Deprotonation of Arginines in S4 is Involved in NaChBac Gating

Deprotonation of Arginines in S4 is Involved in NaChBac Gating Tzur Paldi Abstract Voltage-gated ion channels participate in cell excitability by enabling selective ion flux in response to changes in the membrane potential. The channel senses voltage across the membrane via a voltage-sensing module composed of four membrane-spanning helices (S1-S4). A stretch of positively charged arginines in the fourth transmembrane segment (S4) that traverses the membrane s electric field, is the principal sensing component of the module. Yet, the driving forces behind S4 movement are not fully understood. The prevailing helical-screw model, which describes the movement of S4 along its axis, suggests that salt bridges between positively-charged residues on S4 and negatively-charged residues on S1-S3, alternately break and reform in the course of S4 movement. However, the estimated energy needed to separate the charges in a low dielectric cavity, is incompatible with experimental data (Green ME, J Theor Biol 193:475-483, 1998). Here, it is shown that extracellular titration of the three outermost arginines on S4 stabilizes the bacterial voltage-gated sodium channel (NaChBac) at different states, and enhances the coupling of the outermost arginine on S4, with E43 on S1. It is suggested that salt bridges that stabilize S4 are impaired by arginine deprotonation during voltage-sensing module activity. It is also shown that acid-induced channel blocking is strongly affected by arginine substitutions at S4. The electrostatic interactions of these arginines with acidic residues, exemplified by a structural model of NaChBac, suggests that extracellular acidification induces the retraction of S4, thereby enhancing channel inactivation by reclosure of the channel s activation gate. Keywords Electrostatic interactions Helical screw pk a shift Voltage-dependent ion channels Introduction Voltage-gated ion channels (VGICs) are ubiquitous Author: Tzur Paldi Email: tzur.paldi@outlook.com 2012 Tzur Paldi, All Rights Reserved 1 membrane proteins that are pivotal to the process of cell excitability. These proteins sense and respond to changes in membrane potential by opening, thus enabling selective ion flux (Hille 2001). The VGIC superfamily shares a topology of six trans-membrane segments (S1-S6), in which S5-S6 form the channel pore domain, and S1-S4 constitute the voltage-sensing module (VSM). A stretch of arginines within S4 is the principal sensing component of the VSM, coupling changes in membrane potential to conformational alterations, and transferring its energy to control the pore gate. The four outermost arginines on S4 are critical for channel gating (Seoh et al. 1996) and their interactions with conserved acidic residues on S1-S3 form an electrostatic network that functions in both protein folding, and in the regulation of channel activation (Tiwari-Woodruff et al. 1997; Paldi et al. 2010). Basic or acidic groups in proteins change their ionization state due to exchanges of protons with their environment, thereby modulating the structural and functional properties of the protein (Antosiewicz and Shugar 2011). Low extracellular ph (ph o) reduces the maximal conductance of many VGICs (usually referred as proton induced channel blocking) and modulates their gating properties, such that stronger membrane depolarizations are required to gate the channel open (Hille 2001). Nevertheless, a conserved proton-exchanging site in the VGIC superfamily is still elusive; thus, the effect of low ph o on VGICs remains an open question. The four outermost conserved arginines on S4 are expected to have a pk a of ~13 in water. This high pk a suggests that under normal physiological ph, the arginine residues would be positively charged. However, the pk a of protein residues is subject to dramatic shifts, depending on local microenvironmental conditions such as surface electric potential, solvent polarity, and ionic strength (Fersht 2001). Less polar or polarizable microenvironments will favor the neutral form of a basic residue; therefore, the pk a value of the basic residue will be downshifted, relative to its normal value in bulk water (Isom et al. 2011). Currently, there is very limited experimental knowledge about the ionization state of arginine residues buried in hydrophobic environments. Recently, Harms et al. (2011) showed that arginine residues introduced into internal positions in

