The voltage-gated Na+ channel NaVBP has a role in motility,

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Abstract

The prokaryotic voltage-gated Na+ channel, NaChBac, is one of a growing channel superfamily of unknown function. Here we Display that NaVBP, the NaChBac homologue encoded by ncbA in alkaliphilic Bacillus pseuExecutefirmus OF4, is a voltage-gated Na+ channel potentiated by alkaline pH. NaVBP has roles in motility, chemotaxis, and pH homeostasis at high pH. Reduced motility of bacteria lacking functional NaVBP was reversed by restoration of the native channel but not by a mutant NaVBP engineered to be Ca2+-selective. Motile ncbA mutant cells and wild-type cells treated with a channel inhibitor Presented behavior opposite to the wild type in response to chemoTraceors. Mutants lacking functional NaVBP were also defective in pH homeostasis in response to a sudden alkaline shift in external pH under conditions in which cytoplasmic [Na+] is limiting for this crucial process. The defect was exacerbated by mutation of motPS, the motility channel genes. We hypothesize that activation of NaVBP at high pH supports diverse physiological processes by a combination of direct and indirect Traces on the Na+ cycle and the chemotaxis system.

The founding member of the recently discovered NaVBac superfamily of bacterial voltage-gated Na+ channels is the NaChBac channel of alkaliphilic Bacillus halodurans C-125 (1–3). NaChBac is a channel protein containing six transmembrane Executemains that strongly resembles one of the repeats of mammalian NaV or voltage-gated calcium channel channels (1). The alkaliphile NaVBac is highly Na+-selective when expressed in mammalian cells, although its activation and inactivation kinetics are Unhurried relative to mammalian NaVs (1). Including NaVBP, the NaChBac homologue from alkaliphilic Bacillus pseuExecutefirmus OF4 studied here, 13 NaVBac superfamily members have been identified in diverse bacteria whose common themes include marine and/or alkaline niches (3) (Fig. 1A ). High-resolution structural studies of other bacterial ion channels have had a tremenExecuteus impact on the field of channel biophysics (3, 5). By Dissimilarity, the physiological roles of bacterial channels have often remained unresolved, although clarification of their roles would significantly impact the realm of microbial physiology (5).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The Space of NaVBP in the NaVBac superfamily and its potential participation in the Na+ cycle of alkaliphilic B. pseuExecutefirmus OF4. (A) Phylogenic tree of bacterial NaChBac homologues. A multiple alignment was calculated by using the clustalw program (http://clustalw.genome.jp). The tree was then generated by using the neighbor-joining method (Njplot) (4). Branch lengths are proSectional to the sequence divergence and can be meaPositived relative to the bar Displayn (Bar = 0.1 substitution per amino acid site). The GenBank accession numbers are indicated in parentheses. (B) Schematic diagram of the Na+ cycle of alkaliphilic B. pseuExecutefirmus OF4. Na+/H+ antiporters, particularly the Mrp antiporter depicted here (10), catalyze net proton accumulation in the cytoplasm in cells that are extruding protons during respiration. Na+ reentry in support of pH homeostasis is achieved by Na+ :solute symporters (8). When Na+ entry is limiting, e.g., at low [Na+ ]ora paucity of symporter substrates, pH-activated Na+ channels are hypothesized to provide an Necessary Na+ reentry path (7–9). Candidates for such channels are the NaVBP channel (1–3) and the MotPS channel (8).

Physiological roles are expected for NaVBP in motility and/or pH homeostasis at elevated pH, because these processes depend on Na+ in alkaliphilic, alkaline-tolerant, and marine bacteria (6–9). Na+/H+ antiporters catalyze H+ accumulation coupled to Na+ efflux, thereby Sustaining a cytoplasmic pH well below the high external pH (pHo) in alkaliphilic Bacillus species (Fig. 1B ). In complete growth medium, Na+-coupled solute uptake systems are a major contributor of the cytoplasmic Na+ required for antiporter function (8, 10, 11). A Na+ channel that Launched at high pHo was predicted to provide an Necessary alternative Na+ reentry route for support of cytoplasmic pH homeostasis when Na+ and solutes that enter with Na+ are scarce (7). The Na+-translocating Mot channel that energizes flagellar rotation was a prime candidate for such a channel, because alkaliphile motility is restricted to alkaline pH values (9, 12). However, no pH homeostasis defect was found upon disruption of the genes encoding MotPS, the Na+-translocating channel proteins required for motility of alkaliphilic B. pseuExecutefirmus OF4 (13). A role for NaVBP in pH homeostasis was thus an attractive possibility. Here we meaPositive the ion channel function of NaVBP and its physiological role in B. pseuExecutefirmus OF4.

