplitude and open duration (Tencerova et al., 2012; Faltinova and Zahradnikova, 2013). Under the above ionic conditions, no other channels have similar properties. Detection of RyR channel was followed by perfusion to a basal Ca²⁺ level. The identity of RyR was occasionally further tested at the end of the experiment by addition of 100 μM Ca²⁺.

Currents through the incorporated single RyR2 channels were measured in voltage-clamp mode at 0 mV with MultiClamp 700B amplifier (Molecular Devices, USA) with a 50 GΩ feed-back resistor. The signal was filtered by a 4-pole Bessel filter at 1 kHz and digitized using an A/D converter (Digidata 1440A, Molecular Devices, USA) at a frequency of 4 kHz. Acquisition and analysis were performed using pClamp software (Ver. 10, Molecular Devices, USA) and Origin Pro (Ver. 9, OriginLab Corporation, USA).

Equilibrium open probabilities for different gating models (Equations 1–3) were determined from the gating schemes. Equations were derived using Mathematica (Ver. 10, Wolfram Research, USA) as previously described (Zahradnik et al., 2005). The value of EC50 was calculated in Mathematica from Equations (1) to (3) as the ligand concentration, at which the increase of open probability reached 50% of maximum increase.

Homology models of peptides were made using the I_TASSER server (Roy et al., 2010) (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). Modeling of loops was performed in Modeller (ver. 9.15) (Sali and Blundell, 1993).

Docking of protein structures into electron density maps was performed using the program COLORES from the SITUS 2.7 package (Wriggers, 2010) as described previously (Borko et al., 2014).

Peptide-protein complex formation was modeled using the GRAMM-X public web server (Tovchigrechko and Vakser, 2006). All model structures were further energy-optimized using the Chimera interface of the Molecular Modelling Toolkit (Hinsen, 2000) with the optimization criteria as follows: 200 iterations for steepest descent gradient method followed by maximum 500 iterations for conjugated gradients method, maximum total energy difference at the last iteration of 0.015 kJ/mol per residue. Interactions between amino acids were quantified using the PIC web server (Tina et al., 2007). Hydrogen bonds were analyzed in Chimera with constraints according to Mills and Dean (1996) relaxed by 0.4 Å and 30°.

Statistical analysis and data fitting were performed in Origin Pro (Ver. 9, OriginLab Corporation, USA). Analysis of linear models was performed in Mathematica (Ver. 10, Wolfram Research, USA).

Circular dichroism (CD) measurements were performed using a Jasco J-810 spectropolarimeter (Japan). CD measurements were performed in the 198–250 nm wavelength range, which enabled estimation of the secondary structure of the peptide. The DP_cpvtN2 peptide was dissolved either in 2 mM Na-phosphate (pH 7.0) or in 99.8% ethanol to a final concentration of 1.5–7 μM. CD spectra were expressed as averages of 3–5 consecutive scans. 1 mm cuvette was used for measurements. Typically, a time constant of 2 s, bandwidth of 2.0 nm, scanning speed 200 nm/min and the standard sensitivity were used. The spectra were analyzed using the web server CAPITO (Wiedemann et al., 2013), which provides the percentage of α-helical, β-sheet and random coil in the secondary structure.

