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A Prokaryotic Voltage-Gated Sodium Channel

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Science  14 Dec 2001:
Vol. 294, Issue 5550, pp. 2372-2375
DOI: 10.1126/science.1065635

Abstract

The pore-forming subunits of canonical voltage-gated sodium and calcium channels are encoded by four repeated domains of six-transmembrane (6TM) segments. We expressed and characterized a bacterial ion channel (NaChBac) from Bacillus haloduransthat is encoded by one 6TM segment. The sequence, especially in the pore region, is similar to that of voltage-gated calcium channels. The expressed channel was activated by voltage and was blocked by calcium channel blockers. However, the channel was selective for sodium. The identification of NaChBac as a functionally expressed bacterial voltage-sensitive ion-selective channel provides insight into both voltage-dependent activation and divalent cation selectivity.

Voltage-gated potassium (Kv), sodium (Nav), and calcium (Cav) channels underpin specialized higher order cell functions such as excitability, contraction, secretion, and synaptic transmission (1). Hundreds of Kv, Nav, and Cav channel proteins provide the tremendous functional diversity required for the complex behaviors of eukaryotic vertebrate and invertebrate cell types (2, 3). Ion channels are also widespread in prokaryotes, but their gating and function are poorly understood because few have been functionally expressed in a system in which their properties can be studied.

The primary structural characteristic of ion-selective channels is a pore region surrounded by two-transmembrane (2TM) segments. The first high-resolution images of a bacterial 2TM tetrameric channel revealed the structural basis of K+ ion selectivity encoded by the signature Gly-Tyr-Gly or Gly-Phe-Gly amino acid sequence in the pore region (4). In the primary structure of voltage-sensitive ion channels, an additional four transmembrane segments precede the pore-containing domain. The pore-forming subunits (α1) of Nav and Cav are composed of four similar repeats of 6TM domains (3, 5). It is thought that gene duplication of the 6TM Kv or TRP channels might have provided the precise structural requirements for highly selective Na+ and Ca2+ channels. In particular, selectivity for Ca2+ requires coordination of the Ca2+ ions by four negatively charged glutamic or aspartic acid residues lining the pore. Members of the TRP class of ion channels are presumably tetramers of single 6TM subunits, but only a subset of these channels are moderately Ca2+-selective (6). Here we report that a bacterial channel with strong similarity to the pore domains of Cav(7) is a voltage-dependent Na+-selective channel. The properties of this presumably tetrameric channel closely mimic those of Nav.

We previously isolated a mammalian putative voltage-gated cation channel, CatSper (8). CatSper is unique in that its amino acid sequence, especially in the putative pore region, is similar to that of Cav, even though CatSper is a 6TM protein. CatSper is required for cyclic nucleotide-mediated Ca2+ signaling in sperm and is essential for male fertility. Expression of CatSper in heterologous systems did not yield a detectable current. In a search for CatSper homologs for functional analysis, we discovered a putative gene assembled by shotgun sequencing of B. halodurans[(9), GenBank accession number BAB05220].

Isolation and sequencing of the gene encoding NaChBac (10) revealed an open reading frame (ORF) of 274 amino acids with a predicted molecular size of 31 kD and a calculated isoelectric point of 9.35 (Fig. 1A). NaChBac expressed in bacteria migrated during SDS–polyacrylamide gel electrophoresis with an apparent molecular mass of ∼34 kD (Fig. 1B). Hydrophobicity analysis was consistent with a primary structure containing 6TM domains (Fig. 1C). NaChBac contains an S4 segment characteristic of voltage-gated ion channels, with positively charged amino acids (Lys or Arg) interspersed every third residue (Fig. 1A). A BLAST search against the database revealed that the functional proteins with the closest similarity to NaChBac are Cav channels (7). In contrast to Cav channels that have four negatively charged amino acids in the pore, Navchannels have glutamate or aspartate residues in domains I and II but lysine and alanine in domains III and IV (Fig. 1C). Replacing the lysine and alanine in domains III and IV of Nav with glutamic acid conferred Ca2+ channel properties on the Nav channel (11). A functional Cavor Nav composed of only six transmembrane domains has never been effected, despite several attempts to artificially divide the large four-repeat α1 subunits into single repeats (12, 13).