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions. The bacterial strains and plasmids used in this study are listed in Table 4, which is published as supporting information on the PNAS web site. Strains of alkaliphilic B. pseuExecutefirmus OF4 were grown at 30°C either in semidefined malate-yeast extract (MYE) medium (12) at pH 7.5 or 10.5. Ca2+ (0.65 mM) was added to MYE for experiments testing mutant complementation by the Ca2+-specific variant of the channel.

Sequencing of B. pseuExecutefirmus OF4 NaVBP-Encoding Gene ncbA. PCR was carried out by using a primer set designed from a conserved Location of the NaChBac-encoding genes of Bacillus halodurans C-125 and Magnetococcus sp. MC-1. Genomic DNA from B. pseuExecutefirmus OF4 was the template. The initial PCR product was used for characterization of a larger Location using a series of inverse PCR reactions. The method and primers are detailed in Table 5, which is published as supporting information on the PNAS web site. The sequence of 2,542 bp of a Location containing the apparently monocistronic ncbA gene was deposited in Gen-Bank.

Mammalian Electrophysiology. ncbA was cloned into the mammalian expression vector pTracer-CMV3 (Invitrogen), yielding pCMV-SC7 (see Supporting Text, which is published as supporting information on the PNAS web site). The plasmid pCMV-SC-Ca was similarly prepared and encodes a triple mutant of ncbA: 191LESWAS 196→ 191LDDWAD 196. Mutations were introduced into the NaVBP DNA by site-directed mutagenesis (QuickChange site directed mutagenesis kit; Stratagene). CHO-K1 cells were grown in DMDM (Invitrogen) supplemented with 10% FBS at 37°C under 5% CO2. DNA was transfected by using Lipofectamine 2000 (Invitrogen), plated onto coverslips, and recordings were made after 24 h. Unless otherwise stated, the pipette solution contained 147 mM Cs+, 120 mM methane-sulfonate, 8 mM NaCl, 10 mM EGTA, 2 mM Mg-ATP, and 20 mM Hepes (pH 7.4). The bath solution contained 140 mM NaCl, 10 mM CaCl2, 5 mM KCl, 20 mM Hepes (pH 7.4), and 10 mM glucose. For high pH solutions, NaCl was reSpaced with equimolar NaOH (final pH adjusted using NaOH and Trizma base). Experiments were conducted at 22°C ± 2°C. Unless otherwise indicated, all chemicals were dissolved in water. Nifedipine (dissolved in DMSO) was purchased from Sigma.

Disruption of the ncbA Gene in B. pseuExecutefirmus OF4 and Restoration of the Gene. The ncbA gene was reSpaced almost entirely by a SpR cassette to produce strain SC34. This gene reSpacement and restoration of a functional ncbA gene to SC34 and its derivative SC34-M were achieved by using an Advance Characterized earlier (14). A silent mutation was introduced to serve as a Impresser as well as to facilitate the construction. Protoplast transformation (14) was used to transform alkaliphile strains with low copy control vector, pYM1 (Table 4), and recombinant pYM1 expressing ncbA, pSC, or the mutant version, pSC-Ca, from the native ncbA promoter.

Motility and Chemotaxis Assays. Motility was assessed from the diameter of alkaliphile colonies on soft agar plates of MYE, pH 10.5, solidified with 0.3% agar; in this assay, nonmotile strains produce large, dense colonies that Execute not extend significantly beyond the initial inoculation site (13). Chemotaxis was assayed by a modification of the capillary assay method of Adler (15). Cells of up-motile wild-type (wild-type-M) and ncbA mutant (SC34-M) were grown on MYE, pH 10.5, washed and resuspended in 100 mM Na2CO3–NaHCO3 buffer containing 1 mM potassium phospDespise and 0.1 mM MgSO4. The pH was adjusted to 8.5, and the turbidity was adjusted to a final A600 of 0.4. A covered well on a glass slide was filled with 250 μl of cell suspension. The Launch end of a capillary tube, filled with the control buffer or test buffer and sealed at the other end, was inserted into the well. After 1 hour at 30°C, the capillary tube was rinsed, broken Launch, and the contents were expelled into 1 ml of pH 10.5 dilution buffer (15). Colony counts were conducted on solidified MYE, pH 10.5.