The activity of single RyR2 channels was recorded at cytosolic (100 nM) and luminal calcium (1 mM) concentrations and at a holding potential (0 mV) corresponding to the diastolic conditions. Under these conditions, the open probability of the ryanodine receptor was very low (P_O = 0.0039 ± 0.0012, n = 9) and consisted of short openings (t_O ~ 15 ms). Aliquots of DP_cpvtN2 stock solution were sequentially added into the cis compartment up to final concentrations in the range of 0.5–2 μM. The activity of the channel under control conditions and after each increase of DP_cpvtN2 concentration was monitored for a 2-min. period. Typical current traces are shown in Figure 1A. Within 1 min after addition of DP_cpvtN2, sporadic occurrence of long openings (t_O >100 ms) could be observed. Open probabilities at different concentrations of DP_cpvtN2 obtained from 9 channels are summarized in Figure 1B. Increase in open probability and the presence of long openings could be observed at all DP_cpvtN2 concentrations in the range of 0.5–2 μM; long openings (t_O > 100 ms) were never observed in the absence of DP_cpvtN2. There was a high temporal variation of channel open probability (Figure 1C) as well as of the responses of individual channels (Figure 1B). Therefore, it was not directly apparent whether a maximal effect of DP_cpvtN2 was achieved at the highest DP_cpvtN2 concentration of 2 μM. Higher DP_cpvtN2 concentrations could not be used, because they induced rupture of the bilayer membrane after DP_cpvtN2 addition. The median time to rupture at 1–2 μM DP_cpvtN2 did not depend on the presence of RyR in the membrane, and in the absence of RyR2 the rupture was not preceded by channel-like activity.

We compared the observed calcium dependence of open probability with the predictions of three putative gating schemes (GS1–GS3) describing channel activation, illustrated in Figure 1D, in which DP_cpvtN2 sequentially binds to individual monomers of the RyR2 tetramer. According to GS1, binding of one DP_cpvtN2molecule to the ryanodine receptor is sufficient for channel opening, and the long channel openings correspond to the periods of bound DP_cpvtN2. The concentration dependence of open probability (P_O) is then described by the equation:

where P_O0 is the open probability in the absence of DP_cpvtN2, c_p is the concentration of DP_cpvtN2 and K_p is its dissociation constant.

According to GS2, the channel opens only upon binding of DP_cpvtN2 to all four RyR2 binding sites. The concentration dependence of open probability (P_O) is then described by the equation:

where individual parameters were explained previously.

According to GS3, DP_cpvtN2 increases RyR2 open probability allosterically, as previously observed for the peptide DP_cpvtC (Faltinova and Zahradnikova, 2013). That is, when DP_cpvtN2 is bound to the channel, the free energy necessary for channel opening is decreased. In this scheme, the concentration dependence of open probability (P_O) is described by the equation:

where f_p is the allosteric factor that couples DP_cpvtN2 binding to channel opening, and the remaining parameters were described previously.

The best data fit curves corresponding to Equations (1)–(3) are shown in Figure 1B, and the fit parameters and EC₅₀ values are shown in Table 1.

Visually, the fit of the data by GS1 is acceptable while that by GS2 shows systematic deviation. This was confirmed by analysis of the likelihood of the fit which rejected GS2 (Akaike weight 0.92 and 0.08 for GS1 and GS2). GS1 and GS3 provided acceptable fits and both are in accordance with the effect of DP_cpvt2 on RyR2 open probability (Akaike weight 0.52 and 0.48 for GS1 and GS3, respectively).

However, the data in Table 1 suggest that GS1 and GS3 lead to diametrically opposed mechanism of peptide action: according to GS1, DP_cpvtN2 has very low affinity to the RyR2 monomer that is independent of the number of DP_cpvtN2 molecules bound to the RyR2 tetramer. That is, the free energy of DP_cpvtN2 binding is expected to be positive even when the channel resides in the peptide-induced open state. At the same time, DP_cpvtN2 has a very strong effect on open probability, i.e., the free energy of channel opening is expected to be much lower in the complex than in the free monomer.

On the contrary, according to GS3 (Table 1, Figure 1D), the DP_cpvtN2 has high affinity to RyR2, i.e., the free energy of DP_cpvtN2 binding is expected to be negative. The effect on RyR2 open probability is modest, thus the free energy of channel opening is expected to be somewhat lower for the complex than for the free RyR2 monomer. Affinity of DP_cpvtN2 is higher in the open than in the closed states by a factor of 1/f_p, and thus the free energy of complex formation is expected to be lower in the open than in the closed state.

