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Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

DeCaen PG, Takahashi Y, Krulwich TA, Ito M, Clapham DE - Elife (2014)

Bottom Line: Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential.Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria.This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

View Article: PubMed Central - PubMed

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

ABSTRACT
Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

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Related in: MedlinePlus

A comparison of the voltage-dependence of the bacterial Nav channels.(A) Left, Example traces from HEK 293T cells expressing sodium channels, NsvBa (black), NaChBac (gray) and NavBp (purple). Currents were activated by 0.5 s (NsvBa and NaChBac) or 1 s NavBp prepulses of increasing potential followed by a test pulse to −20 mV. (B) Resulting conductance-voltage and inactivation-voltage relationships were measured by plotting the average prepulse peak current converted to conductance, and reduction of test pulse peak current, respectively as a function of prepulse potential (n = 6–10, Error = ±SEM) and fit to a sigmoid equation. (C) Inactivation rate (τinact) -voltage relationship was measured by fitting the current decay to a single exponential equation for NsvBa (Black), NaChBac (gray) and NavBp (purple).DOI:http://dx.doi.org/10.7554/eLife.04387.004
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fig1s1: A comparison of the voltage-dependence of the bacterial Nav channels.(A) Left, Example traces from HEK 293T cells expressing sodium channels, NsvBa (black), NaChBac (gray) and NavBp (purple). Currents were activated by 0.5 s (NsvBa and NaChBac) or 1 s NavBp prepulses of increasing potential followed by a test pulse to −20 mV. (B) Resulting conductance-voltage and inactivation-voltage relationships were measured by plotting the average prepulse peak current converted to conductance, and reduction of test pulse peak current, respectively as a function of prepulse potential (n = 6–10, Error = ±SEM) and fit to a sigmoid equation. (C) Inactivation rate (τinact) -voltage relationship was measured by fitting the current decay to a single exponential equation for NsvBa (Black), NaChBac (gray) and NavBp (purple).DOI:http://dx.doi.org/10.7554/eLife.04387.004

Mentions: 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 (τinact) 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 Nav channels, the selectivity filter sequence (TLDSWGSG) deviates at the high field-strength site (SiteHFS 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.10.7554/eLife.04387.005Figure 2.Comparison of cation selectivity between the nonselective NsvBa and Na+-selective NaChBac channels.


Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus.

DeCaen PG, Takahashi Y, Krulwich TA, Ito M, Clapham DE - Elife (2014)

A comparison of the voltage-dependence of the bacterial Nav channels.(A) Left, Example traces from HEK 293T cells expressing sodium channels, NsvBa (black), NaChBac (gray) and NavBp (purple). Currents were activated by 0.5 s (NsvBa and NaChBac) or 1 s NavBp prepulses of increasing potential followed by a test pulse to −20 mV. (B) Resulting conductance-voltage and inactivation-voltage relationships were measured by plotting the average prepulse peak current converted to conductance, and reduction of test pulse peak current, respectively as a function of prepulse potential (n = 6–10, Error = ±SEM) and fit to a sigmoid equation. (C) Inactivation rate (τinact) -voltage relationship was measured by fitting the current decay to a single exponential equation for NsvBa (Black), NaChBac (gray) and NavBp (purple).DOI:http://dx.doi.org/10.7554/eLife.04387.004
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225499&req=5

fig1s1: A comparison of the voltage-dependence of the bacterial Nav channels.(A) Left, Example traces from HEK 293T cells expressing sodium channels, NsvBa (black), NaChBac (gray) and NavBp (purple). Currents were activated by 0.5 s (NsvBa and NaChBac) or 1 s NavBp prepulses of increasing potential followed by a test pulse to −20 mV. (B) Resulting conductance-voltage and inactivation-voltage relationships were measured by plotting the average prepulse peak current converted to conductance, and reduction of test pulse peak current, respectively as a function of prepulse potential (n = 6–10, Error = ±SEM) and fit to a sigmoid equation. (C) Inactivation rate (τinact) -voltage relationship was measured by fitting the current decay to a single exponential equation for NsvBa (Black), NaChBac (gray) and NavBp (purple).DOI:http://dx.doi.org/10.7554/eLife.04387.004
Mentions: 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 (τinact) 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 Nav channels, the selectivity filter sequence (TLDSWGSG) deviates at the high field-strength site (SiteHFS 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.10.7554/eLife.04387.005Figure 2.Comparison of cation selectivity between the nonselective NsvBa and Na+-selective NaChBac channels.

Bottom Line: Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential.Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria.This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

View Article: PubMed Central - PubMed

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

ABSTRACT
Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

No MeSH data available.


Related in: MedlinePlus