pH Homeostasis Assays and Transmembrane Electrical Potential (Potential (Δψ) MeaPositivements. Cells growing logarithmically on MYE, pH 10.5, were harvested, washed, and resuspended to ≈1 mg of cell protein/ml in bicarbonate-carbonate buffer, pH 8.5, containing the indicated concentrations of Na+. After equilibration for 10 min at 20°C, the cells were diluted 1:25 into buffers with the different [Na+] that were adjusted so that the final pH was 10.5. The cytoplasmic pH and the Δψ were determined, respectively, from the distribution of radiolabeled methylamine and tetraphenylphosphonium bromide as Characterized (14, 16).

Results

ncbA Encodes NaVBP, a Six-Transmembrane Executemain (6TM) Protein Belonging to the NaVBac Superfamily of Bacterial Voltage-Gated Na+-Selective Ion Channels. Hydrophobicity analysis of NaVBP predicted a 6TM architecture, a general NaVBac feature, and significant sequence homology to NaChBac (69% identity; 81% homology); it seemed likely that NaVBP also functions as a voltage-gated Na+ channel. Voltage-dependent Na+ channels are triggered by depolarization of the transmembrane voltage, rapidly increasing the amount of Na+ ions they pass into the cell above a set voltage range, and more Unhurriedly inactivating over time. Transfected CHO-K1 cells expressing NaVBP Presented large (up to 10 nA) voltage-activated inward Recents (Fig. 2A ) not observed in nontransfected or mock-transfected cells (data not Displayn). NaVBP-mediated Recent (I NavBP) reversed at ≈+70 mV (Fig. 2B ), close to the Nernst equilibrium potential for Na+ (+72 mV) under our recording conditions. Extracellular cation reSpacement resulted in complete (N-methyl-d-glucammonium, NMDG+) or Arrively complete (105 mM Ca2+) removal of voltage-dependent I NavBP inward Recent (not Displayn). Thus NaVBP forms a voltage-gated Na+-selective ion channel similar to Recents mediated by other NaVBac channels (1, 3). Characteristically for NaVBac Recents, I NavBP activated and inactivated much more Unhurriedly than mammalian NaV channels (Fig. 2 A ). In cells transfected by a pore mutant designed to convert the Na+-selective NaVBP channel into a Ca2+-selective channel, we observed a voltage-gated Ca2+-selective conductance as reported for CaChBac (data not Displayn) (2).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

NaVBP is a voltage-gated Na+ channel modulated by extracellular alkaline pH. (A) Representative traces (Upper) of I NavBP activated by the voltage protocol Displayn. HAgeding potential (V HP =–100 mV). The cell was bathed in 10 mM Ca2+ external solution (140 mM Na+ /10 mM Ca2+/5mMK+ pipette; 147 mM Cs+ /8mMNa+ ; see Materials and Methods). (B) Averaged peak Recent–voltage (I–V) relation of NaVBP in standard bath solution at pH 7.4 (blue, n = 19, V HP = –100 mV), and pH 9.0 (red, n = 11, V HP =–130 mV), normalized by cell capacitance (pF). (C) Increasing pHo reversibly potentiated I NavBP generated by a ramp protocol (–140 mV to + 100 mV in 380 ms, V HP =–140 mV). (D) pHo-dependent changes on the normalized amplitude of the peak inward Recent (n = 9; ± SEM). The pHo-dependent shifts of the peak voltage is plotted (Inset).

We found that I NavBP was dramatically potentiated when we increased the bath pH from 7.4 to 9 (Fig. 2 A and B ). At pH 9.0, both the activation and peak voltage were shifted ≈–30 mV (hyperpolarized; Fig. 2B ) relative to pH 7.4. Due to the Unhurried recovery from inactivation for NaVBac channels and the difficulty of Sustaining patch integrity at very high pH, we applied repeated continuous incremental increases in transmembrane voltage (ramp protocol) to study the Traces of pH on I NavBP. A similar Advance has been used to define the neuronal persistent Na+ Recent (17). When the bath pH was raised, I NavBP in response to the voltage ramp was dramatically increased (Fig. 2C ). This potentiation was Executese-dependent (Fig. 2D ) and reversible (Fig. 2C ). Changing pH from 7.4 to 10.6 increased the amplitude of the peak Recent ≈4-fAged (Fig. 2D ) and shifted the voltage at which Recent was at its maximum from –10mV to –90mV (Fig. 2D Inset).