staphylococcal nuclease exhibit no detectable shifts in their pk a; based on their observations they suggested that in small globular proteins the energetic cost for ionization of internal arginines is low, compared to other types of ionizable residues. The authors further proposed that neutralization of arginines is more likely to occur in the core of large membrane proteins. Accordingly, there are several reports of arginine residues that reside near or constitute a membrane channel pore showing unusual pk a values of 8.0 and less than 5.0 (Cymes and Grosman 2008; Cymes et al. 2005; Niemeyer et al. 2007). The putative location of the four outermost arginines on S4, inside the hydrophobic gating pore of the VSM, suggests that there, these residues may be found in their uncharged form, due to large pk a shifts. Changes in the ionization state of these charged residues would likely affect their function in the channel protein and may facilitate breakage of several salt bridges during S4 displacement. Attenuation of electrostatic interactions by discharging of arginines may explain the relatively low temperature dependency of channel gating, by reducing the high negative change in enthalpy produced by salt bridge formation, as has been postulated by Green (1998). To test this hypothesis, the Na + channel of Bacillus halodurance (NaChBac) was used to examine the effect of S4 arginine titration on channel activation properties. The four outermost arginines on S4 (R1-R4) were substituted, and the effect of low ph o on each channel mutant was monitored. The results suggest that the three outermost arginines on S4 undergo deprotonation during channel activation, a process that may form part of an electrostatic switch mechanism in VGIC gating. Materials and Methods Site-Directed Mutagenesis, Cell Line Culture, and Transfection The gene encoding NaChBac from Bacillus halodurans was amplified via PCR, and subcloned into the vector pcdna3.1 (Invitrogen, Carlsbad, CA, USA). Mutants were generated using standard PCR techniques, and their sequence integrity was verified. CHO cells grown in Dulbecco s modified Eagle s medium supplemented with 2 mm glutamine, 10% fetal calf serum, and antibiotics, were maintained at 37 C in 5% CO 2/air. Briefly, cells seeded on poly-l-lysine-coated glass coverslips (13 mm in diameter) in a 24-multiwell plate were cotransfected with pires-cd8 as a marker for expression, along with wildtype or mutant pcdna3.1-nachbac. For electrophysiology, CHO cells were visualized 2 days after transfection using the anti-cd8 antibody-coated beads 2 method (Jurman et al. 1994). All transient transfections were performed with TransIT-LT1 (Mirus, Madison, WI, USA) according to the manufacturer s instructions. Patch-Clamp Electrophysiology Recordings of macroscopic Na + currents mediated by NaChBac-transfected CHO cells were performed, using the whole-cell configuration of the patch-clamp technique, as previously described (Paldi et al. 2010). The patch pipettes were filled with: 5 mm NaCl, 105 mm CsF, 10 mm EGTA-Cs, and 10 mm HEPES. The ph was adjusted to 7.4 with NaOH. The bath solution contained: 150 mm NaCl, 2 mm KCl, 1.5 mm CaCl 2, 1 mm MgCl 2, 10 mm glucose, and 10 mm HEPES. The ph was adjusted to 7.4 or 6.0 with NaOH or methanesulphonic acid, respectively. Currents were measured from the same cell in a bath solution at ph 7.4, followed by perfusion of three bath volumes of bath solution at ph 6.0. All experiments were performed at 30 C, using a heated bath platform (Warner Instrument, Hamden, CT, USA) with a custom-made temperature controller. Data Analysis Data analysis was performed using IGOR Pro 6.1 (WaveMetrics, Portland, OR, USA). Steady-state conductance of the unmodified NaChBac and mutants (G) was calculated from peak currents (I), using the equation G = I/(V-V rev), where V is the test potential, and V rev is the reversal potential for the Na + current calculated from the x- intercept of the linear fit of currents, before and after reversal. G was normalized to the maximal conductance per cell (G/G max). Data points of normalized