Mutations in the central helix decrease the thermal and conformational stability of NTR and/or destabilize the interaction between domains A, B, and C of the N-terminal region (Kimlicka et al., 2013b; Borko et al., 2014). Furthermore, mutation R402G of the skeletal RyR1 isoform, corresponding to R417 in the central helix of NTR in hRyR2, induces changes in the relative orientation of domains A and C of the N-terminal region (Kimlicka et al., 2013a). The “domain switch” mechanism (Ikemoto and Yamamoto, 2000; Yamamoto et al., 2000; Tateishi et al., 2009; Suetomi et al., 2011) assumes that DP_cpvtN2 exhibits its effect by competition between DP_cpvtN2 and central helix for a common binding site that might involve domains A/B. This mechanism would require three conditions to be met:

The conformation of DP_cpvtN2 in solution should be similar to the conformation of the central helix.

Structure of DP_cpvtN2 in solution was examined by CD spectroscopy. The CD spectra of 1.5–3 μM DP_cpvtN2 solution in water and in ethanol are shown in Figures 2A,B, respectively. The CD spectra of DP_cpvtN2 show a positive band with a maximum below the measuring range (<198 nm) and two negative bands with minima at 208 and 225 nm that suggest that a substantial fraction of the peptide is in the α-helix conformation. In ethanol (Figure 2B), the ellipticity was an order of magnitude higher, without a substantial change in the shape of the spectra. The content of α-helix was estimated using the web server CAPITO (http://capito.nmr.leibniz-fli.de/index.php) to be 99%. The high content of α-helix estimated from CD spectra shows that the structure of DP_cpvtN2 in solution is comparable to the structure of the central helix in the RyR2 molecule.

The interaction of DP_cpvtN2 with the N-terminal region was studied in structural models of the closed and open conformation of the NTR (Borko et al., 2014) using the GRAMM-X web server. The model of DP_cpvtN2 was constructed using the I_TASSER server. The model of NTR in the closed conformation was taken from Borko et al. (2014), and the model of NTR in the open conformation was constructed by aligning the A, B, C domains of the NTR model to the respective domains of 4JKQ in the open conformation (Borko et al., 2014), reconstructing the loops between domains in Modeller, and minimizing the energy of the resulting model in Chimera using the built-in Molecular Modelling Toolkit.

The GRAMM-X server provided a total of 29 conformational solutions for the DP_cpvtN2—NTR complex. The models were checked for compatibility with the structure of the whole RyR by alignment with NTR fitted into a cryo-EM map of RyR1 (EMD 2807; Yan et al., 2015). In three conformational models (CM1–CM3), bound DP_cpvtN2 did not sterically interfere with the RyR1 electron density. The conformations CM1–CM3 were further energy-minimized. The positions of DP_cpvtN2 in the grooves of NTR in the closed conformation, predicted by CM1–CM3, are shown in Figures 3A,B. In all three cases DP_cpvtN2 binds to the N-terminal domain at the cytosolic, top face of the RyR (Figure 3C).

Figure 3D shows the distribution of electrostatic potential on the complementary surfaces of NTR and DP_cpvtN2 in the three models. The surface of DP_cpvtN2 is positively charged due to the presence of four arginine residues oriented toward the NTR surface. The surface of the NTR groove that cradles bound DP_cpvtN2 is charged negatively due to the presence of one serine and two glutamate residues (E50, S200, E320) in CM1, two aspartate, two serine and six glutamate residues (E40, N44, E50, N57, S136, S200, E320, E450, E521, E524) in CM2, and one aspartate and two glutamate residues (E50, N293, E320) in CM3. It is therefore apparent that the interaction between the peptide and NTR has electrostatic character.