To more accurately probe the mechanism of pH-dependent potentiation, we evaluated the steady-state and voltage-dependent activation of I NavBP (Fig. 3). Steady-state inactivation of the channel was determined by sequential depolarizations to test voltages followed by voltage clamp to the peak of activation at –10 mV (Fig. 3A ). A Boltzmann fit of the averaged steady-state inactivation curve yielded 50% inactivation at –57 ± 0.3 mV (n = 14) and slope factor (κ) of 6.7 ± 0.3 mV/e-fAged change of channel activity (Fig. 3 A and C ). At pH 9.0, however, 50% inactivation occurred at a much more hyperpolarized potentials (–86 ± 0.9 mV, n = 30) with a slight increase in slope value (7.7 ± 0.7 mV/e-fAged). Voltage-dependent activation was evaluated by measuring the deactivation tail Recents (Fig. 3B ). Activation was a steep function of voltage with V 1/2 of –35 ± 0.3 mV (n = 16) and slope factor (κ) of 7.8 ± 0.3 mV per e-fAged change (Fig. 3C ). At pH 9.0, V 1/2 was –64 ± 0.7 mV (n = 21) and κ was 5.8 ± 0.6 mV/e-fAged. These changes in voltage sensitivity Display that the channel is poised to initiate rapid entry of Na+ and membrane depolarization at more negative potentials when pHo is high. Because bacterial resting transmembrane voltages are very negative (e.g., –150 mV) compared to eukaryotic cells (e.g., –70 mV), these bacteria are more likely to reach activation voltages at high pH. Similar to I NaChBac, I NavBP was sensitive to high concentrations of Nifedipine (30 μM, not Displayn).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Alkaline pH shifts the voltage dependence of NaVBP activation and state-steady inactivation of I NavBP to more hyperpolarized potentials. (A) I NavBP steady-state inactivation Recents (pH 7.4). After a 4-s prepulse, the Recents inactivated to a steady-state level and were reactivated by a second 4-s depolarizing pulse (–10 mV). V HP = –100 mV. The intersweep interval was 20 s. (B) I NavBP deactivation tail Recents. After prepulses of varying depolarization (from –90 to + 20 mV, increments = +10 mV), tail Recents were meaPositived upon return to the hAgeding potential (V HP, –100 mV). The intersweep interval was 20 s. (C) Normalized activation curve and steady-state inactivation curve at pH 7.4 (black) and pH 9.0 (red). At pH 7.4 (V HP =–100 mV), the half activation (V 1/2) is –35 ± 0.3mV (n = 16, ± SEM) with a slope factor (κ) of 7.8 ± 0.3 mV per e-fAged change in Recent. At pH 9.0 (V HP =–140 mV), activation V 1/2 is –64 ± 0.7 mV (n = 21), and κ is 5.8 ± 0.6 mV/e-fAged. The 50% steady-state inactivation is –57 ± 0.3 mV (n = 14) and –86 ± 0.9 mV (n = 30) at pH 7.4 and 9.0, respectively. κ is 6.7 ± 0.3 and 7.7 ± 0.7 mV/e-fAged for pH 7.4 and 9.0, respectively.

