Life essentially runs on electricity: electrical signals cause nerve cells to fire, heart muscles to contract and allow organisms to sense the world around them. These signals are triggered by the movement of positively-charged ions—such as sodium, potassium and calcium—moving into a cell through special ion channels in the cell membrane, which can open and close in response to changes in the voltage across the cell membrane.

With few exceptions, voltage sensitive ion channels usually only let one type of ion pass into the cell. But how do ion channels discriminate amongst ions and how did they acquire this ability during evolution? To address these questions, researchers have studied a family of sodium channels from bacteria for the past decade. Here DeCaen et al. describe a new member from this ion channel family from a bacterium called Bacillus alcalophilus. This ion channel does not discriminate between positively-charged ions and B. alcalophilus needs this ion channel for it to dwell in environments that have high levels of potassium or sodium. DeCaen et al. demonstrate that these ion channels can be made selective for sodium or calcium with as little as two small changes in the gene that encodes the ion channel. Furthermore, making similar genetic mutations in related ion channel genes from other Bacillus species has the same effect. DeCaen et al. suggest that Bacillus ion channel genes are easily adapted to function in a variety of environmental conditions with different levels of positively-charged ions. Thus it is easier for Bacillus channels to evolve to be selective for different ions.

Bacillus bacteria divide rapidly in warm to hot temperatures and under alkaline pH. DeCaen et al. demonstrate that both of these conditions make Bacillus ion channels easier to open in response to voltage. In addition, DeCaen et al. demonstrate that Bacillus ion channels can be targeted by drugs that impair the ability of the bacteria to grow. These findings—together with other work that revealed where drug molecules bind to ion channels—could potentially guide efforts to develop treatments for illnesses caused by other Bacillus strains, which include anthrax and some forms of food poisoning.

Ion selectivity is a defining feature of ion channels. While the structural and biophysical determinants of K⁺ selectivity are well described (Doyle et al., 1998; Jiang et al., 2003), those of Na⁺ and Ca²⁺ are unresolved. To fill this knowledge gap, the voltage-gated sodium channels (Na_v channels) from bacteria have been extensively studied by structural biologists. To date, two full-length (NavAb and NavRh) (Payandeh et al., 2011, 2012; Zhang et al., 2012) and three pore-only (NavMs, NavAe and NavCt) (McCusker et al., 2012; Tsai et al., 2013; Shaya et al., 2014) prokaryotic Na⁺ channel crystal structures have been solved. The full-length structures demonstrate that four identical subunits, each containing 6-transmembrane segments, assemble together to form a functional channel. The first four transmembrane segments form the voltage sensor (S1–S4) while the fifth and sixth transmembrane segments (S5, S6) form the pore domain. The selectivity filter, which forms critical interactions with the permeating hydrated ions, defines the pore and is scaffolded by two pore helices (P1 and P2) from each subunit. The dipole created by the helices, and an acidic residue from each subunit, create an electronegative region that attracts cations. However, the molecular arrangement that enables Na⁺-selectivity in prokaryotes might differ from mammalian voltage-gated Na⁺ channels. Thus, it is not clear if structural features that determine ion selectivity in homotetrameric prokaryotic Na_V reflect the functionally heterotetrameric eukaryotic sodium channels.

The first prokaryotic voltage-gated sodium channel, cloned from Bacillus halodurans C-125, was called NaChBac (Na⁺ Channel of Bacteria) (Ren et al., 2001). Since then, Na_V channels from at least 9 bacterial species have been functionally characterized. All full-length channels exhibit Na⁺-selectivity (Ren et al., 2001; Ito et al., 2004; Koishi et al., 2004; Irie et al., 2010; Ulmschneider et al., 2013). Most bacteria require the ion-motive forces provided by H⁺ or Na⁺ ions that move through the MotAB or MotPS stator to power the rotary motor proteins to drive the motion of the flagella, (Figure 1A) (Ito et al., 2004; Fujinami et al., 2009). In soda lakes and other extremely H⁺-poor (pH 9–12; 1 nM—1 pM [H⁺]) and Na⁺-rich environments (up to 500 mM), alkaliphilic prokaryotes couple motility and ion transporters to Na⁺ rather than H⁺. Growth and motility of the alkaliphilic bacterium Bacillus alcalophilus AV (Vedder, 1934) are better supported in K⁺ rather than Na⁺ conditions (Terahara et al., 2012). In alkaliphilic Bacillus, either ion can drive the flagellar motor, and a single mutation in the MotS protein was identified that converted the naturally nonselective MotPS (Na⁺ or K⁺) stator into a Na⁺-selective stator. Presumably, bacterial Nav channels provide a source of Na⁺ ions that drives the stators and maintains ion homeostasis. However, it is unclear what condition or stimulus would open these channels at the very hyperpolarized resting membrane potentials (Ψ_rest ≈ −180 mV) found in bacteria.

