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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.

No MeSH data available.


Related in: MedlinePlus

The effect of temperature and pH on NsvBa selectivity.(A) Top, Representative NsvBa current traces recorded at 34⁰C under different extracellular cationic conditions with a pH = 9. Currents were activated by increasing potentials from −10 mV to 70 mV from a holding potential of −140 mV. Bottom, Corresponding current–voltage relationships measured under the conditions listed above (n = 4 or 5 for each condition, Error = ±SEM). Peak inward currents were normalized to the Na+ current measured either in 110 or 150 mM extracellular solution. Erev was calculated by fitting the current from 0 mV to 60 mV to a linear equation, and determining the potential at zero current. When corrected for liquid junction potential differences in the salines, Erev under these conditions were 40 mV for 150 mM Na+; 36 mV for 110 mM Na+; 41 mV for 110 mM Ca2+ and 43 mV for 150 mM KCl. (B) The calculated relative permeability for cations measured at 22⁰C and 34⁰C.DOI:http://dx.doi.org/10.7554/eLife.04387.015
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fig6s1: The effect of temperature and pH on NsvBa selectivity.(A) Top, Representative NsvBa current traces recorded at 34⁰C under different extracellular cationic conditions with a pH = 9. Currents were activated by increasing potentials from −10 mV to 70 mV from a holding potential of −140 mV. Bottom, Corresponding current–voltage relationships measured under the conditions listed above (n = 4 or 5 for each condition, Error = ±SEM). Peak inward currents were normalized to the Na+ current measured either in 110 or 150 mM extracellular solution. Erev was calculated by fitting the current from 0 mV to 60 mV to a linear equation, and determining the potential at zero current. When corrected for liquid junction potential differences in the salines, Erev under these conditions were 40 mV for 150 mM Na+; 36 mV for 110 mM Na+; 41 mV for 110 mM Ca2+ and 43 mV for 150 mM KCl. (B) The calculated relative permeability for cations measured at 22⁰C and 34⁰C.DOI:http://dx.doi.org/10.7554/eLife.04387.015

Mentions: 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 V1/2 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 Px/PNa 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 hNav1.1 channel was less temperature- (Q10 = 1.2 and 1.4, Figure 6D) and pH- (Δ V1/2 < 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 Nav channels.10.7554/eLife.04387.014Figure 6.Temperature and pH dependence of sodium 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)

The effect of temperature and pH on NsvBa selectivity.(A) Top, Representative NsvBa current traces recorded at 34⁰C under different extracellular cationic conditions with a pH = 9. Currents were activated by increasing potentials from −10 mV to 70 mV from a holding potential of −140 mV. Bottom, Corresponding current–voltage relationships measured under the conditions listed above (n = 4 or 5 for each condition, Error = ±SEM). Peak inward currents were normalized to the Na+ current measured either in 110 or 150 mM extracellular solution. Erev was calculated by fitting the current from 0 mV to 60 mV to a linear equation, and determining the potential at zero current. When corrected for liquid junction potential differences in the salines, Erev under these conditions were 40 mV for 150 mM Na+; 36 mV for 110 mM Na+; 41 mV for 110 mM Ca2+ and 43 mV for 150 mM KCl. (B) The calculated relative permeability for cations measured at 22⁰C and 34⁰C.DOI:http://dx.doi.org/10.7554/eLife.04387.015
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6s1: The effect of temperature and pH on NsvBa selectivity.(A) Top, Representative NsvBa current traces recorded at 34⁰C under different extracellular cationic conditions with a pH = 9. Currents were activated by increasing potentials from −10 mV to 70 mV from a holding potential of −140 mV. Bottom, Corresponding current–voltage relationships measured under the conditions listed above (n = 4 or 5 for each condition, Error = ±SEM). Peak inward currents were normalized to the Na+ current measured either in 110 or 150 mM extracellular solution. Erev was calculated by fitting the current from 0 mV to 60 mV to a linear equation, and determining the potential at zero current. When corrected for liquid junction potential differences in the salines, Erev under these conditions were 40 mV for 150 mM Na+; 36 mV for 110 mM Na+; 41 mV for 110 mM Ca2+ and 43 mV for 150 mM KCl. (B) The calculated relative permeability for cations measured at 22⁰C and 34⁰C.DOI:http://dx.doi.org/10.7554/eLife.04387.015
Mentions: 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 V1/2 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 Px/PNa 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 hNav1.1 channel was less temperature- (Q10 = 1.2 and 1.4, Figure 6D) and pH- (Δ V1/2 < 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 Nav channels.10.7554/eLife.04387.014Figure 6.Temperature and pH dependence of sodium 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