The energies of NTR, DP_cpvtN2 and of the DP_cpvtN2—NTR complexes are compared in Table 2. It has to be noted that several interactions contributing to the overall energy of the complex between RyR2 and DP_cpvtN2 are not accounted for in CM1–CM3, namely, the energy of interaction between the four NTR monomers and the energy of interaction between the N-terminal region and the remainder of the RyR2 molecule. Nevertheless, the energetic profiles from Table 2 suggest that the transition from the closed to the open conformation of the isolated NTR is energetically highly unfavorable (ΔΔGopeningfree= +1580 kJ/mol), which is consistent with the very low open probability of the whole RyR2 in the absence of channel activators. The energies of the complex in the closed as well as in the open conformations are the smallest in CM2. In CM1 and CM3, formation of the complex in the closed conformation of NTR is energetically unfavorable, while formation of the complex in the open conformation of NTR and opening of the DP_cpvtN2 -bound NTR are energetically favorable, suggesting low binding affinity of DP_cpvtN2 to the closed conformation, high binding affinity of DP_cpvtN2 to the open conformation, and high open probability of the complex. In contrast, in CM2 the energy of complex formation is highly favorable for both, open and closed conformations of NTR, which suggests high binding affinity of DP_cpvtN2. The opening of DP_cpvtN2 -bound NTR is energetically unfavorable, which suggests low open probability of the complex. This analysis confirms that complex formation between DP_cpvtN2 and NTR is energetically plausible and that models CM1 and CM3 on one hand and model CM2 on the other hand predict different thermodynamic properties of DP_cpvtN2—RyR2 binding.

The energy differences between the open and closed conformations of CM1 and CM3 (Table 2) were consistent with high open probability of the complex (ΔΔG_opening < 0), in accordance with GS1. In contrast, the energy difference between the open and closed conformations of CM2 was consistent with low open probability of the complex (ΔΔG_opening > 0), in accordance with GS3.

The energy differences between NTR in the closed conformation with unbound DP_cpvtN2 on one hand and the closed conformations of CM1 and CM3 on the other hand suggest a low binding affinity between DP_cpvtN2 and NTR (ΔΔGbindingC> 0) in accordance with GS1; the energy difference between NTR in the closed conformation with unbound DP_cpvtN2 and the closed conformation of CM2 suggested a high binding affinity (ΔΔGbindingC < 0) in accordance with GS3.

The energy differences between NTR in the open conformation with unbound DP_cpvtN2 on one hand and the open conformation of all three models on the other hand suggested a high binding affinity between DP_cpvtN2 and NTR (ΔΔGbindingO < 0). Since GS1 assumes the same binding affinity for the complex in the closed as well as in the open conformation, CM1 and CM3 are at odds with the results of bilayer experiments. The larger binding energy of CM2 in the open than in the closed conformation (ΔΔGbindingO − ΔΔGbindingC = −720 kJ/mol) is in agreement with the prediction of GS3, which postulates that the change of opening energy induced by ligand binding is reflected in the increase in binding affinity. In other words, only CM2 and GS3 are mechanistically compatible.

To find out whether CM2 is compatible with the “domain switch” mechanism involving domains A and B of the N-terminal region, the inter-domain and peptide-NTR interactions were analyzed further. The interactions between amino acids R417, R298, E40, and D61 were previously found to be important for the stability of NTR (Borko et al., 2014), and are summarized for individual NTR molecules in Table 3. In the closed conformations (Figures 4A,B), interactions present in NTR—the hydrogen bond between amine group of R417 and carboxyl oxygen of E40, and the hydrogen bond between the amine group of R298 and carboxyl oxygen of D61 were disrupted in CM2 due to conformational changes evoked by formation of hydrogen bonds between the main-chain nitrogen of H1 in DP_cpvtN2 and the carboxyl oxygens of D61 in NTR. In the open conformation (Figures 4C,D), the overall number of hydrogen bonds between R417, R298, E40, and D61 was higher for CM2 than for the free NTR: the amine nitrogens of R298 and the carboxyl oxygens of D61 formed two hydrogen bonds in NTR but three hydrogen bonds in CM2, the amine nitrogens of R417 and the carboxyl oxygens of E40 formed one hydrogen bond in NTR but three hydrogen bonds in CM2, and one hydrogen bond was present between the amine nitrogens of R417 and the carboxyl oxygens of E61 in NTR but not in CM2. As in the closed conformation, these changes in hydrogen bonding were accompanied by formation of hydrogen bonds between the main-chain nitrogen of H1 in DP_cpvtN2 and the carboxyl oxygens of D61 in NTR. A major conformational change was also observed in the region encompassing D318 and E320, in which no important inter-domain interactions were present in NTR (Figures 4E,G) but in CM2 a network of H-bonds (Figures 4F,H) was formed between the NTR carboxyl oxygens of D318 and E320 the DP_cpvtN2 amine and imine nitrogens of R5 (5 hydrogen bonds in the closed conformation and 3 hydrogen bonds in the open conformation) and the DP_cpvtN2 imine nitrogen of H1 (1 hydrogen bond in the open conformation).