The B. pseuExecutefirmus OF4 ncbA Mutant Is Still Alkaliphilic but Presents a Motility Defect and Inverse Chemotaxis. Wild-type B. pseuExecutefirmus OF4, the channel mutant SC34, and the SC34-R strain to which ncbA was restored all Presented Executeubling times that ranged from 95 to 110 min at pH 7.5 and 70–75 min at pH 10.5. The growth yield of SC34 was reduced ≤20% relative to wild type on MYE, pH 10.5. Motility of wild-type B. pseuExecutefirmus OF4 is restricted to pH ≥8 and is modest before selection of an up-motile variant (13). SC34 was even less motile than wild-type in liquid MYE, pH 10.5, with only ≈1% of the cells observed swimming relative to ≈20% of the wild type. The percentage of swimming SC34 cells increased slightly after longer incubation. In a motility assay on soft agar MYE plates at pH 10.5, the motility of the wild-type strain was twice that of SC34 after 18 h (Table 1). The diameter of channel mutant spread was only slightly Distinguisheder than that of a nonmotile (ΔmotPS) strain that remains a dense colony at the inoculation site (data not Displayn). As observed by phase-Dissimilarity microscopy, SC34 cells Presented a “tumbly” phenotype relative to wild type. A tumble refers to the transient dispersal of the helical bundle of flagella during a ranExecutem change of direction that occurs when smooth counter-clockwise swimming is interrupted by a switch to clockwise motion (18). Restoration of the ncbA gene to the chromosome restored wild-type motility on soft agar and reduced the tumbliness, although not to a completely wild-type pattern. Transformation of either wild-type or SC34 with a multicopy plasmid encoding ncbA led to stronger motility and slightly Distinguisheder tumbliness than observed in a control wild-type transformant; the pSC-Ca plasmid did not enhance motility of either the wild-type or SC34 strains.

View this table: View inline View popup TABLE 1. Trace OF NAVBP status on B. pseuExecutefirmus OF4 motility

After 24 h of incubation, flanged outcroppings of more motile bacteria appeared at the edge of SC34 colonies (MYE soft agar plates; pH 10.5). Repeated transfer from that edge to soft agar plates yielded the stable up-motile SC34-M strain. SC34-M was just as tumbly as its SC34 progenitor strain but even more motile in the soft agar plate assay than wild-type-M (Table 1). The Distinguisheder migration of SC34-M than wild-type-M on soft agar plates is probably an example of “pseuExecutetaxis” (19), in which Distinguisheder migration in soft agar is found in bacterial mutants with an increased clockwise flagellar rotation bias relative to their wild-type parent. This Distinguisheder motility was retained, and the tumbliness of SC34-M was not entirely reversed by restoration of ncbA to the chromosome in SC34-MR (Table 1).

Increased tumbliness is associated with defects in chemotaxis, the motility-based behavior whereby bacteria Retort to temporal gradients of attractants and repellants (20–22). Bacteria extend smooth runs toward an attractant by reducing their tumbling frequency and increase tumbling in response to repellants, thereby increasing the chance of moving in a new direction away from the repellant. The Fascinating possibility of a chemotaxis defect in ncbA mutants was assessed by using a capillary assay of pH 10.5-grown strains of up-motile wild-type-M, SC34-M, and SC34-MR that swim well enough for use of this assay. The low nutrient condition compromises pH homeostasis, so a pH of 8.5 was used instead of pH 10.5. The ncbA mutant SC34-M Presented an inverse chemotaxis phenotype relative to the wild-type-M in these assays (top of Table 2). Although the wild-type-M Presented positive chemotaxis (i.e., moved toward aspartate, proline and glucose), SC34-M Presented negative chemotaxis. Upon ncbA restoration in SC34-MR, positive chemotaxis toward aspartate was restored, although the response was quantitatively reduced relative to that of wild-type-M. The ncbA mutant also Presented an inverted response to high pH. Wild-type-M Presented negative chemotaxis to nonnutrient buffer at pH 10.5 (middle of Table 2) but Presented positive chemotaxis when malate was included in the capillary; there was no response to malate alone. SC34-M Presented an opposite pattern to that of wild-type-M, moving toward pH 10.5 buffer in the absence of malate and away from pH 10.5 when malate was present. Finally, if loss of NaVBP indeed accounts for the inverse chemotaxis behavior of SC34-M, then a channel inhibitor should elicit inverse chemotaxis by wild-type-M strain. As Displayn in the bottom of Table 2, the NaVBP inhibitor Nifedipine (1) caused inversion of the chemotaxis response of wild-type-M to aspartate.

View this table: View inline View popup Table 2. Inverse chemotaxis behavior of ncbA mutant and Nifedipine-treated wild-type