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Here, we determine the permeation and gating properties of a voltage-gated ion channel (NsvBa) from Bacillus alcalophilus AV. While related to members of the Na_v superfamily (Figure 1B), NsvBa is nonselective among cations. Using mutagenesis, we demonstrate that the nonselective filter from NsvBa can be converted into one that is more selective for Na⁺ or Ca²⁺, and that these features can be conferred onto the NaChBac filter. In addition to selectivity, we characterize the voltage-, pH- and temperature-dependence of Bacillus Na_v channels. We find that a combination of higher temperature and pH are required to reduce the activation threshold of channel opening in Bacillus Na_v channels, which is unique to this family of sodium channels. We also characterize Na_v current antagonism by drugs that impaired the growth and motility of alkaliphilic Bacillus species at corresponding concentrations. These findings shed light on the biophysical requirements for ion channel selectivity, pharmacology, biochemical adaptations among Bacillus species, and the evolution of voltage-gated Na⁺ channels.

Among the first alkaliphilic extremophiles described in the literature, the gram-positive, rod-shaped Bacillus alcalophilus AV was initially isolated from human feces (Vedder, 1934). We cloned NsvBa and generated plasmids for mammalian cell expression to enable measurement by patch clamp methods. Currents from NsvBa-transfected HEK293T cells were robust (≈119 pA/pF in 150 mM extracellular [Na⁺]) with voltage-dependent activation and inactivation similar to NaChBac (Figure 1—figure supplement 1A,B). The time constant of NsvBa Na⁺ current inactivation (τ_inact) measured at 0 mV was 42 ms, ∼2 times faster than NaChBac (78 ms) but ∼6 times slower than NavMs (7 ms, Figure 1—figure supplement 1C). The sequence TLESWxxG is conserved in the selectivity filters of prokaryotic Na⁺ channels (Figure 1C). Although the sequence of NsvBa is homologous to other prokaryotic Na_v channels, the selectivity filter sequence (TLDSWGSG) deviates at the high field-strength site (Site_HFS most extracellular site; see Discussion) with an aspartate residue replacing glutamate (D186) and a flexible glycine at an extracellular position (G189). When aligned, G189 and G191 form a ‘GxG motif’ commonly found in K⁺-selective (Kv) and nonselective ion channels (e.g., TRP and CNG channels, Figure 1C). To determine the selectivity of the NsvBa channel, we patch clamped transiently-transfected HEK cells and measured voltage-dependent currents in the presence of monovalent alkali ions (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺) or divalent alkaline earth metals (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) (Figure 2A,B). Compared to the relatively Na⁺-selective NaChBac channel, NsvBa was nonselective and all cations but Cs⁺ and Mg²⁺ permeated the pore (Figure 2C,D). NsvBa Na⁺ and K⁺ single channel conductances were equivalent (30 ± 3 pS and 36 ± 3 pS, respectively, Figure 2—figure supplement 1), indicating that the channel does not distinguish between these ions.