The peptide formed a number of further hydrogen bonds and ionic and hydrophobic contacts with amino acid residues from domains A and B (Table 4). Out of 11 H-bonds in the closed conformation, 4 remained present in the open conformation while 5 new H-bonds were formed. The hydrogen bonds between the peptide and the domains A, B of NTR (Table 4) did not have a counterpart in equivalent H-bonds between the central helix and the domains A, B of free NTR (column 1 and 3 of Table 5). As an example, the amine nitrogen of R414 formed a hydrogen bond with the main chain oxygen of S126 from the A domain of NTR in the free NTR as well as in CM2, while the amine nitrogens of the corresponding peptide amino acid R5 formed hydrogen bonds with carboxyl oxygens of E320 and D318 from the B domain of NTR. The number of H-bonds between the peptide and domains A, B was comparable to the number of H-bonds between the central helix and domains A, B, which decreased from 12 to 9 upon peptide binding in the closed conformation (columns 1 and 2 of Table 5) and remained at 7 in the free NTR as well as in CM2 in the open conformation (columns 3 and 4 of Table 5).

Hydrophobic and ionic interactions of domains A, B with the domain peptide and with the central helix were also different: In the CM2 complex, DP_cpvtN2 amino acids I10 and V14 formed hydrophobic contacts with the respective amino acids L46 and F48 from the A domain of the NTR, and DP_cpvtN2 amino acid R5 (corresponding to R414 of the central helix) formed ionic contacts with D318 and E320 from the B domain of the NTR. On the other hand, no hydrophobic contacts were present between the central helix and domains A, B, since the hydrophobic side of the central helix was positioned toward domain C; ionic contacts were formed between the central helix amino acid R417 and E40 and D61 from the A domain and between the central helix amino acid E412 and R235 from the B domain. Thus, the nature of hydrogen bonds, ionic and hydrophobic contacts between the central helix or the domain peptide on one hand and domains A, B on the other has no similarities. The similarity between the effect of DP_cpvtN2 and that of central helix mutations on RyR activity is therefore not caused by competition between the domain peptide and the corresponding central helix amino acids for the binding site at the “domain zipper,” as postulated for the “domain switch” mechanism (Ikemoto and Yamamoto, 2000; Yamamoto et al., 2000; Tateishi et al., 2009; Suetomi et al., 2011). It is conceivable, that RyR2 activation by DP_cpvtN2 and its destabilization by central helix mutations arises from similarities between the energy changes caused by complex formation and those caused by mutation of amino acids in the central helix.

To compare the energy changes caused by complex formation and by mutation of amino acids in the central helix, we have created models of NTR, each with one of the seven reported mutations, in the closed and open conformation and optimized them by energy minimization. Total energies of individual molecules of WT and mutant NTRs in the closed and open conformation are compared in Figure 5 (the numerical values are also given in Table 6). Complex formation between NTR and DP_cpvtN2 (Table 2) is energetically favorable since the energy of both, the closed and the open conformation is more negative than the energy of the free NTR and free DP_cpvtN2. This is reflected in the high binding affinity of DP_cpvtN2 to the NTR. On the contrary, all models of mutant NTRs show higher, less favorable energies than the wild type NTR (Figure 5A). Thus, the energy changes induced by complex formation and by amino acid mutations are in the opposite direction.