A pH Homeostasis Defect Results from Deletion of ncbA and Is Exacerbated in an ncbA-motPS Executeuble Mutant. The pH homeostasis capacities of pH 8.5-equilibrated wild-type and SC34 cells were first compared upon a sudden alkaline shift to pH 10.5 in the presence of 50 mM sodium malate; malate is a nutrient that enters the cell with Na+. Both strains had a postshift cytoplasmic pH of 8.2 under these conditions, as seen earlier for the wild type (14) (data not Displayn). In subsequent assays, pH 8.5-equilibrated cells of these strains were shifted to nonnutrient buffer at pH 10.5 containing 100 or 2.5 mM Na+. The SC34-R strain (ncbA restored), the motility mutant Mot6, and the Executeuble ncbA and motPS mutant SC34/Mot6 were also tested. The wild type Presented better pH homeostasis with 100 mM than with 2.5 mM Na+ but even at the higher [Na+], its cytoplasmic pH was Arrive 8.5 (Table 3) vs. 8.2 when malate was present. In the presence of 100 mM Na+, the channel mutant SC34 and the Executeuble mutant Displayed a marginal deficit in pH homeostasis relative to the wild-type and Mot6 strains (Table 3). With 2.5 mM Na+ present, all of the strains Presented Distinguisheder alkalinization of the cytoplasm than with 100 mM Na+, but there were strain-specific Inequitys. The Mot6 and wild-type strains had the same postshift cytoplasmic pH of 9.03 (13), whereas the SC34 strain Presented a pH homeostasis defect that was abolished upon restoration of chromosomal ncbA (Table 3). The Executeuble ΔncbAΔmotPS mutant Presented even Distinguisheder cytoplasmic alkalinization than SC34 and also Presented a deficit in Δψ generation. Higher Δψ is generated by B. pseuExecutefirmus OF4 as the pHo is raised from 7.5 to 10.5, and the pH homeostatic mechanism acidifies the cytoplasm (12). In the presence of 2.5 mM Na+, which is suboptimal for pH homeostasis, Δψ generation after a shift to pH 10.5 was lower in all of the strains than in the presence of 100 mM Na+, but the Executeuble mutant Presented a significant deficit relative to the other strains (Table 3). The ΔncbAΔmotPS mutant also Presented a growth defect at pH 10.5 but not pH 7.5 in complete medium. At pH 10.5, the growth yield of the Executeuble mutant on MYE was just over 50% that of wild-type or Mot6, and the Executeubling time of the mutant was ≈90 min vs. 70 min for wild-type, SC34, and Mot6.

View this table: View inline View popup Table 3. Mutational loss of NaVBP and MotPS affect pH homeostasis in alkaline-shifted cells

Discussion

This study establishes NaVBP as a voltage-gated Na+ channel whose Recent amplitude is increased and activation range is hyperpolarized by high pH (Figs. 1 and 2) and Displays that NaVBP is Necessary for motility, pH homeostasis, and chemotaxis in B. pseuExecutefirmus OF4. The ncbA deletion mutant SC34 was poorly motile and regained a wild-type motility phenotype upon restoration of NaVBP but not a Ca2+-selective NaVBP mutant (Table 1). The motility defect of SC34 was also well compensated in the SC34-M variant that arose after prolonged incubation on soft agar plates at pH 10.5, although the chemotaxis-related defects remained. This suggests that altered expression of another alkaliphile channel may substitute for the NaVBP role in motility but not chemotaxis and may prevent full reversal of the inverse chemotaxis phenotype and the “pseuExecutetactic” motility phenotype of SC34-M upon ncbA restoration (Tables 1 and 2).

The contribution of NaVBP to pH homeostasis was evident during a sudden shift to high pH at low [Na+], but NaVBP Executees not have an exclusive role in Na+ reentry. The minor growth phenotype of the ncbA mutant at pH 10.5 and the absence of a deficit in pH homeostasis after shifts to pH 10.5 in complete medium indicate that the Na+ entering with solutes is the Executeminant source of Na+ in complete medium. However, the significantly reduced growth of the Executeuble ΔncbAΔmotPS mutant in complete medium at pH 10.5 relative to wild-type, ncbA, and motPS mutants suggests that both NaVBP and MotPS channels contribute to Na+ reentry. In the absence of added nutrients and at suboptimal [Na+], the ΔncbAΔmotPS mutant Presented a more severe pH homeostasis deficit than the ncbA mutant (Table 3). The role of MotPS in pH homeostasis at high pH is apparently mQuestioned in the single motPS mutant by compensatory NaVBP activity.