Figure 1

Primary structure of NaChBac. (A) Deduced amino acid sequence. The putative six transmembrane domains (S1 to S6) and the pore region are indicated. The positively charged residues in the S4 region are in bold. (B) IPTG (isopropyl-β-d-thiogalactopyranoside)–inducible His-tagged NaChBac protein expressed in bacteria and detected by Western blot with antibody to His. (C) Hydropathy plot of NaChBac predicts six transmembrane domains (1 to 6) and a pore region (P). (D) Alignment of the putative pore region of NaChBac with that of CatSper and the four domains (I, II, III, and IV) from representative voltage-gated Ca2+ and Na+ channels. GenBank accession numbers for sequences used in the alignment are AF407332 (CatSper), X15539 (Cav1.2),M94172 (Cav2.2), 054898 (Cav3.1), X03638(Nav1.1), and X92184 (Nav1.8). Single-letter abbreviations for amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

We examined whether NaChBac could yield a functional channel current in a heterologous expression system. We transfected NaChBac into CHO-K1 (Chinese hamster ovary) or COS-7 (green monkey kidney) cell lines and recorded whole-cell current 24 to 48 hours after transfection (14). NaChBac-transfected cells displayed large (∼1000 to >10,000 pA) voltage-activated inward currents (Fig. 2A). This large current is unlike the small (50 pA), fast-inactivating, tetrodotoxin (TTX)-sensitive current (15) present in up to 20% of native CHO cells. Similar currents were not recorded in nontransfected or mock-transfected CHO-K1 or COS-7 cells. NaChBac-mediated current (I NaChBac) reversed at +70 mV, close to the Nernst potential of Na+ (E Na = +72 mV). The activation of I NaChBacact = 12.9 ± 0.4 ms at –10 mV,n = 32) was slow relative to currents conducted by Nav channels (τact < 2 ms). Inactivation was also slow (τinact = 166 ± 13 ms at –10 mV, n = 32) relative to the typically fast-inactivating Nav current [τinact< 10 ms (1)].

Figure 2

Activation and inactivation of NaChBac expressed in CHO-K1 cells. Voltage-clamp protocols are shown at the top, with representative traces below. (A)I NaChBac (middle panel) and averaged peak current-voltage (I-V) relation (lower panel). Currents were normalized to cell capacitance (9.0 ± 0.3 pF; n = 18). (B) Middle panel:I NaChBac activation and steady-state inactivation currents. Lower panel: normalized activation curve (n = 21, ±SEM) and steady-state inactivation curve (n = 19, ±SEM). (C) Recovery from inactivation. The time interval between the test pulse (–10 mV, 2000 ms) and the inactivation pulse (−10 mV, 1000 ms) was varied from 250 ms to 16 s. The ratios between currents elicited by the two pulses were used to construct the recovery curve (n = 20, ±SEM). The half-time for recovery was 660 ms. (D)I NaChBac single channels and ensemble average.

We evaluated voltage-dependent activation by measuring the deactivation tail current (Fig. 2B). A Boltzmann fit of the averaged activation curve yielded a midpoint voltage V h of –24 mV. Steady-state inactivation of the channel was determined by sequential depolarization to test voltages followed by clamp to the peak of activation at –10 mV. Steady-state inactivation was a steep function of voltage, with 50% inactivation at –40 mV (Fig. 2B). The channel recovered slowly from inactivation (Fig. 2C), with 50% recovery by 660 ms and 90% recovery by 5.5 s (–100 mV).