The conformation of the amino acids E40, D61, R298, and R417 involved in formation of the hydrogen-bonding network showed no significant differences from the wild-type NTR in the models of mutant NTRs either in the closed or in the open conformation (Figure 6). In the closed conformation, the number of hydrogen bonds remained unchanged (n_HB = 4) in all mutants. In the open conformation, the number of hydrogen bonds remained unchanged (n_HB = 4) in three mutants (I419F, L433P, and R414L), it increased by one in two mutants (R414C and R420W) and it decreased by one in one mutant (T415R) (Table 3). The configuration of the hydrogen-bonding network involving the central helix cannot therefore be the main reason for the increased total energy of mutant NTRs.

To determine, which interactions are most important for the energy changes observed upon the closed/open conformational transition and upon mutation, we have tested linear models incorporating the number of intramolecular interactions (including hydrogen bonds) between and within subdomains (i.e., domains A, B, C and the central helix). The complex of NTR with DP_cpvtN2 was not included into the analysis, since its total energy is heavily affected by intermolecular interactions that are not present in the free NTR molecules. The energy difference between the open and closed conformation of WT and mutant NTRs could not be directly expressed as a function of the number of intramolecular interactions (in all tested models, the prediction for at least two out of 8 molecules had a relative error of >20%). The total energy of NTR and its mutants in both the closed and the open conformation could be satisfactorily approximated (relative error of <5%; R = 0.62, CV = −0.022), assuming a linear dependence on the number of ionic interactions between subdomains (NIE, F = 12.3, P = 0.004) and on the number of hydrogen bonds between amino acid side chains (NHBSC; F = 9.3, P = 0.009):

That is, the increased energies of mutant NTRs were mostly due to a decreased number of ionic interactions between the subdomains and a decreased number of hydrogen bonds between amino acid sidechains. Hydrogen bonds involving main-chain atoms, ionic interactions within subdomains, and hydrophobic, aromatic and cation-π interactions contributed to the total energy of the molecules on average by −26039 kJ/mol. Contributions of these interactions to the total energy could not be further dissected, most probably because of their low number (aromatic and cation-π interactions) or because of a smaller variation between individual studied molecules (hydrogen bonds involving main chain atoms, ionic interactions within subdomains, and hydrophobic interactions).

In the closed conformation, mutations did not change the number of inter-domain ionic contacts (NIE = 15 for WT and NIE = 13.9 ± 0.7, N = 7 for mutant NTRs) but decreased the number of hydrogen bonds between amino acid side-chains (NHBSC = 178 for WT and 157 ± 2.7, N = 7 for mutant NTRs, P < 0.001), which is in accordance with the higher total energy of mutant NTRs when compared with the wild type. In the open conformation, the number of inter-domain contacts was not significantly changed by mutations (NIE = 12 for WT and 11.7 ± 0.2 for mutant NTRs) and the number of hydrogen bonds between amino acid side-chains in mutant NTRs was smaller only slightly albeit significantly (NHBSC = 154 for WT and 151 ± 0.7, N = 7 for mutant NTRs, P < 0.01).

While mutant NTRs differed from CM2 in having a higher total energy than the wild-type NTR, the energy differences between the open and closed conformation of the NTR showed a similar tendency for both, CM2 and mutant NTRs (Figure 5B). With the exception of R414C, the mutant NTRs shared with the complex a lower energy necessary for channel opening (Figure 5B). The value of ΔΔG_opening was 1,091 ± 224 kJ/mol (mean ± SE) for mutant NTRs on average and 860 kJ/mol for CM2, compared with 1,580 kJ/mol for wild-type NTR. These data are consistent with a higher open probability for channels with mutant NTRs as well as for channels with bound DP_cpvtN2.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.