G-V curves were fitted to the two-state Boltzmann function: G/G max=1/{1+exp[zf(v 0.5- V)/RT]}, where G/G max is the normalized conductance relative to G max, V is the test potential, V 0.5 is the voltage required to elicit 50% of G max, z is the effective valence, F is the Faraday constant, R is the gas constant, and T is the temperature in degrees Kelvin. Unless otherwise stated, changes in Gibbs free energy (ΔG) were calculated from steady-state activation and availability curves at reference potentials (V t) of -30 mv and -80 mv, respectively, using the equation G = 0.2389zF(V 0.5-V t), as described in Paldi et al. (2010). The free energy that couples R1 to E43 was calculated by ΔΔG coupling= (ΔG Mut1 - ΔG WT) - (ΔG Mut1+Mut2 - ΔG Mut2). Changes in ΔG upon lowering the ph were estimated by ΔΔG ph 7.4 ph 6.0 = ΔG ph 6.0 - ΔG ph 7.4. The apparent changes in the ΔΔG ph 7.4 ph 6.0 of a mutant channel, as compared to that of a wild-type channel (ΔΔΔG ph 7.4 ph 6.0) were calculated as follows: ΔΔΔG ph 7.4 ph 6.0 = ΔΔG wt(ph 7.4 ph

6.0) mut (ph 7.4 ph 6.0) = ΔΔG mut (ph 7.4 ph 6.0) ΔΔG wt (ph 7.4 ph 6.0). Relative changes in G max when ph o was lowered from 7.4 to 6.0 (RCG max) were calculated and normalized as follows: 1 - [(1 G rel, mut) / (1 G rel, wt)], where G rel indicates maximal conductance values obtained at ph o 6.0, relative to those obtained at ph o 7.4. Statistics All experiments were performed on at least five cells. Unless stated otherwise, group data are reported as mean ± SEM. The Student s unpaired, two-tailed t-test was used in IGOR Pro for statistical comparison. Results R1-R4 Substitutions Affect NaChBac Sensitivity to Low ph o To test possible ionization changes in the four outermost arginines on S4, the effect of titration on channel gating properties, when the arginines were substituted with lysines, was monitored. The rationale for substituting arginine with lysine is based on the following: First, the lower pk a of the lysine s side chain, compared to that of arginine, makes it suitable for titration experiments. Secondly, the conservative nature of this mutation is essential for preventing misleading results arising out of radical changes in channel operation. Indeed, the effect of such substitutions on channel activity was shown to be minimal (Chahine et al., 2004). Lastly, despite the supposed charge conservation, arginine to lysine substitutions markedly reduced the coupling with a negatively charged residue at physiological ph (Paldi et al. 2010). Comparison of steady-state activation curves (G-V) of NaChBac and the mutants upon transition of the ph o from 7.4 to 6.0, revealed substantial differences (Fig. 1). The G- V curve of the unmodified channel shifted 45 mv toward more depolarizing potentials, as reflected in a ΔΔG ph 7.4 ph 6.0 of 3.3 ± 0.2 kcal/mol, and a 34 ± 2% reduction in maximal conductance at the tested voltage range (Fig. 1 and Table 1). Apparent changes in the ΔΔG ph 7.4 ph 6.0 of a channel mutant, as compared to the wild-type channel (ΔΔΔG ph 7.4 ph 6.0 values), indicated that the substitution R1K stabilized the closed state by 1.0 ± 0.3 kcal/mol upon extracellular acidification, and the R2K substitution stabilized the channel open state by 1.9 ± 0.2 kcal/mol (Fig. 2a). Due to very low expression levels of the R3K channel mutant, R3 was substituted with cysteine, as previously described (Chahine et al. 2004). The R3C substitution stabilized the channel open state upon extracellular acidification, by 3.3 ± 0.2 kcal/mol (Fig. 2a). The reduction in maximal conductance in the R1K channel mutant upon transition to ph o 6.0, did not significantly differ from that of the unmodified channel (reduction of 32 ± 4%; p > 0.05; n 5), but was substantially lower for R2K and R3C (reductions of 13 ± 3% and 5 ± 3%, respectively; Fig. 1 and Table 1). The G-V curve for the R4K channel mutant did not reach saturation at the tested membrane potential range (up to +70 mv), where its maximal conductance decreased by 74 ± 4% at ph o 6.0 (Fig. 1). Although the curve parameters for the R4K channel mutant at ph 6.0 could not be obtained, it clearly was the most susceptible to low ph o, as reflected in its low channel open probability. These results demonstrate that the R1-R4 on S4 play an important role in