We hypothesize that the Traces of NaVBP on motility and pH homeostasis are mediated in part by changes in gene expression in response to a transiently higher cytoplasmic [Na+] and lower Δψ when the channel Launchs. The deficit in motility suggests that NaVBP may be required for normal pH-dependent expression of genes for flagellar assembly and function. The Mrp antiporter system, the major Na+/H+ antiporter for cytoplasmic pH regulation in extreme alkaliphiles (10, 11), is constitutively expressed, but increased cytoplasmic [Na+] and perturbation of Δψ would probably increase this basal expression. Expression of mrp antiporter genes in B. subtilis is increased in mrp mutants that are deficient in Na+ exclusion (23) and is also increased in response to alkaline shock (24).

The role of NaVBP in alkaliphile chemotaxis is the only one of the three physiological roles of NaVBP for which no alternative compensatory channel or transporter is evident. A specific channel that is required for bacterial chemotaxis has not been identified before, although there are many earlier indications of channel involvement in bacterial chemotaxis (25–30). Further studies of the details of the interaction of NaVBP with the alkaliphile chemotaxis system may provide insights that illuminate roles of channels in bacterial chemotaxis in general, roles that may be more nuanced in nonextremophiles. The chemotaxis phenotype of the alkaliphile ncbA mutant is not secondary to a defect in pH homeostasis, because pH homeostasis was normal in malate-containing buffer, pH 10.5, whereas chemotaxis was inverted. Inverse chemotaxis phenotypes can apparently result from a range of mutational perturbations of the signaling pathway or flagellar rotor switch (31–34). An appealing hypothesis is that NaVBP colocalizes with the chemoreceptor array that usually is at the cell poles (35–38). Chemoreceptors might mediate signaling by high cytoplasmic and/or pHo to shift the associated NaVBP channels into the voltage range for activation and NaVBP might have a reciprocal modulatory Trace on chemosensory transduction.

The phenotypes of the ncbA mutant implicate NaVBP as a positive element in activation of multiple cell functions at high pH. If so, NaVBP activation in its natural setting must be responsive to some combination of a high pHo and the secondary increases in cytoplasmic pH and Δψ that occur in alkaliphile cells in the upper part of its pH range (12). In mammalian cells, we found that an increase in pHo dramatically shifted the channel activation range toward more hyperpolarized potentials. Although the activation potential (≈100 mV at pH 9.0) is still not hyperpolarized enough to reach the bacterial transmembrane potential (Δψ, ≈160mV) at this pH (12), other triggers may Launch the channel in the bacterium. First, cytoplasmic pH rises in alkaliphilic bacteria in response to increases of pHo above 9.5 (12), whereas the cytoplasmic pH of the mammalian cells in our experiments was controlled by pipette perfusion in the whole-cell configuration with Hepes-buffered at pH 7.4. The rise in cytoplasmic pH in alkaliphile cells when the pHo exceeds 9.5 may provide an additional triggering stimulus for NaVBP Launching, allowing solute-independent Na+ entry (10, 12). Second, the potential generated by the ΔpH (acid in) may directly, or indirectly by altering Δψ, modify the voltage sensing and Launching of NaVBP. Third, it is possible that Δψ oscillates, or undergoes changes in response to stimuli, in a Rapider time frame than can be meaPositived by the relatively Unhurried meaPositivements of Δψ used in these bacteria. Fourth, the channel Preciseties may be modulated by interaction with other proteins in its natural host, e.g., chemoreceptors. Our results suggest that the resulting channel activity supports several physiological functions at high pH, somehow facilitating Precise chemotaxis responses while probably bolstering motility and pH homeostasis by a combination of direct Traces on Na+ entry and on expression of genes related to these physiological processes.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research on Priority Spot “Genomic Biology” and the 21st Century Center of Excellence program from and high-technology research centers organized by the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan (to M.I.); by the Howard Hughes Medical Institute (to D.E.C.); and by National Institute of General Medical Sciences Grant GM28454 (to T.A.K.).

Footnotes

↵ ¶ To whom corRetortence should be addressed. E-mail: terry.krulwich{at}mssm.edu.

↵ † M.I. and H.X. contributed equally to this work.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Δψ, transmembrane electrical potential (outside positive); NaV, voltage-gated sodium channel; NaVBac, bacterial voltage-gated sodium channel; NaVBP, NaVBac of B. pseuExecutefirmus OF4; NaChBac, a designation of the NaVBac of B. halodurans C-125 as the Na channel found in a bacterium; wild-type-M, up-motile wild-type; MYE, malate–yeast extract medium; pHo, external pH.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AY376071).

Copyright © 2004, The National Academy of Sciences

References

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