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Given the 61% shared amino acid identity between NaChBac and NsvBa channels, we were able to introduce the NaChBac selectivity filter (TLESWASG) into the NsvBa channel to examine two relevant residues, D186E and G189A. These changes conferred Na⁺-selectivity onto the channel and abrogated all other monovalent and divalent conductances (Figure 3A,B). When tested separately, the single mutation D186E was sufficient for Na⁺-selectivity (Figure 3—figure supplement 1A,B), whereas the single mutation of G189A substantially reduced, but did not entirely abolish, the permeability of K⁺ and Rb⁺ (Figure 3—figure supplement 1C,D). Furthermore, the G189A mutant channel still conducted Ca²⁺ and Ba²⁺ divalent ions, whereas the D186E mutation did not. These data suggest that a glutamate at Site_HFS selects Na⁺ among other cations while the ‘GxG motif’ likely provides an extracellular K⁺ or Rb⁺ ion coordination site, possibly involving the glycine backbone carbonyls as found in the selectivity filter of the potassium and NaK channels (Doyle et al., 1998; Jiang et al., 2002; Alam and Jiang, 2009a; Alam and Jiang 2009b). Next we sought to determine whether the NsvBa filter sequence could be transferred into Na⁺-selective NaChBac. When the Na⁺-selectivity filter of NaChBac (TLESWASG) was mutated at two positions (E192D:A195 G) to conform to the NsvBa nonselective filter (TLDSWGSG), ion selectivity was identical to that of NsvBa (Figure 3C,D). This finding illustrates the mutual portability of selectivity between the voltage-gated cation channels in Bacillus species halodurans and alcalophilus AV.

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The selectivity for Na⁺ ions in vertebrate Na_v channels is attributed to an asymmetric ring of 4 amino acids (Asp, Glu, Lys, and Ala: DEKA) contributed by each of the pore-lining loops of the 4 domains (Catterall, 2012). Voltage gated calcium channels (Ca_Vs) are thought to achieve Ca²⁺-selectivity by a symmetric ring of 4 glutamate residues (EEEE), each contributed by one domain of the polypeptide (Hess et al., 1986). In contrast, prokaryotes achieve Na⁺-selectivity from an apparent 4-fold symmetry of acidic residues, each from a subunit in the homomer. In previous studies, we demonstrated that the Na⁺-selective NaChBac (TLESWASG) filter could be converted into one that prefers the divalents Ba²⁺ and Ca²⁺ by introducing acidic residues into three positions in the filter sequence (TLDDWADG) (Yue et al., 2002). This filter sequence also was grafted into the NavAb channel (called CavAb), shown to be more Ca²⁺-selective, and the high-resolution structure determined (Tang et al., 2014). We tested the LDDWADG mutated filter (S187D:G189A:S190D) in the NsvBa channel and confirmed that it was divalent permeant (Ca²⁺, Sr²⁺, Ba²⁺) but it also had measurable permeability to monovalent cations (including K⁺ and Rb⁺), demonstrating that Ca²⁺-preference achieved by this filter (P_Ca/P_Na∼30, Figure 4; Figure 4—figure supplement 1) is much lower than mammalian Ca_V channels (P_Ca/P_Na ≳ 1000) (Tsien et al., 1987). Rather, this selectivity is more analogous to some members of the TRP channel family, such as TRPV5, TRPV6 (Owsianik et al., 2006; Wu et al., 2010). A comparison of the filter mutations effects on relative permeability of the NaChBac and NsvBa channels are summarized in Figure 4 and listed in Figure 4—figure supplement 2. We also attempted to convert the NsvBa channel into a K⁺-selective channel by changing the selectivity filter sequence (TLTSWGSG and TLTSWGYG), but these channels either did not express on the plasma membrane or did not conduct cations under our experimental conditions (data not shown).