The single-channel properties of NaChBac were studied in the inside-out patch configuration (16). The unitary single-channel conductance was best fit with a slope of 12 ± 1 pS (n = 7 cells). Consistent with the whole-cell current, single channels were activated by depolarization, and both open and closed times varied as a function of voltage (17). An ensemble average of single-channel currents from five cells resembled whole-cell I NaChBac, with τact = 10 ± 3.5 ms and τinact = 203 ± 43 ms (Fig. 2D).

Cation replacement withN-methyl-d-glutamine (NMDG) resulted in the complete removal of voltage-dependent inward current (Fig. 3A), which suggested that NaChBac was impermeant to anions (18).I NaChBac was weakly permeant to Ca2+; no significant difference in current was observed by sequential perfusion with bath solution containing 1 and 10 mM extracellular calcium ([Ca2+]o) (Fig. 3B). In isotonic [Ca2+]o (cations replaced with 105 mM Ca2+), the inward current was <6% of that in normal [Na+]o (140 mM extracellular Na+) (7.6 ± 0.9 pA/pF at –10 mV, n = 8; Fig. 3B). In contrast, I NaChBac amplitude correlated well with [Na+]o (Fig. 3, C and D). To estimate the reversal potential (E rev) ofI NaChBac, we measured deactivation tail currents (Fig. 3E). Measured reversal potentials plotted as a function of [Na+]o had a slope of 57.8 mV per decade, close to the slope predicted for a Na+-selective pore (58 mV per decade). To estimate the relative ion selectivity of the channel, we measured changes in reversal potential while changing ionic composition. The calculated relative selectivity (±SEM) of NaChBac, as judged by measured E rev, wasP Na/P Ca = 72 ± 10 (n = 12);P Na/P Cs = 383 ± 56 (n = 8);P Na/P K = 171 ± 16 (n = 8). I NaChBac selectivity for Na+ is at least as high as that of traditional Nav channels (1, 19).

Figure 3

Ion selectivity ofI NaChBac. (A) Impermeability ofI NaChBac to Cl as shown by cation substitution with NMDG. (B) Low permeability of I NaChBac to Ca2+. Peak current in isotonic [Ca2+]o was 87 pA (7.6 ± 0.9 pA/pF, n = 8) compared to 1430 pA (135.9 ± 11 pA/pF, n = 8) in 140 mM [Na+]o/1 mM [Ca2+]o. (C)I NaChBac conductances at various values of [Na+]o. Current traces in (A) to (C) were elicited by a test pulse to −10 mV; holding potentialV H = −100 mV. (D) Mean current density plotted as a function of test potential in the presence of 20, 50, and 140 mM [Na+]o(n = 8, ±SEM). (E) Tail currents recorded at various test potentials after depolarizing to 0 mV.I NaChBac tail current amplitudes plotted as a function of test potential were used to determine reversal potentials (inset). [Ca2+]o ≈ 10 μM; capacitance = 9.1, 8.6, and 9.7 pF for (A), (C), and (E), respectively. (F) E rev as a function of [Na+]o. TheE rev/[Na+]o relation was best fit by a line with slope of 57.8 mV/decade (error bars are smaller than symbols).

The pharmacological sensitivity ofI NaChBac to Nav and Cav blockers most closely resembled that of L-type Cav channels. Cd2+ (100 μM; Fig. 4A), Co2+ (1 mM), and La3+ (1 mM) all reduced the channel current to various degrees (Fig. 4F). I NaChBac was most sensitive to two dihydropyridines, nifedipine and nimodipine (Fig. 4, C, D, and G), with half inhibitory concentrations (IC50s) of 2.2 μM and 1 μM, respectively (Fig. 4G). The dose-response curves for dihydropyridines are comparable to those for mammalian Cavs (20). I NaChBac was relatively insensitive to the T-type Cav channel antagonists mibefradil (IC50 = 22 μM) and Ni2+(IC50 = 720 μM) (Fig. 4, B, D, and G). The Cav N-type blocker ω-conotoxin GVIA and the Cav P/Q blocker ω-agatoxin IVA were ineffective even at concentrations (3 μM and 500 nM, respectively) well above those used to block their respective targets (Fig. 4F). The channel was completely insensitive to the Na+ channel blocker TTX (up to 30 μM; Fig. 4, E and F). The sensitivity ofI NaChBac to dihydropyridines is not obvious from a sequence comparison to the known sites for Cavdihydropyridine sensitivity (21–23). Not surprisingly, the residues involved in Nav TTX binding (24–27) do not match identically to residues in NaChBac.