NaChBac modulation, upon changes in the external ph. Substitutions at R1, R2 and R3 Stabilize the NaChBac Open State at Low ph o in Differing Ways In NaChBac, R1 and R2, but not R3 and R4, are accessible to sulfhydryl-reactive reagents applied to the extracellular solution (Blanchet and Chahine 2007), and are likely accessible to extracellular protons. Accordingly, the interdependent modulation of NaChBac activation upon titration of R1 and R2 was examined by comparing the channel activation properties of the R1K:R2K double mutant, with those of each of the single mutants at ph o 7.4 and ph 6.0. Although the differences in G-V curves among the wild-type and mutant channels substantially diminished at ph o 6.0 (Fig. 2b), the R1K:R2K double substitution significantly stabilized the channel open state (p < 0.05; n = 6) in a non-additive manner, relative to each of the individual mutations (Fig. 2c). This finding implies that R1 and R2 cooperate in stabilizing the channel open state at a low ph o. In contrast, changes in the maximal conductance upon extracellular acidification were additive, relative to the changes in each single-channel mutant (Table 1). The R3C substitution stabilized the channel closed state at ph 7.4, but stabilized its open state at ph 6.0 (Fig. 2b, c), resembling the effect of the R1K:R2K double substitution. As the extracellular accessibility of R3 was reported in a voltage-dependent sodium channel (Yang et al. 1996), it is likely that R3 is directly affected by extracellular protons, but stabilizes a channel state different from that observed for titration of R1 or R2. 3

Fig. 1 R1 R4 on S4 determine sensitivity of NaChBac to low pho. Top Representative wholecell current traces in response to the indicated voltage protocol (inset) at ph 7.4, and at ph 6.0 for unmodified and mutant NaChBac. Bottom Steadystate activation curves normalized to the maximal conductance obtained at ph 7.4 (small error bars might be hidden by the data symbols) 4

Table 1 Activation parameters for NaChBac and mutant channels derived from steady-state activation curves (G V) at pho 7.4 and pho 6.0 pho Channel V0.5 (mv) z (e0) Grel DG (kcal/mol) n 7.4 wt -53.5 ± 1 4.2 ± 0.23 1-2.28 ± 0.13 6 wt 1.61 ± 0.15 a R1K -58.2 ± 1.8 2.2 ± 0.17 1 0.59 ± 0.07 a 5 R2K -34.6 ± 1.7 1.6 ± 0.06 1-0.17 ± 0.06 6 R1K:R2K -30.4 ± 1.8 1.3 ± 0.04 1-0.01 ± 0.05 6 E43C -10.6 ± 2.3 1.9 ± 0.05 1 0.87 ± 0.09 6 E43C:R1K -15.0 ± 1.2 1.9 ± 0.07 1 0.65 ± 0.07 5 R3C -20.3 ± 1.8 2.2 ± 0.13 1 0.49 ± 0.07 6 R4K -14.3 ± 2.2 1.4 ± 0.14 1 0.51 ± 0.13 5 6.0 wt -8.0 ± 1 2.1 ± 0.08 0.66 ± 0.02 1.04 ± 0.06 6 wt 2.94 ± 0.12 a R1K -3.3 ± 2.5 1.9 ± 0.11 0.68 ± 0.04 2.96 ± 0.16 a 5 R2K 1.1 ± 1.6 1.7 ± 0.07 0.87 ± 0.03 1.24 ± 0.08 6 R1K:R2K -17.7 ± 1.8 1.6 ± 0.06 0.87 ± 0.03 0.46 ± 0.07 6 E43C -1.7 ± 1.2 2.3 ± 0.10 0.97 ± 0.01 1.49 ± 0.09 6 E43C:R1K -12.7 ± 3.3 1.9 ± 0.08 0.98 ± 0.02 0.75 ± 0.16 5 R3C -20.0 ± 1.6 2.1 ± 0.12 0.95 ± 0.03 0.49 ± 0.07 6 a Calculated at reference potential of -70 mv Unlike the R1-R3 substitutions, R4K does not reduce the sensitivity of the channel to low ph o (Fig. 1). This could be due to the inaccessibility of R4 to the extracellular solution, as was previously reported for NaChBac (Blanchet and Chahine 2007) and for a voltage-dependent sodium channel (Yang et al. 1996) but, quite the contrary, the R4K mutant was hypersensitive to low ph o. This unexpected behavior of the R4K mutant channel is further elaborated upon below. Low ph o Enhances the Coupling of R1 with E43 Low ph o stabilizes the closed state of NaChBac, as is indicated by the voltage shift toward more depolarizing potentials upon channel activation. It was recently shown that the coupling of R1 on S4, with the extracellular E43 on S1, stabilizes the channel closed state at polarized potentials, in a voltage-dependent manner (Paldi et al. 2010). Thus, if R1 is neutralized due to its deprotonation upon channel activation, a low ph o would be expected to recharge it, and fortify ion pairing with E43. This hypothesis was tested by means of double-mutant cycle analysis, based on the data obtained from normalized G-V curves of single- and double-mutant channels at ph o 7.4 and at ph o 6.0 (Fig. 3a). At each ph o examined, the ΔΔG values of the E43C:R1K double substitution, as well as each of the