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Alkaliphilic Bacillus are estimated to have very negative resting membrane potentials (Ψ_rest ≈ −180 mV), although membrane potentials in bacteria are measured from voltage- sensitive dye studies and variability within populations can be large. Nevertheless, the activation threshold for Bacillus sodium channels is ≈ −40 mV, which is extremely depolarized relative to estimates of Ψ_rest. Since alkaliphilic bacteria live in high pH environments, we tested whether their sodium channel gating shifted as a function of pH. As shown for the Na⁺-selective channel from Bacillus pseudofirmus OF4 (NavBp) (Ito et al., 2004), Na⁺ currents from NaChBac and NsvBa are also modulated by high extracellular pH (Figure 5). When extracellular pH was increased from 7.4 to 9.4, the peak current increased twofold to fourfold and the steady state voltage-dependence was negatively shifted by 28–34 mV (Figure 5—figure supplement 1). Basic extracellular pH alone is probably insufficient to reduce this substantial energy barrier to activate these channels from Ψ_rest = −180 mV (≈−3.2 kcal/mol). Thus additional influences are required to bring Ψ_rest and V_1/2 closer together.

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Many alkaliphilic Bacillus species growth rates are temperature-dependent (30–60°C). Thus we tested the effects of temperature (20–37°C) at neutral and basic extracellular pH (7.4 and 9.4, respectively) on the Na⁺ currents conducted by Bacillus NsvBa, NaChBac and NavBp channels (Figure 6A–C). At neutral pH, we observed that increasing the temperature shifted the voltage dependence of activation for these channels by −19 to −24 mV (−62, −55, −51 mV respectively). When tested together, temperature and basic pH effects on V_1/2 for NsvBa, NaChBac and NavBp were additive, converging on ≈−100 mV (−95, −102, −100 mV respectively). Importantly, we determined that the NsvBa remains a non-selective channel at higher pH and temperature, although the P_x/P_Na for K⁺ and Ca²⁺ did increase slightly 2–3 times (Figure 6—figure supplement 1). To quantify the temperature sensitivity of these channels, the relationship between the peak current during a voltage ramp and temperature was fit to a linear equation to determine the 10-degree temperature coefficient (Q₁₀). The Q₁₀ for NaChBac and NavBp peak currents was 3.5–4.4 at neutral pH and 3.5–4.1 at basic pH. In contrast, the voltage-dependence of activation of the hNa_v1.1 channel was less temperature- (Q₁₀ = 1.2 and 1.4, Figure 6D) and pH- (Δ V_1/2 < 8 mV, Figure 5—figure supplement 1) sensitive. Thus, pH and temperature-induced increases of the peak current and reduction of the voltage-dependence of activation are distinct from eukaryotic Na_v channels.

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The stimuli that depolarize these bacteria from −180 mV to the more depolarized range where voltage-gated channels activate (−40 to −100 mV, depending on pH and temperature), are not known. We speculate that Ψ_rest declines as bacterial pumps are starved for internal H⁺ or Na⁺ to supply the hyperpolarizing extrusion pumps. Internal [Na⁺] would be rapidly recharged by activation of the voltage-gated monovalent cation channels. Since cation entry into alkaliphilic bacteria is at least partially dependent on Na_v channels, we hypothesized that Na_v channel antagonists would attenuate bacterial growth. Under voltage clamp, we observed that sodium current from Bacillus channels NavBp, NaChBac and NsvBa are blocked by known Na_v channel antagonists, the local anesthetic lidocaine, the anti-hypertensive nifedipine, and the anti-estrogen tamoxifen, with similar potencies (Figure 7A,B and Figure 7—figure supplement 1). When these drugs were introduced into the culture media, growth of Bacillus species alcalophilus and pseudofirmus were severely impaired as measured by spectroscopic absorbance (Figure 7C,D). The measured half-inhibitory concentrations (IC₅₀) of sodium current and bacterial growth were within a half-log unit (Figure 7—figure supplement 1), suggesting that growth inhibition was not an off-target effect. We also examined the effect of these drugs on two matrices of Bacillus motility: tumbling frequency and swim speed. tamoxifen, nifedipine and lidocaine increased tumbling frequency and decreased swim speed of B. pseudofirmus (Figure 7E, Figure 7—figure supplement 1). However, B. alcalophilus swim speed was not delayed by the three drugs and only tamoxifen and nifedipine increased tumbling frequency (EC₅₀ = 70 μM and 375 μM, respectively).