Figure 4

Sensitivity of I NaChBac to Cav and Nav channel blockers. (A) Representative traces before (control) and after the addition of 100 μM Cd2+. (B)I NaChBac dose-response curves to Cd2+ and Ni2+. (C)I NaChBac was reversibly blocked by nifedipine. (D) I NaChBacsensitivity to the dihydropyridine class (nifedipine and nimodipine) of L-type Cav channel blockers and insensitivity to the T-type Cav channel blocker mibefradil. (E) Insensitivity of I NaChBac to TTX. (F) Summary of I NaChBac inhibition by various Cav-blocking agents. (G) Summary of concentration of agent needed to block I NaChBacby 50% (IC50) as measured from dose-response curves.

NaChBac encodes a 6TM domain, dihydropyridine-sensitive, TTX-insensitive Na+-selective current.I NaChBac differs from traditional 24TM Nav eukaryotic channels in its slower (roughly one-tenth the speed of Nav) activation, inactivation, and recovery from inactivation. The slow inactivation kinetics are similar to those of mammalian persistent sodium current I NaP(28). A noninactivating, TTX-insensitive voltage-gated Na+ channel current has been recorded from mammalian dorsal root ganglion neurons, but its sensitivity to dihydropyridines is not known (29). NaChBac-related mammalian homologs may account for some persistent Na+ currents.

We can make several conclusions from the ability of NaChBac to form functional voltage-gated Na+ selective channels. First, Na+ selectivity does not require the four-domain repeat structure present in Nav channels. Proper orientation of the selectivity filter can presumably be made in homotetramers of NaChBac. Second, the presence of glutamic and aspartic residues in the pore does not ensure Ca2+ selectivity. Third, as is apparent from the defined Cav and Nav sites for dihydropyridine and TTX block, pharmacologic sensitivity need not be correlated with channel selectivity.

NaChBac should facilitate studies of the activation and inactivation gates of the sodium channel. Recently, Hilber et al. (30) showed that when the domain IV 1529 alanine residue of rat skeletal muscle Nav was substituted by aspartic acid, inactivation was drastically slowed and recovery time constants were slowed to the 100-s range, implying that the selectivity filter is involved in channel gating. Interestingly, the corresponding residue in the slowly inactivating NaChBac is a glutamic acid.

Hypothetically, Cav channels may require a flexible tertiary structure around the glutamate and aspartate residues in the pore to enable the channel filter to bind one Ca2+ ion with high affinity or two with lower affinity (31). This Ca2+ binding flexibility may be provided by the similar but nonidentical amino acids surrounding the glutamate and aspartate residues in the four repeats in the pore-forming α1subunit (32). Because all four repeats are presumably identical in a NaChBac tetramer, this flexibility is lost. Heterotetramers of NaChBac homologs might recreate this flexible environment to allow formation of Ca2+-selective channels, considerably increasing the diversity of this channel class. The simple NaChBac channel thus emphasizes the evolutionary utility of the 6TM building block.

We have not tested the biological role of NaChBac in the extremophileB. halodurans. This bacterium lives in extremely high salt (up to 1 M), highly alkaline (up to pH 11) conditions, and thus Na+ influx through the open channel should be large. Na+ drives the flagellar motor used by alkaphilicBacillus (33–36), and NaChBac is a good candidate for control of flagellar activity. Regardless of its prokaryotic function, NaChBac's signal importance will be in providing the protein needed for structural studies of the Na+selectivity filter and voltage sensor.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: dclapham{at}enders.tch.harvard.edu

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