corresponding single substitutions, were not additive and, overall, decreased at ph o 6.0 (Fig. 3b). Calculation of the coupling energy between E43C and R1K at reference voltages ranging from -70 mv to -10 mv revealed that lowering ph o from 7.4 to 6.0 fortified the coupling between E43 and R1, and considerably reduced its voltage-dependent elimination (Fig. 3c). Notably, the observed left shift of R1K substitution at ph o 7.4, diminished upon acidification, was restored on the background of the E43C substitution (Fig. 3a). This demonstrates the contribution of free protons to the electrostatic coupling between R1 and E43, and rules out the protonation/neutralization of E43 at ph o 6.0 (see Discussion). Proton-Induced Channel Blocking is Correlated with S4 Retraction Substitution of charged groups within the gating pore of the VSM may regulate channel gating by changing the electrostatic energy that dominates the movement of S4 along its track (Lecar et al. 2003). Since pore gating (activation and inactivation) is associated with VSM activation (Olcese et al. 1997; Olcese et al. 2001), conformational changes in the VSM are reflected in changes in channel gating. Therefore, normalized ΔΔG values obtained from steady-state activation and inactivation curves (Figs. 1 and Appendix Fig. A1) at ph o 7.4, were compared with the values yielded by a relative change in maximum conductance, obtained upon 5

Fig. 2 Extracellular acidification and substitution of R1, R2 and R3 causes modulations in NaChBac gating. a ΔΔΔGpH 7.4 ph 6.0 values calculated for the indicated substitutions. b Normalized steady-state activation curves at pho 7.4 and pho 6.0. c ΔΔG values calculated for channel activation at pho 7.4 (left) and at pho 6.0 (right). At pho 6.0, values that significantly differ from zero are indicated by an asterisk (p 0.05, n 5) a ph o transition from 7.4 to 6.0 (RCG max; see Materials and Methods for details ). In general, substitutions in the gating pore may exhibit positive ΔΔG values due to changes in S4 mobilization, which could be overcome by using higher potentials. Larger RCG max values indicate channel mutants that were blocked to a lesser extent by extracellular protons. The results show that R1K substitution promotes channel activation (i.e., the outward movement of S4), as is reflected in the negative ΔΔG values (for either activation/inactivation of the channel) of the R1K, and of R1K:R2K mutant channels. However, these changes have only a marginal effect on RCG max (Fig. 4), implying that RCG max is not related to conformational changes associated with enhancing the outward movement of S4 (i.e., VSM activation). The substitutions at R2, R3, E43 and R4 seem to hinder channel activation, but only R4K 6 substitution results in a dramatic, negative change in RCG max. Examination of the expected effect of each substitution on channel gating according to the electrostatic model (Lecar et al. 2003) reveals that substitutions at R2, R3 and E43 would be expected to symmetrically hinder both the outward and inward movement of S4, due to interference with salt bridge formation (Shafrir et al. 2008; Fig. 5). This implies that for these substitutions, ΔΔG values for channel activation also reflect S4 retraction, and thus are correlated with RCG max values. Conversely, R4 stabilizes the channel open state by forming a salt bridge with D60 (Shafrir et al. 2008; DeCaen et al. 2009; Fig. 5); its substitution would be expected to hinder S4 outward movement, and asymmetrically, to Fig. 3 Low pho enhances the coupling between E43 on S1, and R1 on S4. a Normalized steady-state activation curves at pho 7.4

and at pho 6.0. b ΔΔG values calculated for channel activation at pho 7.4 (left) and pho 6.0 (right). At pho 6.0, values that significantly differ from zero are indicated by an asterisk (p < 0.05; n 5). c Voltage dependence of the coupling between E43 and R1 Fig. 4 Proton-induced channel blocking is correlated with parameters that reflect the mobility of S4 in the gating pore. Normalized ΔΔG values calculated from G-V and availability curves of the indicated channel mutants (bars) are compared to normalized changes in maximal conductance when the pho is lowered ( ; RCGmax) promote S4 retraction. Thus, the ΔΔG values for R4K and R1K do not reflect