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We have functionally characterized NsvBa, a cation nonselective voltage-gated ion channel from Bacillus alcalophilus. Our findings demonstrate that this family of prokaryotic voltage-gated Na⁺ channels are not exclusively Na⁺-selective and that the filter sequence that controls passage of cations into the bacteria are transferable within the Bacillus genus. Through a series of mutations, we showed that the selectivity of Bacillus Na_v channels can be converted from nonselective, to relatively higher Na⁺ or Ca²⁺ selectivity, suggesting that the filter is readily mutable in evolution to adapt to ionic conditions. These highly adaptable selectivity filters are critical for the Bacillus alkaliphiles, allowing for the habitation of various cation-rich environments in which they presumably evolved. It is anticipated that the ion-specificity of at least some antiporters will parallel that of the major coupling ions for the voltage-gated channels that provide a significant amount of their cytoplasmic substrate.

Although no Na⁺ ions reside within the filter of the wild type NavAb crystal structures, 3 Na⁺ coordinating sites were proposed: glutamate side chains form a high-field-strength site (Site_HFS) near the extracellular end of the filter, while backbone carbonyls of Leu and Thr comprise central (Site_CEN) and inner sites (Site_IN) (Payandeh et al., 2011). In the CavAb structure, 3 hydrated Ca²⁺ ions were found coordinated within the filter and 3 sites (Sites 1–3) proposed within the filter. Site 1, the one closest to the extracellular surface, is formed by 4 Asp carboxyl side chains, equivalent to positions Ser 196 in NaChBac and Ser 190 in NsvBa. Site 2 (equivalent to Site_HFS in the wt NavAb crystal structure) is formed by four side chain carboxyls and four backbone carbonyls from equivalent residues of Glu 192 in NaChBac and Asp 186 in NsvBa. Our data suggest that the shorter side chain of the NsvBa Asp acidic residue at site 2 is correlated with decreased Na⁺-selectivity, but Na⁺ selectivity can be artificially endowed when replaced by a Glu (D186E) (Figure 3—figure supplement 1). In agreement with this interpretation, a recent report demonstrates that when the longer side chain Glu 192 is mutated to the shorter Asp in NaChBac (E192D), the channel becomes less selective for Na⁺ among Ca²⁺ and K⁺ ions (Finol-Urdaneta et al., 2014). The full-length bacteria Na_V from marine α-proteobacterium Rickettsiales HIMB114 (NavRh) was crystallized containing the selectivity filter TLSSWET-. Although the NavRh channel did not function when heterologously expressed in mammalian and insect cells, the filter was found to be Na⁺-selective when grafted into the NaChBac channel. It is surprising that although the glutamates within the NavRh and NavAb filters originate at distinct positions (See Figure 1), the carboxyl side chains from both channels occupy the same positions in space (Zhang et al., 2012). Thus, it is likely that selectivity for Na⁺ over other cations is partly achieved by the geometry of the arrangement of the carboxylates at Site 2 (Site_HFS) within the filter of bacterial Na⁺ channels. High-resolution crystal structures of Na_v channels in which sodium ions are resolved are needed to confirm this hypothesis.

Among all sequenced living species, naturally occurring nonselective voltage-gated ion channels are rare. It is not clear whether cation-selective or nonselective filters arose first within prokaryotes. Analysis comparing domains of bacteria and mammalian Na_v channels indicate that Na⁺-selectivity was independently acquired in these channels families (Liebeskind et al., 2013). Bacterial Na_v channels are functionally more related to homotetrameric CatSper and Kv channels and thus may not be direct ancestors of mammalian Na_v channels. Our results are consistent with this interpretation. Not surprisingly, we were not able convert Bacillus Na_v channels to K⁺-selective channels by mutating the filter sequence, presumably since the pore architecture between Kv and bacterial Na_v channels (e.g. lumen size, filter length and number of pore helices) are structurally divergent. Our results suggest that the ‘GxG motif’ found in the NsvBa filter evolved convergently within the bacterial Na_v family and is only distantly related to K⁺-selective Kv channels. A recent report on the expression and characterization of Na⁺ channel homologs from the invertebrate sea anemone Nematostella vectensis (NvNa_V2.1), revealed a heterotetrameric Na⁺ channels bearing noncanonical selectivity site (DEEA) that was not selective among K⁺ and Na⁺ ions (Gur Barzilai et al., 2012). Thus it appears that the homomeric prokaryotic NsvBa and functionally-heteromeric eukaryotic NvNa_V2.1 channels are examples of evolution of Na⁺-selectivity to non-selectivity that occurred independently within prokaryotes and metazoans.

