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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.


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NsvBa can be converted into a divalent cation-selective channel.The selectivity mutant NsvBa channel was replaced with the CaVBac selectivity filter sequence TLDDWADG (S187D:G189D:S190D). (A) Representative current traces from the mutant channels under the same conditions described in Figure 2. (B) Resulting current-voltage relationships measured for the mono and divalent conditions. (n = 4–8 for each channel, Error = ±SEM).DOI:http://dx.doi.org/10.7554/eLife.04387.010
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fig4s1: NsvBa can be converted into a divalent cation-selective channel.The selectivity mutant NsvBa channel was replaced with the CaVBac selectivity filter sequence TLDDWADG (S187D:G189D:S190D). (A) Representative current traces from the mutant channels under the same conditions described in Figure 2. (B) Resulting current-voltage relationships measured for the mono and divalent conditions. (n = 4–8 for each channel, Error = ±SEM).DOI:http://dx.doi.org/10.7554/eLife.04387.010

Mentions: The selectivity for Na+ ions in vertebrate Nav 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 (CaVs) 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 (PCa/PNa∼30, Figure 4; Figure 4—figure supplement 1) is much lower than mammalian CaV channels (PCa/PNa ≳ 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).10.7554/eLife.04387.009Figure 4.Summary of the relative permeability of cations from selectivity filter mutations.


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)

NsvBa can be converted into a divalent cation-selective channel.The selectivity mutant NsvBa channel was replaced with the CaVBac selectivity filter sequence TLDDWADG (S187D:G189D:S190D). (A) Representative current traces from the mutant channels under the same conditions described in Figure 2. (B) Resulting current-voltage relationships measured for the mono and divalent conditions. (n = 4–8 for each channel, Error = ±SEM).DOI:http://dx.doi.org/10.7554/eLife.04387.010
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4s1: NsvBa can be converted into a divalent cation-selective channel.The selectivity mutant NsvBa channel was replaced with the CaVBac selectivity filter sequence TLDDWADG (S187D:G189D:S190D). (A) Representative current traces from the mutant channels under the same conditions described in Figure 2. (B) Resulting current-voltage relationships measured for the mono and divalent conditions. (n = 4–8 for each channel, Error = ±SEM).DOI:http://dx.doi.org/10.7554/eLife.04387.010
Mentions: The selectivity for Na+ ions in vertebrate Nav 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 (CaVs) 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 (PCa/PNa∼30, Figure 4; Figure 4—figure supplement 1) is much lower than mammalian CaV channels (PCa/PNa ≳ 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).10.7554/eLife.04387.009Figure 4.Summary of the relative permeability of cations from selectivity filter mutations.

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