the retraction of S4, and therefore show no correlation with RCG max values. Overall, these results are compatible with a ph-dependent reduction in channel maximal conductance that is related to S4 retraction. This possibility is not unlikely, because extracellular acidification stabilizes S4 in the closed state (Campbell and Hahin 1984; Claydon et al. 2007) and accelerates its retraction, as evidenced by the kinetics of the OFF-gating current in Na + channels (Campbell and Hahin 1984), and in K vs deactivation (Jiang et al. 1999; Claydon et al. 2007). It is likely that the hindrance of S4 retraction caused by a substitution, compensates for the reduced conductance mediated by extracellular acidification in the relevant channel mutants. Discussion The tendency of the channel G-V curves to converge upon extracellular acidification, regardless of their inclination to stabilize either the open or closed state upon mutation (Figs. 2b and 3a) suggests that protons play a dominant role in channel gating modulation. It is shown here that a single or double substitution in the gating pore of the VSM may reduce, or entirely eliminate, either the proton-induced 7

Fig. 5 Ribbon representations of the NaChBac VSM. Three putative prevailing conformations of NaChBac are shown: Rest (closed; left), Intermediate-closed (middle), and Open (right) states. The putative titrated region is flanked by dashed lines: S1, cyan; S2, green; S3, yellow; S4, orange. Negatively charged residues on S1-S3 are presented as red sticks. R1-R4 on S4 are presented as blue sticks. The coordinates of NaChBac models (Shafrir et al. 2008) were kindly provided by Dr. H. R. Guy voltage right shift, or channel blocking. These findings contradict the theory of charge screening by protons (Hille 2001) in NaChBac, and suggest that common conformational changes associated with S4 movement underlie these two proton-induced events. Currently, the helical screw model (Guy and Seetharamulu 1986) constitutes a plausible mechanism for the role of electrostatic interactions in channel activation. According to this model, S4 movement along the gating pore is mediated by the sequential formation and impairment of salt bridges between conserved, positively charged residues on S4, and negatively charged residues on S1-S3. R1, however, is exceptional, in that its coupling with E43 at rest exclusively stabilizes the channel closed state (Paldi et al. 2010). This feature of R1 enables more reliable dissection of the electrostatic pairing during channel activation, as well as upon extracellular acidification. It is shown here that the coupling between R1 and E43 is ph-dependent (Fig. 3). This finding suggests that destabilization of the channel closed state upon R1K substitution at ph o 7.4 is related to the lower affinity of lysine to protons, compared to that of arginine. This proposal is corroborated by restabilization of the closed state of R1K mutant channels by extracellular protons exhibiting gating properties similar to those of the unmodified channel (Fig. 3). Moreover, neutralization of E43 against the background of the R1K mutant channel (E43C:R1K) at ph o 6.0, but not at ph o 7.4, destabilizes the channel closed state, restoring the G-V left shift behavior observed upon R1K substitution at ph o 7.4. This suggests that protonation of the lysine that replaces R1, upon lowering the ph o, enhances the lysine s electrostatic coupling with E43. Overall, this ph-modulated doublemutant cycle analysis is consistent with a proton-controlled mechanism underlying the coupling between R1 and E43. The fact that extracellular protons enhance the coupling between E43 and R1 rule out the protonation and neutralization of E43 upon lowering the ph o, since such protonation would preclude salt bridge formation with R1. Furthermore, if E43 is protonated by low ph o, it is expected to destabilize the closed state of the R1K mutant channel when the ph o is lowered. In fact, the opposite occurs, and the closed state of the R1K channel is destabilized only after substitution of E43, as noted above (see E43C:R1K at ph o 6.0 in Fig. 3b). This finding implies that E43 remains deprotonated (charged) under these conditions. In addition, proton-induced enhancement of the coupling between E43 and R1 localizes