In excitable eukaryotic tissues such as nerves and muscle, the half-activation threshold (V_1/2) of voltage-gated sodium channels is within ∼80 mV of their cellular resting membrane potential (Ψ_rest ∼ -60 to −90 mV). At first glance, this appears to contrast with the 140 mV difference between the Ψ_rest and V_1/2 of alkaliphilic Bacillus. But as we have shown, increasing alkalinity and temperature shift bacterial Na_v V_1/2's to approximately −100 mV. At temperatures approaching 40°C, our patch recordings became unstable, thus limiting the temperature range we tested to <37°C. If the sodium current sensitivity is extrapolated to 60°C, the voltage dependence would likely be well within 80 mV of the most negative bacterial resting membrane potential. Since bacterial Ψ_rest is a function of metabolic state (Na⁺/H⁺ pumping) and bacteria occupy more variable ionic environments than neurons, the larger difference between ‘optimal’ a Ψ_rest and V_1/2 makes physiological sense. The high temperature and pH sensitivity of bacterial Na_v channels is similar to TRP and some Kv channels (Patapoutian et al., 2003; Yang and Zheng, 2014), and is not shared by vertebrate Na_v channels. We suspect that the fidelity of neuronal firing in metazoans, which depend on their Na_V channels, led to evolution of less sensitivity to pH and temperature, perhaps by selection of the residues that become exposed to waters within the voltage-sensing S4 and selectivity filter (Chowdhury et al., 2014). In contrast, we propose that alkaliphilic bacteria, which dwell in high temperature (35–60°C) and high pH (9–11) environments, employ homomeric Na_v channels for an entirely different purpose, that of rapidly adjusting internal sodium concentration for metabolic control. This would thus impact growth rates of these Bacillus species as we have observed.

Concentrations of lidocaine, nifedipine, and tamoxifen that inhibit heterologously-expressed bacterial Na_v channels (NavBp and NsvBa) also block the growth of native Bacillus species (B. pseudofirmus and B. alcalophilus). Although all three drugs affected motility for B. pseudofirmus, only tamoxifen and nifedipine increased tumbling frequency of B. alcalophilus. These results suggest that in B. alcalophilus there may be other transporters or channels besides NsvBa that provide a cation source to drive flagellar motion. These finding demonstrate that block of Na⁺ entry via Na_v, which disrupts the sodium cycle, can impair Bacillus motility and growth. The prokaryotic Na_v from Magnetococcus marinus (NavMs), was recently crystalized with a brominated drugs bound and the proposed binding site validated (Bagneris et al., 2014). Thus voltage-gated channels found in pathogenic Bacillus (e.g. Bacillus cereus and anthracis) could potentially be antimicrobial targets.

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Author details

1. Paul G DeCaen

   Department of Cardiology, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, United States

   Contribution

   PGD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Yuka Takahashi

   1. Graduate School of Life Sciences, Toyo University, Gunma, Japan
   2. Japan and Bionano Electronics Research Center, Toyo University, Saitama, Japan

   Contribution

   YT, Acquisition of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

3. Terry A Krulwich

   Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   TAK, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Masahiro Ito

   1. Graduate School of Life Sciences, Toyo University, Gunma, Japan
   2. Japan and Bionano Electronics Research Center, Toyo University, Saitama, Japan

   Contribution

   MI, Conception and design, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

5. David E Clapham

   1. Department of Cardiology, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, United States
   2. Department of Neurobiology, Harvard Medical School, Boston, United States

   Contribution

   DEC, Conception and design, Drafting or revising the article

   For correspondence

   dclapham@enders.tch.harvard.edu

   Competing interests

   The authors declare that no competing interests exist.

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