the accessibility region to extracellular protons inside the VSM (Fig. 5c, dashed lines). Molecular dynamics simulations of the KvAP VSM show that E43 (E45 in KvAP) resides in a water-filled crevice and have no direct contact with the bulk solution (Freites 2006). This suggests that S4 arginine near E43 is exposed to relatively low dielectric surroundings of the membrane protein, while is still accessible to extracellular protons diffusing from the bulk solution. Unlike protonation of R1 and its interaction with E43, the titratable nature of R2 and R3 might appear to be less straightforward. However, in light of the helical screw model, the emergence of S4 causes R2 and R3 to interact 8

with E43 with each helical screw step (Fig. 5; Intermediate closed and Open states, respectively). Thus, their protonation may stabilize the coupling with E43, as shown for R1. Indeed, the interdependency between substitutions at R1 and R2 in stabilizing the open state at a low ph o (Fig. 2b, c) is in agreement with the stabilization of two different closed states (Rest and Intermediate-closed states) by protonation of R1 and R2 (Fig. 5). Accordingly, protonation of R3 alone may affect transitions between two substates of the activated VSM, as was suggested by Villalba-Galea et al. (2008). The results presented herein indicate that regulation of the S4 transitions by ph o not only determines channel gating properties, but also determines the maximal conductance of the channel. The proton-induced channel blocking of K vs is related to enhanced C-type inactivation (Zhang et al. 2003; Claydon et al. 2007). Since NaChBac inactivates via a C-type inactivation mechanism (Kuzmenkin et al. 2004), it would be expected that RCG max values of NaChBac mutants would be correlated with channel steady-state inactivation. The lack of such a correlation implies that RCG max may be correlated with a C-type enhancing process, rather than with C-type inactivation itself. Shin et al. showed that reclosure of the activation gate by decoupling the VSM from the pore, enhances current decay in HCN cation channels (Shin et al. 2004); Accordingly, reclosure of the activation gate, by ph o-induced retraction of S4, is likely an inactivation enhancer in NaChBac. The strong electrostatic interactions at low dielectric environments, compared to those occurring in bulk aqueous environment, imply the central role of salt bridge formation in the VSM gating pore in voltage sensing (Lecar et al. 2003). Hence, changes in the ionization state of arginines participating in these salt bridge interactions, suggest that the electrostatic switching mechanism may have a crucial role in both channel activation, and its regulation. Acknowledgments The author thanks Dr. Asher Peretz for valuable technical assistance, and for critical reading of the manuscript. References Antosiewicz JM, Shugar D (2011) Poisson-Boltzmann continuum-solvation models: applications to ph-dependent properties of biomolecules. Mol Biosyst 7:2923-2949 Blanchet J, Chahine M (2007) Accessibility of four arginine residues on the S4 segment of the Bacillus halodurans sodium channel. J Membr Biol 215:169-180 Campbell DT, Hahin R (1984) Altered sodium and gating current kinetics in frog skeletal muscle caused by low external ph. J Gen Physiol 84:771-788 Chahine M, Pilote S, Pouliot V, Takami H, Sato C (2004) Role of arginine residues on the S4 segment of the Bacillus halodurans Na + channel in voltage-sensing. 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Appendix Fig. A1 Voltage-dependence availability (steady-state inactivation) of NaChBac and mutants at ph o 7.4. The fit of the data points to a two-state Boltzmann function (smooth lines), was used to calculate ΔΔG values for NaChBac inactivation (see Fig. 4, in the main text). Curve parameters are listed in Table S1, below. Table A1 Availability curve parameters for NaChBac and mutant channels at ph o 7.4 Channel V 0.5 (mv) z (e 0) ΔG (kcal/mol) a n wt -79.2 ± 1.3 6.7 ± 0.2 0.1 ± 0.2 7 R1K -98.8 ± 1.3 5.4 ± 0.3-2.3 ± 0.2 8 R2K -76.8 ± 0.9 6.5 ± 0.2 0.5 ± 0.1 5 R3C -61.3 ± 1.0 4.6 ± 0.3 3.0 ± 0.1 7 R4K -67.1 ± 0.8 4.5 ± 0.4 1.3 ± 0.1 9 R1K:R2K -87.2 ± 0.8 5.0 ± 0.4-0.8 ± 0.1 5 E43C -66.4 ± 1.4 4.1 ± 0.2 1.3 ± 0.1 5 a calculated at reference potential of -80 mv 11